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Microbiology Notes Based on INC Syllabus.

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Two Marks

Different shape of Bacteria (2 Mark)

Bacteria is found in different shapes

1. Cocci – Round or oval cells.

2. Bacilli - Rod shaped cells.

3. Spirilla – Non flexuous spiral forms.

4. Vibrios – Curved or comma-shaped rods.

5. Spirochaetes- Slender and flexuous spiral forms.

6. Mycoplasmas – Cell wall absent, So, bacteria found as round or oval cells.

7. Actinomycetes – Branched filamentous bacteria.

Name four Bacteria (2 Mark)

1. Staphylococci

2. Streptococci

3. Mycobacterium

4. Shigella

Name four virus (2 Mark)

1. Hepatitis

2. Picorna viruses

3. Retroviruses

4. Reoviruses

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Name four Fungi (2 Mark)

1. Aspergillus

2. Histoplasma

3. Rhizopus

4. Trichophyton

Name four Protozoa (2 Mark)

1. Plasmodium

2. Leishmania

3. Entamoeba

4. Balantidium

Name four worms (2 Mark)

1. Necator

2. Ascaris

3. Taenia

4. Trichuris

Name four bacteria causing food poisoning ?

1. Clostridium

2. Staphylococci

3. Salmonella

4. Listeria

Name four viruses causing food poisoning ?

1. Hepatitis A

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2. Norovirus

3. Rotovirus

4. Adenovirus

Types of Flagella

1. Monotrichous

2. Amphitrichous

3. Lophotrichous

4. Peritrichous

Examples for Transport Media

1. Stuart’s Transport Medium

2. Cary-Blair Medium

3. Ames Transport Medium

4. Venkatraman Ramakrishnan Medium

Examples for Anaerobic Media

1. Thioglycollate Broth

2. Cooked Meat Broth.

Examples for RNA viruses

1. Picorna viruses

2. Arenaviruses

3. Retroviruses

4. Filoviruses

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Examples for DNA viruses

1. Parvo viruses

2. Herpes viruses

3. Pox viruses

4. Papova Viruses
Example for Anti viral Drugs

1. Ribavirin

2. Protease inhibitors

3. Antisense RNA

4. Nucleoside RT

Different Shape of viruses

1. Binal Virus

2. Helical virus

3. Polyhedral virus

4. Spherical virus

Examples for Anti bacterial drugs

1. Penicillin

2. Cephalosporins

3. Monobactams

4. Rifampin

5. Tetracycline

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Examples for Antifungal Drugs

1. Polyenes

2. Nystatin

3. Amphotericin B

4. Azoles

Examples for Antiprotozoal Drugs

1. Quinine

2. Atovaquone

3. Pyrimethamine

4. Mefloquine

Examples for Anti Helminthic Drugs

1. Benzimidazoles

2. Mebendazole

3. Albendazole

4. Piperazine

Organism causing Sexual Transmit Disease

1. Treponema

2. Herpes Virus

3. HIV

4. Chlamydia

5. Mycoplasma

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Organisms affecting Central Nervous System

1. Treponema

2. Plasmodium

3. Rabies

4. Poliomyelitis

5. Toxoplasma

Organisms causing Nosocomial Infections

1. Staphylococcus

2. Acinetobacter

3. Enterococci

4. Klebsiella

5. Pseudomonas

Types of Immunoglobulins

1. IgG

2. IgD

3. IgA

4. IgM

Types of Vaccines

1. Live Vaccines

2. Toxoid Vaccines

3. Attenuated Vaccines

4. Killed Vaccines

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Examples for Dermatophytes

1. Microsporum

2. Trichophyton

3. Epidermophyton

Examples for Plasmodium species

1. Plasmodium vivax

2. Plasmodium ovale

3. Plasmodium malariae

4. Plasmodium falciparum

Types of Hepatitis Viruses

1. Hepatitis A

2. Hepatitis B

3. Hepatitis C

4. Hepatitis D

5. Hepatitis E

Screening and Confirmative Test for HIV

1. Enzyme Immunoassay

2. Western blot

3. Nucleic acid based assay

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Diagram – Structure of Bacteria (2 Mark)

Diagram – Trichomonas Vaginalis (2 Mark)

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Diagram – Immunoglobulins (2 Mark)

Microbiology ?

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Microbiology is the study of living things so small that they cannot be seen with the
naked eye. Microorganisms are microscopic organisms that exist as single cells or
cell clusters.

Main objective of microbiology is to learn and excel in knowing information about

microorganisms, their disease, treatment and preventive measures.

Antiseptic: (2 Mark)

It is a chemical agent of disinfection that is mild enough to used on human skin or

tissues.

Sterilization: (2 Mark)

The process by which all micro organisms present on or in an object are

destroyed or removed. Sterilization is classified into physical methods and chemical
methods. Physical methods include sunlight, drying, heat, radiation and chemical
methods includes gases and chemical compounds such as ethylene oxide,
formaldehyde, halogens, surface active compounds etc.

Vaccination: (2 Mark)

It is a practice of using modified (killed or attenuated) microorganisms, or portions, to

induce immunity to a particular disease without actually causing a disease.

Disinfection: (2 Mark)

The elimination or inhibition of pathogenic micro organisms in or on an object. Its

include chemical agents such as gases and chemical compounds such as ethylene
oxide, formaldehyde, halogens, surface active compounds etc.

Antibiotic: (2 Mark)

Microbially produced substance that has antimicrobial properties. E.g. Penicillin,

cephalosporin, Tetracycline.

Nosocomial infection: (2 Mark)

Infections are acquired in hospitals.

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Antibodies: (2 Mark)

Antibodies are proteins produce by the immune systems in response to infection.

Pasteurization: (2 Mark)

 A mild heating used to destroy pathogens and spoilage organisms present in

food and drink. Pasteurization include conventional and modern methods.
Conventional methods of pasteurization mean that the heating of milk to a
temperature between 63 and 65 o C for a period of about 30 minutes, and then
cooling it to room temperature. Recently, they revised of that process, milk can also
be “flash pasteurized” by raising temperature to about 71o C for a minimum of 15
seconds. Ultra-high-pasteurization is the process uses even higher temperature of
about 90-130 o C. for periods of a second or more next. Flash pasteurization uses
higher temperature than conventional pasteurization, but the temperature
maintained for a shorter time. The product is then rapidly cooled to below 10 o C, a
temperature at which it can then be stored. The use of flash pasteurization is to
eliminate harmful microorganisms while maintaining the product to its natural state.

Food poisoning: (2 Mark)

A condition that occurs following the ingestion of a preformed toxin produced by

microorganisms present in food.

Edward Jenner

(1749-1823)

 At age Thirteen, he was apprenticed to a surgeon. Then in 1770, he moved to London

to work with John hunter, an eminent Scottish surgeon who encouraged Jenner to be
experimental in his approach to medicine.

 In 1773, he returns to Berkeley and practice as a country doctor.

 During his period, there were severe outbreak of small pox and large number of
people were severely affected. Jenner noted that dairy workers who had been
exposed to low pox, seemed immune to severe infection.

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 On May 14, 1796, he removed the third from a low pox lesion from dairy maid and
inoculated into eight year old boy with small pox. The be remained healthy.

 He called his method vaccination, using the latin word vacca meaning low and
vaccinia meaning low pox.

 As the demand for the vaccine exceeds he discovered that lymph from small pox
pustule can be used and dried in glass tube to be used later.

 He died at the age of 73. Nearly two centuries after Jenner’s experimental
vaccination, the WHO declared endemic small pox to be eradicated.

Joseph Lister

(1827 – 1912)

 Joseph Lister had made fundamental revolution in surgery wih the introduction of
antiseptic method.

 Lister was born in England and studied medicine at University college.

 Lister was concerned with high mortality rate of post-amputation patients and the
high rate of gangrene after surgery.

 He applied knowledge that bacteria caused disease and existence of airborne

microorganisms. He concludes that airborne bacteria could cause infection in
surgical wounds.

 He used carbonic acid in the operating room and use it to sterilize the surgical
instruments and his hands.

 Carbolic acid is extremely harmful to those who came in contact with it.

 Later, Lister found milder antiseptics and heat sterilization for the surgical
instruments.

 The mortality rate dropped drastically with the use of an antiseptic method.

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Alexander Fleming

(1881-1955)

 Fleming was born in 1881 in Scotland.

 In 1901 he began his medical career in St.Mary’s Hospital Medical School.

 Fleming showed that, in deep wounds, the bacteria survive treatment by antiseptics
while WBC in the wound is destroyed. This creates worse condition in which
bacterial infection spread rapidly. So, he decided to focus on safe antibacterial
substances.

 In 1921 he discovered that nasal mucus destroyed bacteria in a petric dish and
isolated compound responsible for antibacterial action. He called this compound
“Lysozyme” found in saliva, blood, tears, pus, milk and in egg whites.

 In 1928, he made greatest discovery. While he was growing cultures of bacteria in

petric dishes for experiment, he accidently, left uncovered certain plates for several
days. Fleming notices mold growing in the dishes and begun to discard them when
he noticed, that bacteria near the molds were being strain of penicillium and deduced
an antibacterial compound from mold and named it penicillin.

 He shared the Nobel Prize with Florey and chain in 1945.

 The discovery of antibiotic Penicillin saved millions of life around the world.

MMR Vaccine

The MMR vaccine is very safe, and it is effective at preventing measles, mumps, and
rubella. MMR vaccine has been linked with a very small risk of febrile seizures
(seizures or jerking caused by fever). Febrile seizures following MMR are rare and
are not associated with any long-term effects. Because the risk of febrile seizures
increases as infants get older, it is recommended that they get vaccinated as soon as
recommended.

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Some people may experience swelling in the cheeks or neck. MMR vaccine rarely
causes a temporary low platelet count, which can cause a bleeding disorder that
usually goes away without treatment and is not life threatening.

DTP Vaccine

Diphtheria, tetanus, and pertussis vaccine (DTaP) can help prevent these
diseases. Most children who are vaccinated with DTaP will be protected throughout
childhood. Many more children would get these diseases if we stopped vaccinating.
DTaP is a safer version of an older vaccine called DTP.

BCG Vaccine

BCG, or bacille Calmette-Guerin, is a vaccine for tuberculosis (TB) disease. Many

foreign-born persons have been BCG-vaccinated. BCG is used in many countries with
a high prevalence of TB to prevent childhood tuberculous meningitis and miliary
disease. The BCG vaccine should be considered only for very select persons who meet
specific criteria and in consultation with a TB expert. BCG vaccination should not be
given to persons who are immunosuppressed (e.g., persons who are HIV infected) or
who are likely to become immunocompromised (e.g., persons who are candidates for
organ transplant).

BCG vaccination should not be given during pregnancy. Even though no harmful
effects of BCG vaccination on the fetus have been observed, further studies are
needed to prove its safety.

Drugs for HIV Treatment

Antiretroviral Therapy - HIV treatment involves taking medicines that slow the
progression of the virus in your body. HIV is a type of virus called a retrovirus, and the
drugs used to treat it are called antiretrovirals (ARV). These drugs are always given in
combination with other ARVs; this combination therapy is called antiretroviral therapy
(ART). ART reduces the amount of virus (or viral load) in your blood and body fluids.
ART is recommended for all people living with HIV.

Name four bacterial diseases ?

Meningitis

Bacterial vaginosis

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Leptospirosis

Lyme disease

Name four viral diseases ?

Rabies

Meningitis

Viral Hemorrhagic fever

Ebola viral disease

Varicella

Name four fungal diseases ?

Fungal meningitis

Pneumocystis pneumonia

Ring worm

Candidiasis

Name four Protozoal diseases ?

Trichomoniasis

Amebiasis

Toxoplasmosis

Cryptosporidiosis

Name four helminthic Diseases ?

Schistosomiasis

Ascariasis

Taeniasis

Trichuriasis

Steps in Gram Staining ?

Steps:

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1. Prepare bacterial smear on a glass slide

2. Heat fix using a spirit lamp

3. Bacterial cells fixed on slide

4. Put a drop of crystal violet and wait for 2-3 minutes and remove stain with tap
water.

5. Bacterial cells are stained

6. Stain with Gram’s iodine and wait for 1-2 minutes; wash stain with tap water.

7. Bacterial cells are stained

8. Put slide in a beaker containing 95% ethanol to decolourise the stain.

9. Stains may or may not be decolurised.

10. Stain with safranin and wait for 2-4 minutes.

11. The bacterial may or may not be stained with safranin.

12. Wash with tap water and observe using oil immersion in microscope.

 If the bacterial cells take crystal violet and appear dark purple color, they are called
Gram-positive bacteria.

 If the bacterial cells take safranin and appear pink in colour, they are called
Gram-negative bacteria.

Hydatid cyst ?

Hydatid cysts containing the larval parasites grow large enough to cause discomfort,
pain, nausea, and vomiting. The cysts grow over the course of several years before
reaching maturity and the rate at which symptoms appear typically depends on the
location of the cyst. The cysts are mainly found in the liver and lungs but can also
appear in the spleen, kidneys, heart, bone, and central nervous system, including the
brain and eyes. Cyst rupture is most frequently caused by trauma and may cause mild
to severe anaphylactic reactions, even death, as a result of the release of cystic fluid.

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Name two viruses causing diarrhea ?

Rota virus

Norovirus

Name two spore bearing bacteria ?

Bacillus

Clostridium

Name two fungi causing infection in skin ?

Candida

Dermatophytes

Bile staining ova ?

Trichuris trichura
Ascaris lumbricoides
Taenia saginata and T solium

Bile staining depends on the lipid content

Some eggs also have receptors for bile on their surface (shell).

CSSD ?

The central sterile services department (CSSD), also called sterile processing
department (SPD), sterile processing, central supply department (CSD), or central
supply, is an integrated place in hospitals and other health care facilities that
performs sterilization and other actions on medical devices, equipment and
consumables; for subsequent use by health workers in the operating theatre of the
hospital and also for other aseptic procedures, e.g. catheterization, wound stitching
and bandaging in a medical, surgical, maternity or paediatric ward.

Mantoux Test ?

The Mantoux test or Mendel-Mantoux test (also known as the Mantoux screening
test, tuberculin sensitivity test, Pirquet test, or PPD test for purified protein
derivative) is a tool for screening for tuberculosis (TB) and for tuberculosis diagnosis.
It is one of the major tuberculin skin tests used around the world, largely replacing
multiple-puncture tests such as the tine test.

IgM ?

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Immunoglobulin M, or IgM for short, is a basic antibody that is produced by B cells.

IgM is by far the physically largest antibody in the human circulatory system. It is the
first antibody to appear in response to initial exposure to an antigen. The spleen,
where plasmablasts responsible for antibody production reside, is the major site of
specific IgM production.

IgD ?

Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in

the plasma membranes of immature B-lymphocytes where it is usually coexpressed
with another cell surface antibody called IgM. IgD is also produced in a secreted form
that is found in very small amounts in blood serum, representing 0.25% of
immunoglobulins in serum. Relative molecular mass and half-life of sIgD is
185 kDa and 2.8 days, respectively. Secreted IgD is produced as
a monomeric antibody with two heavy chains of the delta (δ) class, and two Ig light
chains.

IgG ?

Immunoglobulin G (IgG) is a type of antibody. Each IgG has two antigen binding sites.
Representing approximately 75% of serum antibodies in humans, IgG is the most
common type of antibody found in the circulation. IgG molecules are created and
released by plasma B cells. IgG is the main type of antibody found in blood
and extracellular fluid allowing it to control infection of body tissues. By binding many
kinds of pathogens such as viruses, bacteria, and fungi, IgG protects the body from
infection.

DISEASES CAUSED BY DIFFERENT MICROBES

Bacterial Diseases of the skin

Disease Pathogen
Impetigo Staphylococcus aureus; Streptococcus
pyogenes

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Folliculitis Staphylococcus aureus

Toxic shock syndrome Staphylococcus aureus
Necrotizing Fascititis Streptococcus pyogenes
Erysipelas Streptococcus pyogenes
Pseudomonas dermatitis Pseudomonas aeruginosa
Otitis extema Pseudomonas aeruginosa
Acne Propionibacterium acnes

Viral Diseases of the skin

Disease Virus

Warts Papilloma virus spp.

Small pox Small pox virus
Chicken pox Varicella –zoster virus
Shingles Varicella –zoster virus
Herpes simplex Herpes simplex virus type -2
Measles Measles virus
Rubella (German measles) Rubella virus
Fifth disease (erythema injectiosum) Human parvovirus B19
Rosela Human herpesvirus 6

Fungal disease and its causative agent

Disease Fungus
Ringworm (tinea) Microsporum, trichophyton,
epidermophyton spp.
Sporotrichosis Sporothrix schenckii
Candidiasis Candida albicans

Parasitic infestation of the skin

Disease Parasite
Scabies Sarcoptes scabiei (mite)

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Pediculosis (lice) Pediculus humanus capitis

Microbial Disease associated with the Eye

Bacterial Diseases

Disease Organism
Bacterium
Neonatal gonorrheal ophthalmia Neisseria gonorrhoeae
Inclusion conjunctivitis Chlamydia trachomatis
Trachoma Chlamydia trachomatis

Virus
Herpetic keratitis Herpes simplex type 1 virus

Protozoan
Acanthamoeba keratitis Acanthamoeba spp.

Microbial Disease of the Nervous System

Disease Organism
Bacterium
Haemophilus influenzae meningitis Haemophilus influenzae
Meningococcal meningitis Neisseria meningitis
Pneumococcal meningitis Streptococcus pneumoniae
Listeriosis Listeria monocytogenes
Tetanus Clostridium tetani
Botulism Clostridium botulinum
Leprosy Mycobacterium leprae

Virus
Poliomyelitis Poliovirus
Rabies Rabies virus
Arboviral encephalitis Arbovirus

Fungus
Cryptococcosis Cryptococcus neoformans

Protozoan
African trypanosomiasis Trypanosoma brucei
Naegleria meningoencephalitis Naegleria fowleri

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Microbial Disease of the Cardiovascular and Lymphatic system

Bacterial Disease Bacterium

Puerperal sepsis Streptococcus
Endocarditis subacute bacterial Mostly alpha-hemolytic streptococci
Acute bacterial Staphylococcus aureus
Pericarditis Streptococcus pyogenes
Rheumatic fever Group A beta-hemolytic Streptococci
Tulanemia Francisella tularensis
Brucellosis Brucella spp
Anthrax Bacillus anthracis
Gangrene Clostridium perfringens
Cat – scratch disease Bartonella henselae
Plague Yersinia pestis
Relapsing fever Borrelia spp.
Lyme disease Borrelia burgdorferi
Ehrlichiosis Ehrlichia spp.
Epidemic typhus Rickettsia prowazekii
Endemic Murine typhus Rickettsia typhi
Rocky mountain spotted fever Rickettsia rickettsii

Viral Disease virus

Burkitt’s lymphoma Epstein-Barr virus (EBV)
Injectious mononucleosis EBV
Cytomegalovirus Cytomegalovirus (CMV)
Yellow fever Arbovirus (yellow fever virus)
Dengue Arbovirus (dengue fever virus)
Viral hemorrhagic fever Filovirus
(Marburg, Ebola, Lassa)
Hantavirus pulmonary syndrome Sin Nomne hantavirus

Protozoan Disease Protozoan

American trypanosomiasis Trypanosoma cruzi
Toxoplasmosis Toxoplasma gondii
Malaria Plasmodium spp.
Leishmaniasis Leishmania spp.
Babesiosis Babesia microtic

Helminthic Disease Helminth

Schistosomiosis Schistoma spp.
Swimmer’s itch Larvae of schistosomes

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Microbial Diseases of the Upper Respiratory system

Bacterial Disease Bacterium

Streptococcal pharyngitis Streptococci pyogenes
(strep throat)
Scarlet fever Erythrogenic toxin-producing strains of
Streptococcus pyogenes
Diphtheria Corynebacterium diphtheriae
Otitis media Staphylococcus aureus, Streptococcus
pneumoniae and Haemophilus influenzae

Viral Disease virus

Common cold Coronaviruses, rhinoviruses

Microbial Diseases of the Lower Respiratory system

Bacterial Disease Bacterium

Pertussis (whooping cough)
Tuberculosis
Pneumococcal pneumonia
Haemophilus influenzae pneumonia
Mycoplasmal pneumonia
Legionellosis
Psittacosis (ornithosis)
Chlamydial pneumonia
Q fever
Bordetella pertussis
Mycobacterium tuberculosis
Streptococcus pneumoniae
Haemophilus influenzae
Mycoplasma pneumoniae
Legionella pneumophila
Chlamydia psittaci
Chlamydia pneumoniae
Coxiella burnetii

Viral Disease virus

Respiratory syncytial virus Respiratory syncytial virus
Influenza Influenza virus

Fungal Disease fungus

Histoplasmosis Histoplasma capsulatum

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Coccidioidomycosis Coccidioides immitis

Pneumocystis pneumonia Pneumocystis jiroveci
Blastomycosis Blastomyces dermatitidis

MICROBIAL DISEASE OF THE DIGESTIVE SYSTEM

Bacterial Disease of the Mouth

Disease Bacteria
Dental caries Primary Streptococcus mutans
Periodontal disease Porphyromonas spp.

Bacterial Disease of the Lower Digestive System

Disease Bacteria
Staphylococcal food poisoning Staphylococcus aureus
Shigellosis (bacillary dysentery) Shigella spp
Salmonellosis Salmonella enterica
Typhoid fever Salmonella typhi
Vibrio gastroenteritis Vibrio cholerae
Vibrio parahaemolyticus gastroenteritis Vibrio parahaemolyticus
Vibrio vulnifucus gastroenteritis
Enterotoxigenic E.coli gastroenteritis Vibrio vulnificus
Enteroinvasive E.coli gastroenteritis Escherichia coli
Enteroinvasive E.coli gastroenteritis Escherichia coli
Enterohemorrhagic E.coli gastroenteritis Escherichia coli
Campylobacter gastroenteritis E.coli 0157:H7
Helicobacter peptic ulcer disease
Yersinia gastroenteritis Camphylobacter jejuni
Clostridium perfringens gastroenteritis Helicobacter pylori
Bacillus cereus gastroenteritis Yersinia enterocolitica
Clostridium perfringens
Bacillus cereus

Viral Disease of the Digestive System

Disease Virus
Mumps Mumps virus
Hepatitis A Hepatitis A virus
Hepatitis B Hepatitis B virus
Hepatitis C Hepatitis C virus
Hepatitis D Hepatitis D virus
Hepatitis E Hepatitis E virus
Viral gastroenteritis Rotavirus, calciviruses

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Fungal Disease of the Digestive System

Disease Fungus
Ergot poisoning Claviceps purpurea
Aflatoxin poisoning Aspergillus flavus

Protozoan Diseases of the Digestive System

Disease Protozoa
Giardiasis Giardia lamblia
Cryptosporidiosis Cryptosporidium parvum
Cryclospora dirarrheal infection Cryclospora cayctanensis
Amoebic dysentery (amoebiasis) Entamoeba histolytica

Helminthic Diseases of the Digestive System

Disease Helminths
Taeniasis Taenia saginata
Taenia solium
Diphyllobothrum latum (fish tapeworm)
Hydatid disease Echinococcus granulosus
Pin worms Enterobius vermicularis
Hook worms Necator americanus
Ancyclostoma duodenale
Ascariasis Ascaris lumbricoides
Trichinosis Trichinella spiralis

Microbial Diseases of the Urinary and Reproductive System

Bacterial Diseases of the Urinary system

Bacterial Diseases Bacteria

Cystitis (urinary bladder infection) Escherichia coli, staphylococcus.

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Pyelonephritis (kidney infection) E.coli

Leptospirosis (kidney infection) Leptospira interrogans

Bacterial Diseases of the Reproductive system

Diseases Bacteria
Gonorrhea Neisseria gonorrhoeae
Nongonococcal urethritis Mycoplasma hominis, Ureaplasma
Pelvic inflammatory disease N.gonorrhoeae
Syphilis Treponema pallidium
Chancroid (soft chancre) Haemophilus ducreyi
Bacterial vaginosis Gardnerella vaginalis

Viral Diseases of the Reproductive System

Diseases Virus
Genital herpes Herpes simplex virus type 1 and 2.
Genital warts Papillomavirus

Fungal diseases of the Reproductive System

Diseases Fungus
Candidiasis Candida albicans

Protozoan disease of the Reproductive System

Diseases Protozoa
Trichomoniasis Trichomonas vaginalis

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UNIT - 1

Difference between Prokaryotes and Eukaryotes.

(5 Mark).

Cellular structure contains two kinds of cells. These are prokaryotic and Eukaryotic
cells.

Bacteria are prokaryotic organisms. Animals, plants, algae, Fungi and protozoa
are eukaryotic organisms.

Prokaryotic cells:

A Prokaryotic cell is a cell that does not have a true nucleus. The nuclear
structure is called a nucleoid. The nucleoid contains most of the cell’s genetic
material and usually a single circular molecule of DNA. A prokaryotic organism such
as bacterium is a cell that lacks membrane-bound nucleus or membrane-bound
organelles. The outside of the cell contain glycocalyx, flagellum, fimbrial and pili.

Eukaryotic cells:

A Eukaryotic cell is larger and more complex than prokaryotic cells. It is found in
animals, plants, algal, fungi and protozoa. Eukaryotic cell has highly organized
structure of organelles that are bound by a membrane. Each organelle performs a

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specialized function. It also contain a membrane-bound nucleus where the cell’s

DNA is organized into chromosomes.

Eukaryotic cell and Prokaryotic cell.

Charactertics Eukaryotic Cells Prokaryotic Cells

Cell wall Chemically simple Peptidoglycan chemically

Plasma membrane Contain Carbohydrate, No carbohydrate, No

sterols sterols

Glycocalyx Contained in cell lack cell Contain capsule and slime

wall layer

Flagella Multiple microtubules Protein building blocks

Cytoplasm Contain cytoskeleton No cytoplasmic streaming

contain cytoplasmic
streaming

Membrane bound Endoplasmic recticulum, None

organelles Lysosomes, Golgi complex,
mitochondria chloroplast

Ribosomes 80s Ribosomes present in 70s

organelles are 70s

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Nucleus Have nucleus, nclear No nuclear membrane, No

membrane, nucleoli, nucleoli 0.2-2.0mm in
10-100mm in diameter diameter

Chromosomes Multiple linear Single circular

chromosomes have chromosomes , No
histones histones

Cell division Mitosis Binary fission

Sexual reproduction Meiosis No meiosis DNA

transferred in fragments.

Louis Pasteur ( 5 Mark)

(1822-1895)

Louis Pasteur born in France, Pasteur received degree of bachelor of letters from
the college Royal de Beacon. For the next three years. He tutored younger students
and prepared for teacher training college in Paris. For his studies he investigated
chemical and optical properties of tartaric acid. Later, he was appointed as an
assistant professor of chemistry. Meantime, he start investigates process of
fermentation, his interest slide to microorganisms and exploring role of microbes in
fermentation.

 His scientific contribution includes an understanding of how microorganisms carry on

biochemical process of fermentation.

 He established relationship between microorganisms and disease, and concept of

destroying microorganisms to stop the transmission of communicable disease.

 Fermentation is a form of cellular respiration carried by yeast cells, to get energy for
cells when oxygen is not present. He showed yeast cells produce alcohol and carbon

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dioxide from sugar during fermentation. Pasteur found that fermentation would take
place only when living yeast cell present.

 Pasteur tackle problems of the French beverage industry from avoiding wine turn
sour by heating the wine gently at 42oC would kill the bacteria that produced lactic
acid.

 He introduced the idea of pasteurization, process of heating to kill microorganisms in

fluids such as milk and fruit juices. Conventional methods of pasteurization mean that
the heating of milk to a temperature between 63 and 65 o C for a period of about 30
minutes, and then cooling it to room temperature. Recently, they revised of that
process, milk can also be “flash pasteurized” by raising temperature to about 71o C for
a minimum of 15 seconds. Ultra-high-pasteurization is the process uses even higher
temperature of about 90-130 o C. for periods of a second or more next. Flash
pasteurization uses higher temperature than conventional pasteurization, but the
temperature maintained for a shorter time. The product is then rapidly cooled to
below 10 o C, a temperature at which it can then be stored. The use of flash
pasteurization is to eliminate harmful microorganisms while maintaining the product
to its natural state.

 He disprove the idea of spontaneous generation that living things do not arise
spontaneously but rather come from other living things. When he performing his
experiment, invented sterile techniques, boiling or heating of instruments.

 He postulated the germ theory of disease that infectious diseases are due to the
activities of microorganisms.

 Pasteur developed techniques for culturing and examining several disease-causing

bacteria. He identified staphylococcus pyogenes bacteria in boils and streptococcus
pyogenes in puerperal fever.

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 He cultured the bacteria that cause cholera and injecting cholera in healthy chickens.
The chickens failed to get the disease, but received immunity against cholera. He
discovered that weakened microbes make a good vaccine with out coursing diseases.

 Later, Pasteur began work on a vaccine for anthrax, he weaken or attenuating the
bacteria that cause anthrax by incubating high temperature condition and produced a
vaccine.

 He developed successful treatment for rabies, a disease contracted from bites of an

infected, rabid animal. He produced rabies vaccine from the spinal fluid of infected
rabbits.

 The Pasteur Institute was established in 1888 to teat cases of rabies.

 The term vaccination was developed by Louis Pasteur, taken from Latin word for cow
(vacca)

 He coined the term Microbiology, the study of living organisms of microscopic size.

 He establishes optimum condition like temperature, PH and oxygen requirement for

growth of bacteria.

 In regard to major contribution to the field of Microbiology, he regarded as father or

Microbiology.

 When Pasteur died in 1895, he was well recognized for his achievements in science.

In the same course of time, Medicine enters into microbiological aspects such as the
nature of contagious disease. Although, the significance of contagion known with
respect to disease such as small pox, its nature and relationship to microorganism was
not understood different people came close to devising a germ theory of disease
finally Robert koch provided with evidence in 1867, the final evidence proving the
germ theory.

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Robert Koch (5 Mark)

(1843 – 1910)

Robert Heinrich Hermann Koch was born in a small town near Hanover, Germany.
In 1848, Robert taught himself to read and write with the aid of the newspapers.
After graduation, in 1862, Robert decided to study natural sciences at Gottingen
University. Later, he transferred his field of study to medicine. In 1866, he finished
the final exams for medical school and graduated with highest distinction.

 Koch examined under the microscope the blood o infected sheep and confirmed that
anthrax was caused by a bacillus.

 In 1877, Koch used different techniques to isolate Bacillus anthracis; he used dry-fixed
bacterial cultures onto glass slides, stained the cultures with dyes to observe them
and photographed through the microscope.

 In 1881, he has given the importance of pure cultures in isolating disease-causing

organisms. These methods and theory are still considered fundamental to the field
of modern bacteriology.

 Several Scientists had made claims that tuberculosis was infectious but Robert Koch
succeeded in isolating a bacillus from tissues of humans and animals infected with
tuberculosis.

 In 1905, he was awarded the Nobel Prize for physiology and Medicine for his work on
tuberculosis.

 He used sterile needles or platinum wires to draw minimum inoculum from

suspensions and made lines on the surface of the solid medium.

 Koch used Agar-Agar as Solidifying agent to make solid medium to obtain pure
culture.

 He invented hot air oven and steam sterilizer towards sterilization methods.

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 He introduce four basic criteria known as Koch’s postulates, are essential to be

identified as pathogenic or capable of causing disease.

 In 1919, he died at Baden Baden at the age of 67.

Koch’s Postulates (5 Mark)

Microbiologist Robert Koch proposed the postulates in 1919. It is a conditions

that microorganism to be considered the cause of a disease.

Four basic criteria are

1. The organism must be found in the tissues of animals with the disease and not in
disease – free animals.

2. the organism must be isolated from the diseased animal and grown in a pure
culture outside the body, or in vitro.

3. the cultured organism must be able to be transferred to a healthy animal, which

will subsequently show signs of infection.

4. The organisms must be able to be isolated from the infected animal.

Steps:

A. Blood is drawn from a sick animal

B. Blood brought to the laboratory.

c. A sample of the blood reveals bacteria.

d. The bacteria from the blood are cultivated in a pure culture in the laboratory.

e. A sample of the pure culture containing only one kind of bacteria is injected into
a healthy animal.

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If the animal becomes sick and displays the same symptoms as the original
animal, then evidence suggest that this particular disease is caused by this particular
organism.

These postulates have number of limitations. For example, infections organisms

such as bacterium Mycobacterium leprae, some viruses and prions cannot be grown
in artificial laboratory media. In addition the use of an animal that should mimic the
human infection.

Another limitation are microorganism are normally part of normal flora of a host
becomes capable of causing disease when introduced into a different environment in
the host (e.g.) staphylococcus aureus) or when the host’s immune system is
malfunctioning e.g., serratia marcescens.

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IMPORTANCE OF MICROBIOLOGY FOR NURSES (5 Mark)

Microbiology is the science which covers various information regarding infection, host
system and control methods. In nursing point of view, they are involved in control of
infection in hospitals. Main objective was to learn and excel in knowing information
about microorganisms, their disease, treatment and preventive measures. In this
topic, we have list out the application of microbiology in nursing practice.

Nosocomial Infection:

Nosocomial Infection is otherwise known as hospital infections. Nearly, most

people are infected after hospitalization. If we know infection transmission, these can
be kept in control.

 We need to know information about patients susceptible like child; old people and
Immune compromised individuals are hugely affected.

 Most common causative agents their environment and control method like
Handwashing, clearing medical equipment like respirators, etc.

 Common infections are urinary tract infection, wound infections, Respirator-related

preumonia and catheters related infections.

Immunization

 Basic information about history of Immunization.

 Vaccine types- attenuated, inactivated, sub unit and toxoid vaccines.

 Safety issues and risk.

Immune Hypersensitivites

 Information regarding hypersensitivity, allergic, reaction and types of reaction

Immune Response

 Basic information about Nonspecific and specific defenses of host.

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 Nonspecific - 1st line

Skin, mucous membrane, chemicals

 Nonspecific - 2nd line

Interferon, inflammation, fever.

 Specific- 3rd line

Lymphocytes, antibodies.

Specific Immune Response and lab techniques

 Basics of humoral and cell-mediated immunity.

 Information about microbial antigens, different classes of antibodies their general

role.

 General information about agglutination and precipitation reactions in serological

test. e.g. Blood typing and ELISA test.

Basic Epidemiology

 Information learn regarding incidence, prevalence and Reservoir.

 Disease transmission like contact, vehicle and vector.

Basic Disease Information

 Body system affected respiratory disease.

 Longevity/severity-Acute, chronic. Sub acute and latent.

 Transmission – communicable, Non communicable.

 Extent of body affected – Local and systemic.

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 State of host when infected – Primary and secondary.

Controlling Microbial Growth

 Controlling microbial growth through chemical methods like their actions, types and
general precaution.

 Physical methods like Heat, cold, filtration and Radiation their actions, advantage
and disadvantage.

 Antibiotics – their mechanism of actions, toxicity, spectrum of activity and antibiotic

resistance.

These elements of information and details in microbiology are essential to be

successful nurses. There were major scientific advances made for the past new
decades, nurses with knowledge of basic sciences can able to compete with other in
the present world. To make up this task, they need ideas with scientific approach and
able to problem – solve in logical and systematic way.

UNIT – 2

STRUCTURE OF BACTERIA (10 Mark)

Bacteria are prokaryotic cells. Bacteria are classified into gram positive and gram
negative cells. Bacterial cell covered by a chemically complex cell wall. Inside the cell

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wall, plasma membrane present. The genetic material is found in the centre called
nucleoid. Ribosomes and larger masses are called inclusion bodies are scattered
around in the cytoplasmic matrix. Outside to the cell wall, capsule or slime layer is
present.

Glycocalyx

Glycocalyx is a sticky, sugary envelope composed of polysaccharides and/or

polypeptides that surround the cell. Glycocalyx is found in one of two states

1. Capsule – capsule firmly attach to cell surface. Capsule is composed of

polysaccharides made of single or multiple types of sugar residues. Capsule can
protect bacteria. Capsule promotes adherence and colonization. Capsule is identified
by negative staining and Quellung reaction.

Identification:

1. Negative staining or special capsule stains are used to identify capsule. When
suspending the organisms in India ink, the India ink completely stain bacterial cell
except capsule that visualize colourless against stained background.

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2. Quellung reaction. It is the reaction by which bacterial cell react with specific
antibody thus results in swelling of the capsule. This is called quelling reaction. It is
mainly used to identify capsular serotypes.

2. Slime layer – a slime layer is a zone of diffuse, unorganized material of gelatinous

layer. It is loosely attach to cell surface.

Cell wall

The cell wall is the rigid structure that lies outside the plasma membrane. Cell wall
gives shape and protect from osmotic lysis. Cell wall is made of peptidoglycan which
gives shape and strength to the cell. The cell wall has components that contribute to
their pathogenicity. The cell wall provides a barrier against certain toxic chemical
and biological agents. It protects the cell from mechanical disruption and from
bursting results from hypertonicity of the cell. In Streptococcus, it provides a
protection from phagocytosis and helps in the binding to eukaryotic cell hosts.

Christian Gram developed the gram stain method in 1884, from this gram stain
procedure, he identified structural difference in the bacterial groups and divided into
two major groups of bacteria – Gram positive and Gram negative cell. The gram
positive cell wall consists of a single 20-80 nm thick homogenous peptidoglycan or
murein layer lysing outside the plasma membrane. In contrast, gram negative cell wall
has a 2-7 nm peptidoglycan layer surrounded by a 7-8 nm thick outer membrane. The
cell walls of gram positive cells are stronger than those of gram negative bacteria
because of the thicker peptidoglycan layer. In gram negative bacteria, a space is seen
between the plasma membrane and the outer membrane and similar gap may
observe between the plasma membrane and cell wall in gram positive bacteria. This
space is called the periplasmic space. The periplasmic space is filled with gel that
substance is called periplasm. The periplasmic shape of gram negative bacteria
contains many proteins that involved in nutrient acquisition. E.g. binding proteins
involved in transport of materials into the cell. The periplasmic space contains
enzymes involved in perptidoglycan synthesis and the modification of toxic
compounds that could harm the cell. These enzymes are often called exoenzymes.

Components of Bacterial Cells

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Structure Composition Gram (+) cell Gram (-) cell

Capsule Polysaccharide + or - + or -

(slime layer) Or polypeptide

Wall + +

Outer membrane Proteins, + -

phospholipids, and
lipopolysaccharide

Cell membrane or plasma membrane

Cell membrane contains both protein and lipids. In plasma membrane, lipids form a
bilayer. The outer surface of lipid is hydrophilic and inner region are hydrophobic.
Plasma membrane serves as a selectively permeable barrier. It allows particular ions
and molecules to pass, either into or out of the cell.

Function of Bacterial Cell Structures

1 Plasma membrane Selectively permeable barrier, mechanical

boundary of cell, nutrient and waste transport,
location of may metabolic processes (respiration)

2 Ribosomes Protein synthesis

3 Inclusion bodies Storage of carbon, phosphate and other

substances

4 Nucleoid Localization of genetic material (DNA)

5 Periplasmic space Contains hydrolytic enzymes and binding proteins

for nutrient processing and uptake.

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6 Cell wall Gives bacteria shape and protection from lysis in

dilute solutions

7 Capsules and slime layer Resistance to phagocytosis adherence to surface

8 Iimbriae and pili Attachment to surfaces, bacterial mating

9 Flagella Movement

10 Endospore Survival under harsh environmental conditions

Cytoplasmic matrix

The cytoplasmic matrix is the substance lying between the plasma membrane and the
nucleoid. The matrix contains water and ribosomes. Substance found in the matrix is
inclusion bodies, ribosomes and plasmid.

a. Inclusion bodies – it contain granules of organic or inorganic material. These bodies

are used for storing carbon compounds, inorganic substances and energy.
b. Ribosomes – ribosomes are complex objects made of both protein and ribonucleic
acid (RNA). They are the site of protein synthesis. Ribosomes found in bacteria are
called 70S ribosomes. 70S divided into two subunits such as 50S and 30S.

Components of Bacterial Cells

Structure Composition Gram (+) cell Gram (-) cell

Capsule Polysaccharide + or - + or -

(slime layer) Or polypeptide

Wall + +

Outer membrane Proteins, + -

phospholipids, and
lipopolysaccharide

Peptidoglycan Murein (+ teichoate + +

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Layer in gram (+) cell)

Periplasm Proteins and + -

oligosaccharide

Call membrane Proteins, + +

phospholipids

Pili (fimbriae) Protein (pilin0 + or - + or -

Cytosol Polyribosomes, + +
proteins,carbohydrat
e (glycogen)

Nucleoid DNA with proteins + +

Flagella Proteins (flagellin) + or - + or -

Plasmids DNA + or - + or -

Endospore Dipicolinate and - + or -

special components

Nucleoid

The bacterial chromosome is located in an irregularly shaped region called nucleoid.

Nucleoid contains DNA.

Pili and fimbriae

The short, fine, hair like appendages is called fimbriae. They are only visible in an
electron microscope. They are only visible in an electron microscope due to their
small size, a cell may be covered with up to 1000 fimbriae. They are composed of
molecules of a protein called pilin arranged to form a tube with a minute hollow core.
They are about 3-10 nm in diameter and up to several micrometres long. General

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classes of pili are common pili and sex pili. Common pili cover the surface of the cell
and in same cases, adhesions, which are responsible for the ability of bacteria to
colonize surface of the host cell. E.g. the pili of Neisseria gonorrhoeae are necessary
for the attachment of the urethral epithelial cells prior to penetration into the cell.
Gram negative bacteria have five types of pili. They are Type 1,2, 3,4 and 5. Sex pili
are similar appendages, about 1 to 10 per cell, that differ from fimbriae. Sex pili are
required for bacterial mating, the transfer of genetic material from donor cell to
recipient. This process is termed a conjugation. This conjugation process is carried
with the help of pili, which have intimate contact between donor and recipient cells.

Flagella:

Flagella are threadlike locomotor appendages extending outward from the

plasma membrane and cell wall. Most bacteria move with use of flagella is termed
motility. Flagella are slender, rigid structures, about 20 nm width and upto 15 or 20
micrometre long. The detailed structure of a flagellum can only be seen in the
electron microscope.

Structure of flagella:

The bacterial flagellum is composed of three parts

1. the filament – longest portion of flagella, which extends from the cell surface to the
tip.

2. A basal body – embedded in the cell.

3. The hook – a short, curved segment links the filament to its basal body and acts as
a flexible coupling.

 Filament is a hollow, rigid cylinder consists of a single protein called flagellin, which
ranges in molecular weight from 30,000 to 60,000. Some bacteria has membranous
sheath that surrounds the filament e.g. Bdellovibrio and Vibrio cholerae .

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 The hook is made of different protein subunits and slightly wider than the filament.

 The basal body is the complex structure in the flagellum found inside the cell
structure.

 In E.coli and most gram negative bacteria, basal body has four rings connected to
central rod. They are M, S, P and L rings. The outer L and P rings associate with the
lipopolysaccharide and peptidoglycan layers respectively.

 The inner M ring contacts the plasma membrane and S ring found adjacent to the M
ring.

 Gram positive bacteria has only two basal body rings, an inner ring connected to the
plasma membrane and outer ring attached to the peptidoglycan.

Spore

Spore is resting stage of bacteria. Spores are resistant to environmental stresses such
as heat, ultraviolet radiation, gamma radiation, chemical disinfectant and desiccation.
Sporulation is the process of development of spores.

CULTURE MEDIA AND ITS TYPES (10 MARK)

The study of microbiology depends on the ability to grow and maintain

microorganisms in the laboratory and this is possible only if suitable culture media are
available. A culture medium is a solid or liquid preparation used to grow, transport
and store microorganisms. The medium must contain all the nutrients the
microorganism requires for growth. The precise composition of medium depends on
the species because different organisms has different nutritional requirements. To
select the appropriate medium, detail about microorganisms are oftern useful.

History of Culture Media

Preparation of culture media for growing microorganisms in the laboratory was

stated by Louis Pasteur. He used liquids which obtain naturally in the environment

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such as urine and meat broth (Broth mean liquid). Although liquid medium is helpful
to study the microorganisms but these medium found with few disadvantages. For
instance, bacteria grow in liquid media may not echibit specific charactertics for their
identification. Secondly, difficulties in isolating different types of bacteria from mixed
culture. Advantage of using liquid media is

1. Preparation of bulk cultures for antigens and vaccines.

2. Preparation of inoculum for biochemical test.

Few disadvantages make difficult to isolate organisms from mixed culture which
are overcome by Robert Koch. He cultured organisms in solid media. At first , he used
pieces of potato to prepare solid medium. But potato hydrolyzed by some bacteria.
Thus, potato become liquefied after introduction of bacteria cells into the solid
medium. Secondly, he used gelatin to prepare solid media. But gelatin liquefies at 24
degree celcius (optimum temperature for pathogenic bacteria is 37 degre celcius) and
by the action of few bacteria. Lastly, he used agar-agar in place of gelatin for
solidification.

Agar-agar:

It is prepared from a variety of seeweeds and supplied as a powder form. Agar

chemical structure contains a long-chain polysaccharide composed of
D-galactopyranose units and few percentages of inorganic salts, protein and trace
amount of fatty acid. The important properties of agar-agar are

1. It doesn’t have nutritive properties in the medium.

2. It is not affected by the growth of bacteria.

Usually, bacteria grows on surface of the agar medium (solid medium),

generally 1-2% of agar used to prepare perfect gel.

Preparation:

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Appropriate amount of agar powder is added to the liquid medium an dissolved in a

steamer at 100 degree celcius for few minutes. When agar dissolved, it give a clear
solution.

Properties:

Solid media melt at 95 degre celcius and solidify when temperature reduce to about
42 degree celcius. Once solid media is sterilized, the media pour into petric plate ( A
special container used to grow microorganisms). Around 15-20 ml poured into the
plate. When pH of the solid medium is low, medium does not solidify on cooling.

Culture media can be classified into different types based on their charactertics. Few
ways are

1. Solid, Semisolid and Liquid medium

2. Simple (basal), Complex, Synthetic and Special media.

Special media are further classified into enriched, selective, enrichment, indicator, or
differential, sugar media and selective media.

3. Aerobic and anaerobic media.

Peptone:

It contains water-soluble products obtained from lean meat and other protein
sources like casein, soya flour and heart muscle. It is the most common ingredient in
most media.

Simple media:

It is also called basal media. Simple media contain peptone water and nutrient
broths which used in the study of the common pathogenic bacteria. Types of nutrient
broth are

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1. Meat Infusion broth:

It contains watery extract of lean meat with peptone water.

2. Meat Extract broth:

It is a mixture of commercial peptone and meat extract.

3. Digest broth:

It contains watery extract of lean meat which digested with proteolytic

enzymes.

TYPES OF SOLID MEDIUM:

Generally, broth is made into solid by adding 1-2% agar. In semi solid agar, the
concentration of agar reduced to 0.2-0.5%, if it is raised to 6% called hard agar.

Complex Media:

Media that contain some ingredients of unknown chemical composition are called
complex media. It meets nutritional requirements of many different microorganisms.
Complex media contain undefined components like peptones, meat extract and yeast
extract. Peptones are protein hydrolysated prepared by partial proteolytic digestion
of meat, casein and other protein sources. Beef extract and yeast extract are aqueous
extract of lean beef and brewer’s yeast. Beef extract contain aminoacids, peptides,
organic acids, vitamins and mineral, yeast extract contain Vitamin B as well as
nitrogen and carbon compounds. E.g. Nutrient broth and tryptic soy broth.

Enriched Media:

These media used for the growth of fastidious organisms by addition of substances
such as blood, serum and egg to a basal medium. Examples are blood agar medium
used of isolation of Streptococus, chocolate agar for isolation of Neisseria and

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Haemophilus, Bordet-Gengou for isolation of Bordetella and Loeffler’s serum slope

for the isolation of Corynebacterium diptheriae.

Selective Media:

Selective media helpful for the growth of wanted bacteria, at the same time inhibit
the growth of unwanted bacteria by the addition of substance into the solid medium.

Examples: Mac Conkey agar for E.coli, Deoxycholate citrate agar (DCA) for Salmonella
and Shigella, Wilson and Blair’s medium for Salmonella and Lowenstein –Jensen
medium for Mycobacterium tuberculosis.

Indicator or Differential Media:

Medium contain substance which would produce a visible change in the

medium following the growth of a particular organism it is called Indicator medium.

Examples: 1. Mac Conkey medium contain lactose and neutral red. When lactose
fermenting organisms grows in the medium, organisms produce acid and medium
become acidic pH thus neutral red become red in colour. Organisms like E.coli
produces red or pink colonies in this medium.

2. Christensen’s medium contains urea and phenol red. Organisms like Proteus and
Klebsiella grows in the medium produce urease enzyme to breakdown urea into
ammonia and carbon dioxide. Ammonia changes the pH of the medium into alkaline,
thus phenol red make pink colour formation in alkaline pH condition present in the
medium.

Transport Media:

During transportation of the clinical sample to the laboratory, delicate organisms do

not survive or normal flora (e.g. E.coli) may overgrow pathogenic organisms
(Salmonella, Shigella and V.cholerae). Few bacteria is sensitive to drying, exposure
to sunlight and changes in pH. In these situations, samples should be put into the
culture medium immediately to avoid the consequences; if it is not possible then the

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samples should be transported in transport media. Such problems can be overcome

by using media to maintain the viability of the pathogen.

Examples: Stuart’s transport medium, Venkatraman-Ramakrishnan (VR) medium,

Cary-Blair medium and Amies transport medium.

Venkatraman-Ramakrishan Medium: It is prepared by dissolving 20g NaCl and 5g

peptone in 1 litre of distilled water and pH is adjusted to 8.6 – 8.8. It is put into screw
capped bottles in 10-15ml amounts. Add 1-3 ml of stool specimen to the bottle. This
is used to transfer stool samples mainly V.cholerae. In this medium, V.cholerae do not
multiply but remain viable for several weeks. Medium prevent overgrowth of other
organisms.

Cary-Blair’s Medium: This medium contains disodium hydrogen phosphate, sodium

thioglycollate, sodium chloride and agar-agar. The pH should be adjusted to 8.4.
Medium is used for transport cultures containing Salmonella, Shigella and V.cholerae.

Enrichment Media:

Enrichment medium helpful to the growth of wanted bacteria, at the same time
inhibit the growth of unwanted bacteria by the addition of substance into the liquid
medium.

For Example: Tetrathionate and Selenite broth for Salmonella and Shigella and
Alkaline peptone water for Vibrio cholerae.

Storage Media:

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Storage media used to preserve bacteria for long periods by lyophilization. If

bacteria have to be store for a few months, they can use stab inoculated on semisolid
agar or on Dorset egg medium followed by incubation. After growth of organisms in
the medium, they can be stored in refrigerator.

Defined Synthetic media:

Defined synthetic media are prepared from pur chemical substances, their exact
composition is known. It is divided into

1. Simple synthetic media: It contain carbon and energy source such as glucose or
lactate, an inorganic source of nitrogen such as ammonium chloride, phosphate or
sulphate and inorganic salts are used.

2. Complex synthetic media: In addition to simple synthetic media, it contain certain

aminoacids, purine, pyrimidines, and other growth factors.

Sugar Media:

It is the media used to identify the organisms based on utilization of sugars. To

prepare media, 1% of the particular sugar is added to peptone water with a suitable
indicator. To detect gas production by the organisms, a small tube (Durham’s tube) is
kept inverted in the tube contain sugar medium.

Anaerobic Media:

To grow anaerobic organism, the media should contain reducing substances. In

addition to the reducing substances, the media contain methylene blue or resazurin
to act as an oxidation-reduction potential indicator.

Example: Thioglycollate broth and cooked meat broth medium.

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STRUCTURE OF FLAGELLA (5 Mark)

Flagella are threadlike locomotor appendages extending outward from the plasma
membrane and cell wall. Most bacteria move with use of flagella is termed motility.
Flagella are slender, rigid structures, about 20 nm width and upto 15 or 20
micrometre long. The detailed structure of a flagellum can only be seen in the
electron microscope.

The bacterial flagellum is composed of three parts

1. the filament – longest portion of flagella, which extends from the cell surface to the
tip.

2. A basal body – embedded in the cell.

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3. the hook – a short, curved segment links the filament to its basal body and acts as a
flexible coupling.

 Filament is a hollow, rigid cylinder consists of a single protein called flagellin, which
ranges in molecular weight from 30,000 to 60,000. Some bacteria has membranous
sheath that surrounds the filament e.g. Bdellovibrio and Vibrio cholerae.

 The hook is made of different protein subunits and slightly wider than the filament.

 The basal body is the complex structure in the flagellum found inside the cell
structure.

 In E.coli and most gram negative bacteria, basal body has four rings connected to
central rod. They are M, S, P and L rings. The outer L and P rings associate with the
lipopolysaccharide and peptidoglycan layers respectively.

 The inner M ring contacts the plasma membrane and S ring found adjacent to the M
ring.

 Gram positive bacteria has only two basal body rings, an inner ring connected to the
plasma membrane and outer ring attached to the peptidoglycan.

Types of Flagella or Distribution of Flagella:

Bacterial species differ distinctively in their patterns of flagella distribution.

Types of flagella are

1. Monotrichous – Bacteria have one flagellum located at an end or

pole. This is called monotrichous.

2. Amphitrichous – (amphi means “on both sides”) Bacteria have a single

flagellum at each pole.

3. Lophotrichous – (Lopho means tuft) Bacteria have a cluster of

flagella at one or both ends.

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4. Peritrichous – ( peri means around) Bacteria have flagella that

spread fairly evenly over the surface of the cell.

Generally, flagellation patterns are useful in identifying bacteria.

Bacterial Motility or Bacteria Movement:

 In most bacterial species, motility is the property of swimming by means of flagellar

propulsion.

 Bacteria flagellum contain filament which is rigid helix structure and the bacterium
moves when this helix rotates.

 The direction of flagellar rotation determines the nature of the movement. To move
forward, flagella rotate counter clockwise ( viewed from outside the cell), whereas
cell itself rotates slowly clockwise.

 When reversing the direction of flagellar rotation (clockwise), bacteria stop and
tumble randomly.

 The bacteria can swim from 20 to almost 90 micrometre per second.

 Spirochaetes are helical bacteria that travel through viscous substance by flexing
and spinning movement caused by a special axial filament composed of periplasmic
flagella.

 Gliding motility is a different type of motility and has no visible external structure for
movement. E.g. Mycoplasma.

The Bacterial Growth Curve (5 MARK)

Bacterial Growth is a complex process involving different stages.

1. Entrance of basic nutrients into the cell.

2. Conversion of these nutrients into energy and vital cell constituents.

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3. Replication of the chromosome.

4. Increase in size and mass of the cell.

5. Division of the cell into two daughter cells, each containing a copy of the genome
and other vital components.

Growth defined as an increase in cellular constituents. Growth leads to a rise in cell

number when microorganisms reproduce by budding or binary fission. In binary
fission, sequential events are

1. The cell elongates and chromosomal DNA is replicated.

2. The cell wall and cell membrane move inward and begin to divide.

3. The cell wall forming a cross wall completely around the divided DNA.

4. The cells separate into two individual cells.

In budding, a small outgrowth or bud emerges from bacterium and

enlarges until it reaches the size of the daughter cell. Cells separates, forming two
identical cells. Some bacteria called filamentous bacteria reproduce by producing
chains of spores located at the tips of the filaments. The filaments fragment and these
fragments initiate the growth of new cells.

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Population growth is studies by analyzing the growth curve of a microbial culture.

When microorganisms are cultivated in liquid medium, they are usually grown as
batch culture or closed system (incubate in a closed vessel with single time medium
given). During incubation, no fresh medium is provided, as a result nutrient
concentrations decline and concentration of wastes increase. The growth of
microorganisms reproduced by binary fission can be plotted as the logarithm of the
number of viable cells versus the incubation time. The resulting curve has four distinct
phases.

LAG PHASE:

 In lag phase, microorganisms are introduced into fresh culture medium, no increase
in cell number.

 Cell division does not take place quickly and there is no net increase in mass, the cell
is synthesizing new components.

 Before cell division, lag phase are essential for a variety of reasons. The cells may be
old and depleted of ATP, essential cofactors and ribosomes; they must be synthesized
before growth can begin.

 Microorganisms need to adjust to conditions like medium may be different. So, new
enzymes would be needed to use different nutrients.

 The cells retool, replicate their DNA, begin to increase in mass and finally divide.

 The length of lag phase varied with the condition of the microorganisms and the
nature of the medium.

 The lag phase may long if the inoculum is from an old culture or inoculation of a
culture into a chemically different medium.

 When a young, vigorously growing exponential phase culture is transferred to fresh

medium of the same composition, the lag phase wil be short or absent.

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 The period between inoculation and beginning og multiplication is known as lag

phase.

EXPONENTIAL PHASE OR LOG PHASE:

 During the log phase, microorganisms are growing and dividing at the maximal rate
based on genetical potential, the nature of the medium and the condition like
temperature etc.

 Microorganisms are dividing and doubling in number at regular intervals, their rate of
growth is constant.

 The population of cells is most uniform in terms of chemical and physiological

properties during this phase; so, this phase cultures are usually used in biochemical
and physiological studies.

 Exponential growth is balanced growth. In this growth, all cellular constitutents are
manufactured at constant rates, if nutrient level or other environmental conditions
change unbalanced growth results.

 To synthesize cellular constituents, the cells construct new ribosomes to enhance

their capacity for protein synthesis. This is followed by increases in protein and DNA
synthesis. Finally, reproductive rate takes place.

 The time required for one bacterial division during this phase is called generation
time. The number of organisms present in each generation period is almost twice
than previous period. In graph, bacterial count plotted against time a straight line is
obtained.

 Log phase is of short duration because of

1. Exhaustation of nutrients.

2. Accumulation of toxic metabolic end products.

3. Rise in cell density.

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4. Change in pH.

5. Decrease in oxygen tension.

STATIONARY PHASE:

 Stationary phase is the period the growth curve become horizontal due to cease in
population growth.

 During this phase, bacteria population level is around 10 9 cells per ml. but other
microorganisms do not reach to this densities, protozoan and algal cultures has
maximum concentrations of about 10 6 cells per ml.

 In the stationary phase, the total number of viable microorganisms remain constant
because of balance between cell division and cell death or cells cease to divide though
remaining metabolically active.

 Factor limits log phase period are indirectly responsible to enter the stationary phase.

 Batch culture usually enter stationary phase in response to starvation. As the result of
starvation only few bacteria have morphological changes such as endospore
formation, remaining only decrease in overall size, protoplast shrinkage and nucleoid
condensation.

DEATH OR PHASE OF DECLINE:

Environmental changes like nutrient deprivation and the buildup of toxic wastes lead to
the decline in the number of viable cells.

The death of a microbial population is usually logarithmic (a constant proportion of cells

dies every hour). Thus rate of death exceeds the rate of reproduction and the number
of viable cell declines.

Finally, all the cells die and culture become sterile.

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FACTORS AFFECTING MICROBIAL GROWTH (5 MARK)

The growth of microorganisms is greatly affected by the chemical and physical nature
of their surroundings. An understanding of environmental influences help in the
control of microbial growth and the study of the ecological distribution of
microorganisms.

Solutes and Water:

Microorganisms can be affected by changes in the osmotic concentration of their

surroundings. Selectively permeable membrane separated microorganisms from their
environment. If a microorganism is placed in a hypotonic solution (one with a lower
osmotic concentration), water will enter the cell and cause it to burst. Prokaryotes
can contain pressure-sensitive channels that open to allow solute escape when the
osmolarity of the environment becomes much lower than that of the cytoplasm.

Most bacteria, algae, and fungi have rigid cell walls that maintain the shape and
integrity of the cell. When microorganisms with rigid cell walls are placed in a
hypertonic environment, water leaves and the plasma membrane shrinks away from
the cell, a process known as plasmolysis. This dehydrates the cell and may damage
the plasma membrane.

The amount of water available to microorganisms can be reduced by interaction

with solute molecules (the osmotic effect) or by adsorption to the surfaces of solids
(the matric effect). Because the osmotic concentration of a habitat has such profound
effects on microorganisms.

Microorganisms must expand extra effort to grow in a habitat with a osmotic

concentration because it must maintain a high internal solute concentration to retain
water. These organisms are called osmotolerant. Osmotolerant will grow over wide
ranges of osmotic concentration.

Halophiles adapted to hypertonic, saline conditions that they require high levels of
sodium chloride to grow about 2.8 M concentrations.

pH:

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pH is a measure of the hydrogen ion activity of a solution and is defined as the

negative logarithm of the hydrogen ion concentration. Like other factors, pH also
affects microbial growth. Each species of microorganisms has a definite pH growth
range and growth optimum conditions. According to pH conditions, microorganisms
can be classified into

1. Acidophiles are organisms grow between pH 0 to 5.5;

2. Neutrophiles are organisms grow between pH 5.5 to 8.0;

3. Alkalophiles are organisms grow between pH ranges of 8.5 to 11.5.

Extreme alkalophiles has growth optimum at pH 10 or higher.

Most bacteria and protozoa are neutrophiles. Most fungi prefer slightly acid
surroundings, about 4 to 6; algae seem to favor slight acidity. Although,
microorganisms will often grow over wide ranges of pH and far from their optimum
pH. But complete variations in cytoplasmic pH can harm microorganisms by disrupting
the plasma membrane or inhibiting the activity of enzymes and membrane transport
proteins. Microorganisms die if the internal pH drops below 5.0 to 5.5. Changes in the
external pH also might alter the ionization of nutrient molecules and thus reduce their
availability to the organisms.

Temperature:

Environmental temperature affects microorganisms like all other microorganisms.

Microorganisms are susceptible because they are usually unicellular and their
temperature varies with of the external temperature. High temperatures damage
microorganisms by denaturing enzymes, transport carrier and other proteins.
Microbial membranes are also disrupted by extreme temperatures; the lipid bilayer
simply melts and disintegrates. The microorganism’s growth is inhibited because the
damage cannot be repaired. At very low temperatures, membranes solidify and
enzymes don’t work rapidly. When organisms are above or below the optimum
temperature, both function and cell structure is affected.

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Microorganisms has the minimum and maximum growth temperatures and the range
over which growth is possible. Microorganisms can be placed in one of five classes
based on their temperature ranges for growth.

1. Psychrophiles

2. Facultative Psychrophiles

3. Mesophiles

4. Thermophiles

5. Hyperthermophiles

Psychrophiles grow well at 0 degree celcius and have an

optimum growth temperature of 15 degree celcius or lower; the maximum is around
20 degree celcius. Examples for psychrophiles are Pseudomonas, Vibrio, Bacillus and
Anthrobacter. Psychrophiles begin to leak cellular constitutents at temperatures
higher than 20 degree celcius because of cell membrane disruption.

Many bacterial species grow at 0 to 7 degree celcius, they have optimum conditions
between 20 and 30 degree celcius and maximum at about 35 degree celcius. These
called psychrotrophs or facultative psychrophiles.

Mesophiles are microorganisms with optimum temperatures around 20 to 45

degree celcius; minimum temperature of 15 to 20 degree celcius and maximum is
about 45 degree celcius or lower. Almost all human pathogens are mesophiles; they
have constant temperature 37 degree celcius.

Thermophiles are microorganisms grow at temperature of 55 degree celcius or

higher. Their growth minimum is around 45 degree celcius and optimum temperature
between 55 and 65 degree celcius. These organisms found in composts, self- heating
hay stacks, hot water lines and hot springs.

A few thermophiles can grow at 90 degree celcius or above and maximum

temperature about 100 degree celcius. Organisms have optimum temperature for
growth between 80 degree celcius and 113 degree celcius called hyperthermophiles.

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Moisture:

Moisture is important factor require for the growth of bacteria. About 80% of the
bacteria weight contains water, so drying microorganisms are affecting their structure
and functions.

Oxygen Concentration:

Aerobe is an organism able to grow in the presence of atmospheric oxygen, whereas

organisms that can grow in absence of oxygen is called anaerobe. Organisms strictly
require or completely dependant on atmospheric oxygen is called obligate aerobes.
Facultative anaerobe are organisms able to act like aerobes in the presence of oxygen
but able to survive when conditions become anaerobic. Aerotolerant anaerobes are
organisms that are basically anaerobic; but they are not inhibited by atmospheric
oxygen. E.g. Enterococcus.

Strict or obligate anaerobes (e.g. Bacteriodes, Fusobacterium and Clostridium)

do not tolerate oxygen levels below the range of 2 to 10% for growth but that are
damaged by the normal atmospheric level of oxygen (20%).

Hanging Drop Slide Method: (5 MARK)

Direct examination of unstained preparations is suitable for rapid diagnosis in the

laboratory. Many pathogens have a characteristic appearance, e.g. parasites in faeces
or bacteria together with white cells in the urine. Unstained preparation is used to
study the bacterial shape, size, arrangement and motility by using hanging drop
method.

Objectives:

To observe living bacteria motility and study the shape, size and arrangement.

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Principle:

Many bacteria show no motion and are termed nonmotile. In an aqueous

environment, these same bacteria appear to be moving erratically. This erratic
movement is due to Brownian movement. Brownian movement results from the
random motion of the water molecules bombarding the bacteria and causing them to
move.

True motility (self-propulsion) has been recognized in other bacteria and involves
several different mechanisms. Bacteria that possess flagella exhibit flagellar motion.
Helical-shaped Spirochetes have axial fibrils (modified flagella that wrap around the
bacterium) that form axial filaments. These spirochetes move in a corkscrew and
bending-type motion. Other bacteria simply slide over moist surfaces in a form gliding
motion.

These types of motility or non motility can be observed over a long period
in a hanging drop slide. Hanging drop slide are useful in observing the general shape
of living bacteria and the arrangement of bacterial cells when they associate together.
A ring of Vaseline around the edge of the cover slip keeps the slide fro drying out.

Requirements:

Bacterial culture, microscope, cavity slide, cover slip, Vaseline or petroleum jelly,
inoculating needle and Bunsen burner.

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Procedure:

1. Apply Vaseline on the edges of the coverslip to provide seal.

2. Use the inoculating loop to transfer a small drop of bacterial suspensions in the
center of a coverslip.

3. Invert cavity slide and move slide onto the coverslip. Press gently to form a seal.

4. Turn the slide over and place on the stage of the microscope.

5. Examine the hanging drop under low power objective and switch to 90 to 100x
objective, using immersion oil.

GRAM STAINING (5 MARK)

Use more than one stain and used for separation of groups an observation of
cellular structures. E.g. Gram-stain, acid-fast stain, flagella stain, capsule stain, spore
stain and nuclear stain.

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Bacterial Smear Preparation:

A bacterial smear is a dried preparation of bacterial cells on a glass slide. In a

bacterial smear, (1) the bacteria are evenly spread out on the slide in such a
concentration that they are adequately separated from one another. (2) The bacteria
are not washed off the slide during staining and (3) bacterial form is not distorted.

In making a smear, bacteria from either a broth culture or an agar slant or

plate may be used. If a slant or plate is used, a small amount of bacterial growth if
transferred to a drop of water on a glass slide and mixed. The mixture is then spread
out evenly over a large area on the slide. If the medium is liquid, place one or two
loops of the medium directly on the slide and spread the bacteria over a large area.
Allow the slide to air dry at room temperature. After the smear is dry, the next step is
to attach the bacteria to the slide by heat fixing. This is accompanied by gentle
heating, passing the slide several times through the hot portion of the flame of a
Bunsen burner. Most bacteria can be fixed to the slide and killed in this way without
serious distortion of cell structures.

Objectives:

1. Differentiate a mixture of bacteria into gram-positive and gram-negative cells.

2. Understanding Gram reaction.

Principles:

Bacteria differ from one another chemically and physically and may react differently
to a gram staining procedure. This is the principle of differential staining. Differential
staining can distinguish between types of bacteria.

The Gram stain (named after Christian Gram, Danish scientist and
physician, 1853-1938) is the most useful and widely employed differential stain in
bacteriology. It divides bacteria into two groups- gram positive and gram negative.

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The first step in the procedure involves staining with the basic dye crystal
violet. This is the primary stain. It is followed with an iodine solution, which functions
as a mordant; that is, it increases the interaction between the bacterial cell and the
dye so that the dye is more tightly bound or the cell is more strongly stained. The
smear is then decolorized by washing with an agent such 95% ethanol; Gram-positive
bacteria retain the crystal violet-iodine complex. When washed with the decolorizer,
whereas gram-negative bacteria lose their crystal violet –iodine complex and become
colorless. Finally, the smear is counter stained with a basic dye, usually safranin. The
safranin will stain the colorless, gram-negative bacteria pink but does not alter the
dark purple color of the gram-positive bacteria.

Gram Reaction:

In the Gram reaction, it describes reaction takes place inside gram-positive

and gram-negative cell during gram staining method. The smear on the slide is
prepared, stained with crystal violet and then treated with iodine solution as a
mordant. The crystal violet-iodine complex impart purple-black colour to the cells. In
Gram-positive cells, this complex binds to the magnesium-ribonucleic acid component
of the cell wall, forming complex, which is difficult to remove. The intensely stained
cells are then washed with ethanol. This serves as a lipid solvent and a dehydrating
agent for protein. The Gram-positive bacteria contain low lipid content; hence the low
amount of lipid is easily dissolved by alcohol. This makes minute pores in the cell wall
that are closed by dehydration effect of alcohol. In Gram-negative cells, large pores
are formed that do not close appropriately hence, dehydration of cell wall protein
does not occur completely. This facilitates the release of the unbound crystal violet
complex leaving the cells colorless or unstained. If the smear is counter stained with
safranin, the Gram-negative cells are easily seen due to absorption of safranin and
imparting the cells red colour; while Gram-positive cells retain the blue colour of the
primary stain.

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Steps:

1. Prepare bacterial smear on a glass slide

2. Heat fix using a spirit lamp

3. Bacterial cells fixed on slide

4. Put a drop of crystal violet and wait for 2-3 minutes and remove stain with tap
water.

5. Bacterial cells are stained

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6. Stain with Gram’s iodine and wait for 1-2 minutes; wash stain with tap water.

7. Bacterial cells are stained

8. Put slide in a beaker containing 95% ethanol to decolourise the stain.

9. Stains may or may not be decolurised.

10. Stain with safranin and wait for 2-4 minutes.

11. The bacterial may or may not be stained with safranin.

12. Wash with tap water and observe using oil immersion in microscope.

 If the bacterial cells take crystal violet and appear dark purple color, they are called
Gram-positive bacteria.

 If the bacterial cells take safranin and appear pink in colour, they are called
Gram-negative bacteria.

Requirements:

Bacterial slant cultures, crystal violet, Gram’s iodine, ethyl alcohol (95%), safranin,
Bunsen burner, inoculating needle, staining tray, glass slide and microscope.

Procedure:

1. Prepare a bacteria smear and heat fixed on the slide using standard procedure.
Pour a few drops of crystal violet on the smear.

2. Wait for 1 minute and wash with tap water. Now, flood the smear with the Gram’s
iodine for 1 minute and again wash with tap water.

3. Decolourise the stain with ethyl alcohol (95%) by dropping the reagent drop wise.

4. Wash it with tap water and counter stain with safranin for 45 seconds and wash
again with water.

5. After drying, examine under oil immersion.

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Results:

The bacteria that take stain and appear dark violet are called Gram-positive
bacteria. The bacteria appear pink or red coloured do not take crystal violet and
called Gram-negative bacteria.

THE ACID-FAST STAIN (5 MARK)

Acid fastness is a property of the mycobacteria and related organisms. Acid-fast

organisms generally stain very poorly with dyes, including Gram stain. However, they
can be stained by prolonged application of more concentrated dyes, and staining is
facilitated by heat treatment. Their unique feature is that once stained, acid fast
bacteria resist decolorization by concentrations of mineral acids and ethanol that
remove the same dyes from the other bacteria. This combination of weak initial
staining and strong retention once stained is probably related to the high lipid
content of the mycobacterial cell wall. Acid-fast stains are completed with a
counterstain to provide a contrasting background for viewing the stained bacteria.

Objectives:

1. To understand the biochemical basis of the acid-fast stain.

2. Differentiate bacteria into acid-fast and non-acid-fast groups.

Use of Acid-fast Stain:

In the clinical laboratory, the acid-fast stain is important in identifying

bacteria in the genus Mycobacterium; mainly M.leprae (Leprosy) and M.tuberculosis
(tuberculosis). This differential stain is also used to identify members of the aerobic
actinomycete genus Nocardia; mainly, the opportunistic pathogens N.brasiliensis and
N.asteroides that cause the lung disease nocardiosis. The water borne protozoan

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parasite cryptosporidium that causes diarrhea in humans (Cryptosporidiosis) can also

be identified by the acid-fast stain.

Principles:

A few species of bacteria in the genera Mycobacterium and Nocardia, and the
parasite Cryptosporidium do not readily stain with simple stains. However, these
microorganisms can be stained by heating them with carbol fuchsin. The heat drives
the stain into the cells. Once the microorganisms have taken up the carbol fuchsin,
they are not easily decolorized by acid-alcohol, and hence are termed acid-fast. This
acid-fastness is due to the high lipid content (Mycolic acid) in the cell wall of these
microorganisms. The Ziehl-Neelsen acid-fast staining procedure (developed by Franz
Ziehl, a German pathologist, in the late 1800s) is a very useful differential staining
technique that makes use of this difference in retention of carbol fuchsin. Acid-fast
microorganisms will retain this dye and appear red. Microorganisms that are not
acid-fast, termed non acid-fast, will appear blue or brown due to the counterstaining
with methylene blue after they have been decolorized by the acid-alcohol.

Requirements:

Culture, carbol fushsin, methylene blue, decolourising solvent (acid-alcohol), Bunsen

burner, inoculation loop, glass slide, staining tray.

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Steps:

1. Prepare bacterial smear on a glass slide

2. Heat fix using a spirit lamp

3. Bacterial cells fixed on slide.

4. Apply carbol fuchsin to smear and heat for 5 minutes in hot plate, cool and rinse
with water.

5. Bacterial cells are stained.

6. Decolorize with acid-alcohol until pink (10-30 seconds), rinse with water.

7. Stains are may or may not decolorized.

8. Counter stain with methylene blue for about 2 minutes. Rinse with water.

9. Bacterial cells may or may not stain with methylene blue.

10. Examine slide under oil immersion.

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 Bacteria retain carbol fuchsin and appear red are called Acid-fast bacteria.

 Bacteria do not retain carbol fuchsin but take up methylene blue and appear blue
called non acid-fast bacteria.

Procedure:

1. Place the slide with an air-dried and heat fixed smear on a hot plate until the steam
rises with carbol fuchsin stained smear. Keep the preparation moist with stain and
steaming for 5 minutes.

2. Wash the film with gentle steam of water until no colour appears.

3. Decolorize with acid-alcohol and immediately rinse with water. Stain should appear
faintly pink.

4. Now, counter stain with methylene blue for about 2 minutes and rinse with water.

5. After drying, observe slide under oil immersion.

Results:

Acid-fast bacteria appear red, and non acid-fast appear blue in color.

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UNIT – 3

Sources and Reservoirs of infection (5 Mark)

Pathogens are endogenous, arising from the host’s own flora or exogenous,
arising from an external source. Reservoir of infection is the pathogen existence in
human or animal population or environment and from which pathogen can be
transmitted.

Endogenous infections

It is also known as autoinfection, example. Normal flora present inside the

body, usually non pathogenic but occasionally they may led to infection.

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Exogenous infections

Infections are arise from outside of the body, So it is referred as exogenous,

Few sources of exogenous infections are

1. Human
2. Animal
3. Insects
4. Environment
Human as carriers

Source of human infection comes from patient or a carrier.

Patients – patients are ill persons from them infection may acquired. A carrier is a
person who harbours the pathogenic microorganisms without suffering from it.

Few types of carriers are

Healthy carrier – people harbours the pathogen but has never suffered from the
disease. Convalescent carrier – people recovered from the disease but continue to
harbour the pathogen on his body. Temporary carrier – when carrier state lasts for
less than six months in the individual.

Chronic carrier – when carrier state lasts for years or may be for the life of the
patient. Parodoxical carrier – person acquires the organisms from another carrier.
Contact Carrier – person acquires the organisms from a patient.

Animal as carrier

Animals are source of infections. Infections is animals may be asymptomatic and

serve as reservoir for human infections are known as reservoir hosts.

Infections diseases transmitted from animals to man are known as zoonoses.

Insects transmitting pathogens are known as vectors. Thus insects act as a source
of a number of human and animal infections. Few examples for insects are
mosquitoes, ticks, nites, files and lice. Vector is of two types Mechanical vectors and
biological vectors.

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Mechanical vectors

Vectors which carry the organisms on their legs, wings and body and transmit
then to the food which acts as a source of infections e.g Salmonellosis and shigellosis.
(Domestic fly).

Biological vectors

Vectors carry pathogens undergo multiplication of developmental changes with

or without multiplication inside their body. Biological vectors are classified into three
types.

1. Propagative Vector:
When a pathogen undergoes no cyclic change but multiplies in the body of the vector
e.g. plague bacilli in rat fleas.

2. Cyclo – Propagative Vector:

In this pathogen undergoes a cyclic change and multiplies in the body of the
vector. E.g. malarial parasite in female anopheles nosquito.

3. Cyclo – developmental vector:

Pathogen undergoes cyclic changes but does not multiply in the body of the vector.
E.g. filarial parasite in culex mosquito and guinea worm larvae in Cyclops.

Each parasite require extrinsic incubation period to make them infective to cause
disease. Extrinsic incubation period is the interval of time required for the biological
vector to become infective from the time of entry of the pathogen into it.

Environment

Environment is the major source of infection. Environment includes soil, water

and food. Soil contains different organisms Such as clostridium, bacillus, roundworms,
hookworms and few fungal species. They are responsible for cousing disease to the
humans. Like soil, water also found with different pathogen such as Salmonella,
Shigello, Vibrio, Polio, hepatitis and larvae of worms. Food also contains organisms
causing food poisoning, gartroenteritis, diarrhea and dysentery.

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Spread or transmission of infection (5 Mark)

Pathogenic organisms can spread from one host to another by various ways.

Inhalation

 Most infections are transmitted by the respiratory route by aerosolization of

respiratory secretions when inhaled by others.
 Spread of infection depends on extend and method of propulsion of discharges from
the mouth and nose, the size of the aerosol droplets and the resistance of the
infectious agent to desiccation and inactivation by ultraviolet light.
 Droplets are very in size; In still air, a particle of 100mm in diameter requires seconds
to falls down but a 10mm particle remain airborne for about 20 minutes, small
particle even longer.
 When droplet particles with a diameter of 6mm or greater are usually trapped by the
mucosa of the nasal turbinates, where as particles of 0.6 to 5.0 mm attach to mucous
sites at various levels along the upper and lower respiratory tract and may initiate
infection. These “droplet nuclei” are most important in transmitting many respiratory
pathogens e.g. M.tuberculosis.
 Respiratory secretions are often transferred on hands or inanimate objects (tomites)
and may reach the respiratory tract. For example, spread of the common cold may
involve transfer of infectious individual, with transfer to other by hand – to – hand
contact and then from hand to nose by the unsuspecting victim.
 Clothing, handkerchiefs, bedding, floors, furniture and household articles become
contaminated with secretions and act as a reservoir of infection.
Ingestion (Fecal – Oral spread)

 Fecal – oral spread involves direct or finger – to mouth spread, the use of human
feces as a fertilizer or fecal contamination of food or water.
 Food handlers who are infected with an organism transmissible by this route, when
their personal hygienic practices are very poor.
 Some viruses transmitted by fecal – oral route infect and multiply in cells of the
oropharynx and then transmit to other body sites to cause infection. Generally,
organisms that are spread in this way commonly multiply in the intestinal tract and

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may cause intestinal infection. To cause intestinal infection they must able to resist
the acid in the stomach, the file and the gastric and small – intestinal enzymes.
Members of the interobacteriaceae and undeveloped viral intestinal pathogen are
more likely to survice.
Contact

 Infection may be acquired by direct to indirect contact with the patients.

 Sexually transmitted diseases are acquired by direct contact, usually termed as
contagious diseases. E.g Syphilis, gonorrhoea, and AIDS.
 Some infections such as herpes simplex and infectious mononucleosis can be
transferred directly by contact with infectious saliva through kissing.
 In some cases, rapid spread is possible when infectious secretion transmitted by
direct contact with the nasal mucosa or conjunctiva. E.g respiratory syncytial virus and
adenovirus.
Skin-to-skin transfer

 Skin-to-skin transfer occurs with a variety of infections in which the skin is the portal
of entry. E.g. spirochaete of syphilis, strains of group A Streptococci that cause
impetigo and the dermatophyte fungi that cause ringoworm and athelete’s foot.
 An inapparent break in the epithelium is probably involved in infection.
 In most cases, diseases may be spread through fomites. Such as shared towels and
inadequate cleaned shower and bath floors.
Blood born transmission

 Insect vectors are responsible for blood born transmission; vector requires a period of
multiplication or alteration within an insect vector before the organism can infect
another human host. E.g. mosquito and the malarial parasite.
 Presently, direct transmission from human to human through blood are increased by
the use of blood transfusions and blood products and the increased
self-administration of illicit drugs by intravenous or subcutaneous routs, using shared
nonsterile equipment. E.g. Hepatitis B and C viruses as well as HIV are frequently
transmitted in this way.
Eye – to – Eye transmission

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Infections of the conjuctiva may occur in epidemic or endemic form. Epidemics of

adenovirus and Haemophilus conjunctivitis may occur and are highly contagious. The
major endemic disease in trachoma is caused by Chlamydia, which remains a frequent
cause of blindness in developing countries. These diseases may be spread by direct
contact via ophthalmologic equipment or by secretions passed manually or through
tomites such as towels.

Zoonotic Transmission

 Zoonotic infections are spread from animals to humans; some zoonotic infections
such as rabies are directly contracted from the bite of the infected animal, while
other are transmitted by vectors mainly anthropods.
 Some infections and contracted from animals and then transferred between humans.
For example, plague has a natural reservoir in rodents. Human infections contracted
from the bites of rodent fleas, many produce pneumonia which may then spread to
other humans by the respiratory droplet route.
Vertical Transmission

 Organisms such as rubella virus can spread from the mother to the fetus through the
placental barrier is known as vertical transmission.
 Another form of transmission from mother to infant occurs by contact during birth
with organisms such as group B streptococci, C.trachomatis and N.gonorrhoeae which
colonize the vagina.
 Herpes simplex virus and CMV can spread by both vertical methods as it may be
present in blood or may colonize the cervix.
 The third way of vertical transmission is transmitted by breast milk. E.g. CMV.

Nosocomial infections or Hospital infection (10 Mark)

Introduction

The hospital is ideal environment for the transmission of pathogens, because

patients with similar diseases and susceptibilities are present in an enclosed
community patients share contact with many health care workers each day. In this

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setting, the ward, patients and workers become colonized by organisms adapted to
the special environment. New susceptible patients are frequently added to the
population and are at risk of colonization and infection.

Respiratory tract, urinary and wound infections are common in all hospital
patients. In addition, immuno compromised patients readily develop infection with
organisms of low virulence patients in the intensive therapy unit, where many
antibiotics are used, can be colonized with naturally resistant organisms which may
cause pneumonia or bacteraemia.

Organisms with multiple antibiotic resistances can cause problems on general

words as well as in special units. E.g. Methicillin – resistant staphylococcus aureus
(MRSA). These organisms and indwelling devices such as intravenous cannulae and
winary catheters. If they cause infections, prolonged treatment may be necessary,
using expensive or toxic drags such as teicoplanin or vancomycin.

Patient Predispositions to hospital infection

 Pre-existing Condition (Chronic chest disease, obstructed urinary outflow or previous

immune suppression).
 Need for invasive devices (intravenous cannulae, urinary catheters, etc).
 Effect of surgery (skin wound, tissue trauma, opening colonized viscus, anaesthesia,
immobilization, introduction of foreign material. Such as joint prosthesis or arterial
graft).
 Effect of antibiotic treatment (antibiotic – associated diarrhea, colonization by
resistant organisms, predisposition to superficial tungal infections).
 Effect of immunosuppressive treatment (corticosteroids, cancer chemotherapy or
transplant immunosuppression.
 Exposure to healthcare workers and other patients who may carry or transmit
pathogens – T.Exposure to pathogens in the environment, especially bedding and
food.

Infection due to intravenous infection

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 Many kinds of intravenous devices can placed in the vascular system for varying
methods in period to Intravenous devices placed for particular time depends.
Significantly on the likelihood of infection in each site.
 Peripheral venous catheters are readily colonized by organisms of the skin flora.
 Infection with mild inflammation is common but more invasive disease with
organisms such as aureus can cause significant morbidity and mortality.
 Venous catheters are used for intravascular monitoring or to give intravenous feeding
or drugs. E.g.Swan-Ganz catheters used. For long-term intravenous theraphy,
Hickman or portacath catheters can be inserted via a subcutaneous thack or funnel.
Infection in these devices is serious.
 The common pathogen of intravenous catheters is flora from the skin of the host,
particularly S.epidermidis and S.aureus. Corynebacterium Jeikeium may cause
line-related sepsis in leukaemic patients. Acinetobacter Spp. And enterococci can
cause line infections in intensive care. Clinical features – Infection are usually mild
until septicaemia found. Monitoring is important in detecting this early stage, So that
treatment will be successful. As a result of infection, there may be signs of
inflammation at the site of the skin entry with tenderness, cellulites or bacteraemia is
usually continuous and fever is often present but usually mild. Line-related sepsis are
detected by examining patient for signs of metastatic infection or endocarditic.
 To control line-related sepsis, need education and training in medical and nursing
schools.
 A strict protocol of skin disinfection and sterile technique must always be used when
inserting intravenous access devices.
 The choice of device is important. Side ports are phone to colonization, when no flow
occurs. Giving sets may also provide chance for colonization contamination can be
introduced into intravenous fluids by giving sets by repeated addition of drugs to the
intravenous system. This can be prevented by incorporating additive drugs into the
intravenous fluids, under strike and controlled conditions in the pharmacy.
 The most important factor in preventing line-related sepsis is the regular view of
inserted lines.
In some cases, intravenous feeding encourage infection by providing a nich supply of
nutrients for pathogens within the catheter lumen

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Infection associated with urinary Catheters Indwelling urinary Catheters

Indwelling urinary catheters provide easy access for ascending infection of the
urinary tract. After a number of days, organisms will reach the bladder often by
ascending between the catheter and the urethral wall. Permanent bladder
catheterization is always associated with bacterial colonization of the urine.

Gram negative organisms are the commenest colonizers of the catheterized

bladder. E.g. Escherchia coli, klebsiella pneunoniae and pseudomonas spp. Proteus
spp. are also found in chronically stagnant urine and their ability to metabolize urea
and produce alkaline ammonia predisposes to the deposition of calcium as stones.
These deposits can act as a reservoir of infection.

After trans-urethral prostatectomy S.aureus or coagulase – negative

staphylococci can cause urinary colonization catheter – related urinary colonization is
often asymptomatic but there is a risk of ascending infection or bacteraemia if the
catheter becomes blocked. Generally, treatment is not needed unless there is
evidence of infection. If fever, urinary tract symptoms, appropriate antibiotic therapy
should be given. The catheter should usually be replaced before chemotherapy is
discontinued, to ensure adequate drainage of the bladder and to remove a possible
reservoir of infection.

Patients in intensive therapy unit

In the intensive therapy unit (ITV) Patients are susceptible to infection for various
reasons

1. Immune responses are diminished by the stress and metabolic effects of existing
disease.
2. Many patients in intensive care have recently. Undergone anaesthetic and surgical
procedures.
Many of the barriers provided by innate immunity are breasted because of the need
for complex intravenous therapy, invasive monitoring, urinary catheters, artificial
ventilation and extracorporeal procedures such as dialysis or haemofiltration

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Tracheal intubations and Artificial Ventilation

Bacterial lung infections are the commonest complication of admission to
intensive care. The endotracheal tube allows organisms in the pharynx to bypass the
defensive mucociliary blanket and gain direct access to the lower respiratory tract.
When a patient has been in hospital for some time and may have received several
courses of antibiotics the normal upper respiratory tract flora has been replaced with
multidrug-resistant Gram-Negative organisms such as Acinetotacter,
Stenotrophomonas or Enterococcus faccium. These organisms can easily invade the
respiratory tract. Artificial ventilation usually imposes muscular paralysis; this inhibits
the normal sighing and coughing reflexes, further reducing the ability of patients to
resist bacterial invasion of the lungs.

Organisms causing lung infections in hospital

Streptococcus pneumoniae, MRSA, Moraxella catarrhalis, Klebsiella pneumoniae,

Escherichia coli, Enterococci, pseudomonas, candida and other yeasts.

Surgery related infection

Modern anaesthetic and surgical techniques has made dramatic increase in the
rate of infection. Several factors influence the occurrence of infection in surgical
patients: the patient, the operation.

Environmental factors in hospital infection

The hospital environment is very different from the general environment and
poses particular changes to patients ill-equipped to resist infection. Hospitals are
often crowded with much movement of patients, healthcare workers, volunteers and
visitors, providing person-to-person transmission by contact. Hospital food may
transmit food-borne disease if kitchen hygiene or food handling is unsatisfactory. The
hospital environment may allow for the transmission of aerosol-borne organisms.

1. Hospital water supplies

In hospitals, there are great demands for water which must be delivered to a
very large number of sites and for widely differing uses. The Legionella spp. will

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colonize warm water when stagnant water without chlorine then sporadic or
outbreaks of legionellosis may occur.

Inadequately cleaned and disinfected air-conditioning systems or cooling towers

are also common source of legionellosis. It is necessary to provide sterile water to
prevent infection in immuno compromised patients.

2. Hospital air supplies

Hospitals accumulate dust or other which contain sporing organisms and fungi can
thrive in these conditions. They cause respiratory or systemic disease in susceptible
individuals. Operating theatnes, laboratories and individual patient isolation rooms
are sites of particular risk.

3. Operating theatres
Main features in operating theatres are easy-to-clean, impermeable surfaces and
movable equipment. Air should be supplied to the theatre through filters.

4. Hospital equipment and the spread of infection

Disposable hospital equipment such as syringes, needles, blood lancets, scalpels,

intravenous cannulae and winary catheters makes the introduction of infection by
often-repeated procedures. Blood borne infections can be transmitted by inoculating
accidents with used sharp instruments. Use disposable and apply strict protocols to
the decontamination of all operating theatre equipment. Same equipment include
endoscopes can come into contact with body fluids, wound exudates or infected
tissues. These have been involved in hospital outbreaks of infection.

Simple material is mattresses, linen and beds can contribute to infection. All of
these have caused outbreaks of skin infection, sometimes systemic when patients
with burns, skin grafts or immunosuppressive conditions.

Control of infection in hospitals

Appropriate organization and effective management protocols are essential to

the control of hospital infection. Few control measures are

1. Sterilization and Disinfection

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2. Proper hospital waste disposal

3. Public awareness
4. Education and training to hospital staff

Hospital waste Management or BioMedical Waste Management (10 Mark)

Hospitals regularly generate waste which may be a potential health hazard to

health care workers, public and the environment.

We use Bio-Medical waste instead of hospital waste because it is a general or

broader term to define waste generated in the diagnosis, treatment, in research
mainly production or testing of biological molecules.

Any medical waste which has potential to transmit viral, bacterial or parasitic
diseases is known as infectious wastes. Infectious waste includes waste generated in
laboratories, human and animal infectious waste, and veterinary practice. Infectious
waste usually Hazardous in nature. Any waste with a potential to pose a threat to
human health is called hazardous waste.

Classification of Hospital waste

Generally, hospital waste are classified into Hazardous and non-hazardous waste.
Non hazardous waste is non infectious waste, it can be dump directly without any
pretreatment. But Hazardous waste can be further classified into Infectious waste and
other Hazardous wastes. Infectious waste contains Non-Sharp, Sharps, Plastic
disposables and liquid waste. Other Hazardous include radioactive, cytotoxic, glass
waste and incineration waste. Both Infectious and non-hazardous should be clarified
and pretreated before dump the waste. In the hospitals, enormous bulk of waste are
should be clarified based on their treatment and safe handling.

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Detailed Categories of Bio-Medical waste

1. Human anatomical waste – Human tissues, Oxgans and body parts.

2. Animal waste – Animal tissues, Organs, body parts, fluid, blood, experimented
animals.
3. Microbiology and biotechnology waste – waste from laboratory cultures, stocks,
specimens live or attenuated vaccines and cell cultures.
4. Sharp waste – Needles, Syringes, Scalpels, Blades, glass etc.
5. Discarded medicines and cytotoxic drugs.

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6. Solid waste – Items contaminated with blood and body fluids includes cotton,
dressing, beddings.
7. Solid waste – waste generated from disposable items such as tubings, catheters,
intravenous sets etc.
8. Liquid waste – waste generated from laboratory.
9. Incineration ash – Ash from incineration of bio-medical waste.
10. Chemical waste – chemicals used in disinfection etc.

Colour coding and type of container for Bio-Medical waste

Colour Code Waste Categories Type of container

Yellow 1, 2, 3 and 6 Plastic bag

Red 3, 6 and 7 Plastic bag

Blue / White 4 and 7 Puncture proof bag

Black 5, 9 and 10 Plastic bag

Treatment

Medical waste segregated in bags of different colour and appropriate treatment

need to carry out and disposed. Few treatment options are listed here.

Chemical disinfection

Disinfection is done for the following categories of waste such as sharp waste,
Disposable infectious plastics, Infectious glass wares and blood specimen. E.g for
effective disinfectant is hypo chlorites sharp waste taken in a puncture proof
container and filled with and put into puncture proof container for transferring to
shredding machine.

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Autoclave

Waste need to be undergoes autoclaving at 121 degree Celcius for 60 minutes.

Waste arising from laboratories such as cultures and stocks must be autoclave before
disposal by incineration.

Incineration

Pathological waste such as body parts of humans and animals are incinerated.
Incineration is a process by which combustible materials are converted into
non-combustible residue or ask.

Microwave

It is a special treatment to treat most infectious waste except body parts,

cytotoxic waste etc.

Deep burial

A pit should be dug about 2 meters deep. It should be filled with waste, the
covered with time and remaining covered with soil. It must be ensured that animals
do not have access to burial site.

Chemical treatment

Chemical waste must first be neutralized with appropriate reagents and then
flushed into a sewer system.

STERILIZATION (10 Mark)

It is defined as the process by which the article, a surface or a medium is freed of all
microorganisms either in vegetative or spore state.

Although many microorganisms are beneficial and necessary for human well-being
but few microbial activities may have undesirable consequence such as disease.

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Therefore, it is essential to be able kill a wide variety of microorganisms or inhibit

their growth to minimize their destructive effects. Purpose of these methods of
control is

1. To destroy pathogens and prevent their transmission.

2. To reduce or eliminate microorganisms which are responsible for the

contamination of water, food and other substances.

Ancient people have practiced disinfection and sterilization, but

existence of microorganisms was long unsuspected. Today the ability to destroy
microorganisms is most important, aseptic techniques used for different purposed
such as prevention of disease.

Application: It is used to prevent contamination of culture media; surgery for

maintenance of asepsis.

Agents used in sterilization are divided into

1. Sunlight

2. Drying

3. Heat

4. Filtration

5. Radiation.

Sunlight:

It is the natural method of sterilization. Sunlight is the ancient method to sterilize

different objects, contain ultraviolet ray’s acts as germicide.

Drying:

Water is essential for the survival of bacteria because 80% of the body mass of
bacteria contain water. Thus , drying has severe effect on many bacteria but spores
survive.

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Heat:

The application of heat is a simple, cheap and effective method of killing

pathogens. The time required for sterilization by heat is inversely proportional to the
temperature of exposure. High temperature is given for short period of exposure.
Sterilization by heat is depends on microorganism’s population, spores, species,
strains and nature of the material for heating. Generally, heat is of two types, they are
dry heat and moist heat.

Dry heat kills microorganisms by oxidation of essential cell constituents.

Dry heat less is effective than moist heat. The moist heat causes denaturation and
coagulations of proteins. Moist heat is in the form of water or steam is far more rapid
and effective in sterilization than dry heat. In dry heat, the destruction of
microorganisms including spores, possible after exposure to 160 degree celcius for 2
hours in a sterilizing oven.

Dry Heat:

Examples for dry heat are red heat, flaming, incineration and hot air oven.

Red Heat:

It is used to sterilize inoculating wire loops, forceps and spatulas. Sterilization is done
by holding material in a Bunsen burner flame until red hot. It is simple and rapid
sterilization.

Flaming:

It can be used effectively for emergency sterilization of a scalpel blades, needles, and
mouth of culture tubes, glass slides and cover slip. Flaming carried out by passing the
objects through the Bunsen burner without allowing them to become red hot.

Incineration:

Disposable material like soiled dressings and pathological materials are rapidly and
effectively decontaminated by incineration. It is rapid and effective method.

Hot air oven:

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This is the most widely used method of sterilization by dry heat. Holding period for
sterilization in hot air oven is 160 degree celcius for one hour. It is used to sterilize
glassware, forceps, scissors, glass syringes, swabs, test tubes, petric plates, pipettes
and flasks. Hot air oven is electrically heated and is fitted with a thermostat that
maintains the chamber air at a chosen temperature and a fan that distributes hot air
in the chamber. Hot are is bad conductor of heat and its penetrating power is low.
Objects should be arranged in a manner which allows free circulation of air in
between the objects. Glassware should be dried before being placed in oven. Test
tubes, flasks, etc should be wrapped in kraft paper. Rubber material should be used
because it cannot withstand the temperature. The oven must be allowed to cool
slowly for about two hours before the door is opened, since the glassware get cracked
by sudden or uneven cooling.

Efficiency: Dry heat efficiency was checked by keeping paper strips impregnated with
106 spores are placed in envelopes and inserted into suitable packs. After sterilization
is over, the strips are removed and inoculated into thioglycollate or cooked meat
media and incubated under anaerobic conditions for five days at 37 degree celcius.

Another method is using Browne’s tube is placed in the load. After

sterilization, a green colour is produced which indicated proper sterilization.

Moist Heat:

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Moist heat readily kills viruses, bacteria and fungi. Exposure to boiling water for
10 minutes is sufficient to destroy vegetative cells and eukaryotic spore. But the
temperature of boiling wate (100 degree celcius) is not high enough to destroy
bacterial endospores thay may survive hours of boiling. Moist heat sterilization
must be carried out at temperatures above 100 degree celcius in order to destroy
bacterial endospores. A process by which substances that is treated with controlled
heating at temperatures well below boiling is known as pasteurization. Thus moist
heat divided into three forms.

1. Temperature below 100 degree celcius.

2. Temperature of 100 degree celcius.

A. Boiling water. B. Steam.

3. Temperature above 100 degre celcius.

4. Low temperatures.

Temperature below 100 degree celcius:

Pasteurization:

High temperature can cause damage to the taste, texture and nutritional valve of
many food substances and in such cases, it is sufficient to destroy vegetative cells by a
process of pasteurization. Milk can be pasteurized in two ways. In the older method,
the milk is held at 63 degree celcius for 30 minutes. Nowadays, large quantities of
milk are subjected to flash pasteurization of high-temperature short-time (HTST)
pasteurization, which consists of quick heating to about 72 degree celcius for 15
seconds, then rapid cooling. In diary industry also uses Ultrahigh-temperature (UHT)
sterilization. Milk and milk products are heated at 140 to 150 degree celcius for 1 or 3
seconds. UHT-processed milk does not require refrigeration and can be stored at
room temperature for about two months without flavor changes.

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Using pasteurization, non sporing organisms such as Mycobacteria,

Brucella and Salmonella are destroyed. Generally, heat labile fluids e.g. serum may be
disinfected by heating at 56 degree celcius for one hour and patient clothing may be
disinfected by washing in water at 70-80 degree celcius for several minutes.

Temperature of 100 degree celcius:

A. Boiling at 100 degree celcius – Boiling at 100 degree celcius kills all vegetative
bacteria and some spores. Items like metals, glass and rubber are boiled and removed
with sterile forceps, dried and used.

B. Free steam at 100 degree celcius – In this method, steam at 100 degree celcius
under normal atmospheric pressure are employed. Substances like sugars and gelatin
may be decomposed on long heating, this can be avoided by Tyndalization.
Tyndalization is used for those substances or materials that might be damaged by the
high temperature. The material is heated to between 90 and 100 degree celcius for
about 30 minutes on each of three successive days and left at 37 degree celcius in the
intervening periods. Vegetative cells are killed during the heating period and during
the 37 degree celcius incubation, any endospores that have survived will germinate.
Once these have grown into vegetative cells, they too are killed in the next round of
steam treatment. This is long procedure and it is therefore reserved of those
materials which might be harmed by steam sterilization.

Temperature above 100 degree celcius:

Moist heat sterilization at temperature above 100 degree celcius are employed to
destroy bacterial endospores. For this requires the use of saturated steam under
pressure. Saturated steam has more advantages than hot air because it provides
greater lethal action, quicker in heating up articles, good penetration into cotton wool
stoppers paper, surgical linen, cloth wrappes and hollow apparatus.

Autoclave:

Principle and working of Autoclave:

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Autoclave is a device somewhat like a fance pressure cooker. The development of

the autoclave by Chamberland in 1884 stimulated the growth of microbiology. Water
boiled to produce steam, which is released through the jacket and into the
autoclave’s chamber is forced out until the chamber is filled with saturated steam and
the outlets are closed. Hot, saturated steam continues to enter until the chamber
reaches the dersired temperature and pressure, usually 121 degree celcius and 15
pounds of pressure. At this temperature saturated steam destroys all vegetative cells
and endospores in a small volume of liquid within 10 to 20 minutes. Time is extended
for about 15 minutes because larger containers of liquid such as flasks will require
much longer treatment times. Moist heat is thought to kill so effectively by degrading
nucleic acids and by denaturing enzymes and other essential proteins. It also may
disrupt cell membranes.

Autoclaving must be carried out properly or the processed materials

will not be sterile. If all air has not been flushed out of chamber, it will reach 121
degree celcius but it will not reach the appropriate pressure. The chamber should not
be packed too tightly because the steam needs to circulate freely and contact
everything in the autoclave. Bacterial endospores will be killed only if they are kept at
122 degree celcius for 10 to 12 minutes. When a large volume of liquid is sterilized, an
extended sterilization time will be needed because it will take longer for the center of
the liquid to reach 121 degree celcius. The velocity of killing increases logarithmically
with arithmetic increases in temperature, so a steam temperature of 121 degree
celcius is vastly more effective than 100 degree celcius. For example, the spores of
Clostridium botulinum may survive 5 hours of boiling, but can be killed in 4 minutes at
121 degree celcius in the autoclave.

Autoclaves can be used for sterilizing any materials that are not damaged by
heat and moisture, such a heat-stable liquids, swabs, nost instruments, culture
mediua and rubber gloves. When sealed containers of liquids are sterilized, it is
essential that the autoclave cool without being opened or evacuated; otherwise, the
containers may explode as external pressure are varied with inside pressure. If the
articles are kept for a long time after the notmal atmospheric pressure has reached

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inside the autoclave, an excessive amount of liquids will be evaporated from the
media.

“Flash” autoclaves, which are widely used in operating rooms, often use
saturated steam at a temperature of 134 degree celcius for 3 minutes. Air and steam
are removed mechanically before and after the sterilization so that metal instruments
may be available rapidly.

Indicator:

Biological indicator is often autoclaved along with other material. This indicator
commonly consists of a culture tube containing a sterile ampule of medium and a
paper strip covered with spores of Bacillus stearothermophilus or Clostridium
PA3679. After autoclaving, the ampule is aseptically broken and the culture incubated
for several days, if the test bacterium does not grow in the medium, the sterilization
run has been successful. Chemical indicator contain either special tape that spells out

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the word sterile or paper indicator strip that changes color upon sufficient heating is
autoclaved with a load of material. If the word appears on the tape or if the color
changes after autoclaving, the material is supposed to be sterile. These approached
are convenient and save time but are not as reliable as the use of bacterial
endospores.

Low Temperatures:

The most convenient control technique for microorganisms is to inhibit their growth
and reproduction by the use of either freezing or refrigeration. Freezing items at -20
degree celcius or lower will stops microbial growth because of the low temperature
and the absence of liquid water. Some microorganisms will be killed by ice crystal
disruption of cell membranes. Freezing is a very good method for long-term storage
of microbial samples and many laboratories have a low-temperature freezer for
culture storage at -30 or -70 degree celcius.

Filtration:

Many liquids such as solutions of antibiotics or certain components of culture media

became chemically altered at high temperatures, so use of heat methods is not
appropriate. Instead of killing microorganisms using heat methods, an alternative
approach is simply to isolate microorganisms. This can be done for liquids and gases
by passing them through filters of an appropriate pore size. Although different filters
are available but commonly used are membrane filter made of nitrocellulose or
polycarbonate. Filtration has two advantages.

1. Chemical alternation prevented in heat-labile solutions.

2. Reduce microbial populations in solution by removing them.

There are two types of filters. 1. Depth filters 2. Membrane filters.

Depth filters:

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It consists of fibrous or granular materials that have been bonded into a thick layer
filled with twisting channels of small diameter. The solution containing
microorganisms is sucked through this layer under vacuum, and microbial cells are
removed by physical screening or entrapment and also by adsorption to the surface
of the filter material. Depth filters are made of diatomaceous earth (Berkefield filters,
unglazed porcelain (Chamberlain filters), asbestos filters or other similar materials. 2.
Membrane filters.

These filters are porous membranes, 0.1mm thick, made of cellulose acetate,
cellulose nutrate, polycarbonate, polyvinylidene fluoride or other synthetic materials.
It available in different pore sizes, membranes with pores about 0.2 micrometre in
diameter are used to remove most vegetative cells.

HEPA filters:

HEPA filters sterilize air. Laminar flow biological safety cabinets contain
high-efficiency particulate air filters (HEPA), which remove 99.97% of 0.3 micrometre
particles. Laminar flow air through HEPA filters and projects a vertical curtain of
sterile air across the cabinet opening. This protects a worker from microorganisms
being handled within the cabinet and prevents contamination of the room. This
cabinet is used in research labs and industries to provide a sterile working surface.

Radiation:

Radiation is used to control the growth of microorganisms. Radiation is classified

into two types. 1. Ionizing radiation 2. Non-ionizing radiation.

Non-ionizing Radiation:

The most widely used form of non-ionizing radiation is ultraviolet (UV) light.
Generally, wavelength around 260 nm is used because these are absorbed by the
purine and pyrimidine components of nucleic acids and aromatic aminoacid in
proteins. The absorbed energy caused a rupture of the chemical bonds, so cellular
function is damaged. UV light causes the formation of thymine dimers, thus inhibit

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DNA replicaton. UV lamps are commonly used in food preparation areas, operating
theatres and tissue culture facilities to prevent contamination. It is lethal but does not
penetrate glass, water and other substances very effectively. UV lamps are placed on
the ceilings of rooms or in biological safety cabinets to sterilize air. People working in
such areas must be certain that UV lamps are off when areas are in use because UV
radiation burns the skins and damages eyes.

Ionizing Radiation:

Ionizing radiation has a shorter wavelength and much higher energy, giving them
greater penetrating powers. It will destroy bacterial endospores and vegetative cells,
both prokaryotic and euckaryotic; but it is not always as effective against viruses.
Gamma radiation from a Cobalt 60 source is used in sterilization of antibiotics,
hormones, sutures and surgical supplies like syringes, catheters and rubber gloves.
When heat is not employed to sterilize objects then this type of sterilization with
these radiation are known as cold sterilization.

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Unit – 4

Candidia albicans (5 Mark)

It is caused by candida albicans. Candidiasis occurs in localized and disseminated

forms. Localized disease is seen as erythema and white plaques in moist skin folds or
on mucosal surfaces. Immunocompromised patients are often susceptible to deep
tissue and disseminated disease.

Charactertics

Candida species grow as round or oval yeast cells. During infections, it can form
and chlamydoconidia. Most candida species grow rapidly on Sabouraud’s agar and
blood agar. C. albicans and occur as different morphologic forms, mostly as a yeast
with budding by formation of blastoconidia.

C.albicans cell wall is made up of a mixture of the polysaccharides mannan,

glucan and chitin.

Reservoir and Transmission

C.albicans is a normal flora found in oropharyngeal, gastrointestinal and female

genital. Mostly infections are endogenous but in some cases there is direct mucosal

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contact with lesions in others (e.g through intercourse). It is a common cause of

nosocomial infections. It is transmitted through invasive procedures and indwelling
devices.

Pathogenesis

C.albicans usually found on mucosal surfaces. C.albicans becomes pathogenic

potential when there are changes from the yeast to a hyphal form. These changes are
controlled by the environmental conditions.

C.albicans has ability to attach to human epithelial cells. Fungus contains surface
hyphal wall protein which is found on the surface of germ tubes and hyphae, helpful
in binding to host cells. Hyphae secrete proteinases and phospholipases that are able
to digest epithelial cells and help in invasion.

C.albicans has protein surface receptors that bind the C3 component of

complement, thus prevent opsonization. Enhanced production of these receptors
under high glucose concentration and aid in resistance to phagocytosis. Factors allows
C.albicans increase their proporation the flora, during antibacterial therapy, that
compromise immune capacity of the host or that interfere with T-lymphocyte
function are often associated with local and invasive function. Disruptions of the
mucosa by indwelling devices and cancer chemotherapy may enhance the invasion
process. Diabetes mellitus also predisposes to C.albicans infection because of greater
production of the surface mannoproteins in the presence of mannoproteins.

Clinical features

Superficial invasion of the mucous membranes by C.albicans produces a white,

cheesy plague that is loosely adherent to the mucosal surface. The lesion is usually
painless, when plaque torn away then it will be painful. Oral lesions occur will be
painful. Oral lesions occur on the tongue, palate and other mucosal surface as single
or multiple ragged patches. This lesion is called thrush. C.albicans infect Vagina and
cause vaginal candidiasis produces a thick, curd-like discharge and itching of the
vulva.

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C.albicans skin infections occur in crural folds and macerated skin surfaces other
infections of the skin folds and appendages occur due to recurrent immersion in
water. The intial lesions are erythematous, papules or confluent areas with
tenderness, erythema and fissures of the skin.

Persons with specific defects in T cell – mediated immune defence develops

achronic, relapsing form of candidiasis known as chronic mucocutaneous candidiasis.

Inflammatory patches may develop in the esophagus which looks similar to

thrush painful swallowing, chest pain, Ulcerations, deformity and perforation of
esophagus. In immuno compromised patients, similar lesions may also develop in the
stomach and deep ulcerative lesions in the small and large intestine.

Infection of the urinary tract by hematogenous route may produce cystitis,

pyelonephritis, abscesses or fungal ball lesions in the renal pelvis. Endophthalmitis has
the characteric appear as white cotton ball expanding on the vetina or floating free in
the vitreous humor, thus infections of eye structures can lead to blindness.

Diagnosis

To detect superficial C.albicans infections, exudate or epithelial scrapings are

examined by potassium hydroxide preparations or Gram stain show budding yeast
cells; if hyphae present confirm the presence of C.albicans. To detect lung infection, it
requires biopsy or bronchoalveolar lavage for examination. For candida endocarditis,
arterial blood cultures are needed.

Treatment

Superficial infections are treated with topical nystatin or azole preparations,

which help to decrease moisture and chronic trauma in treating skin infections.
Persistent relapsing or disseminated candidiasis is treated with amphotericin B,
flucytosine, fluconazole or combined amphotericin B with other drugs.

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Aspergillosis (5 Mark)

Aspergillosis is caused by Aspergillus species. Aspergillus species are rapidly

growing molds with branching septate hyphae and arrangement of conidia on the
conidiophore. Species are identified based on structure of conidiophore and the
arrangement of the conidia.

Reservoir and Transmission

Aspergillus species are widely distributed in nature and they able to adapt to a
wide range of environmental conditions. Inhalation of conidia is the mode of
infection.

Pathogenesis

After inhalation, Aspergillus conidia are small enough to reach the alveoli, mostly
happens in immuno compromised individuals. The conidia contain protein on their
surface will bind to fibrinogen and laminin. Production of extracellular elastase,
proteinases and phospholipases are help in causing injury.

Clinical features

Aspergillus can cause clinical allergies or invasive infection. In both cases, lung is
primarily involved. Allergic aspergillosis occur in patients with asthma is characterize
by transient pulmonary infiltrates, eosinophilia and IgG Abs. In this, anatomic
abnormalities may serve as a site for growth for organisms.

Invasive aspergillosis occurs to the individuals with preexisting pulmonary

disease, (chronic bronchitis, asthma and tuberculosis) or immunosuppression. In
patients with chronic pulmonary disease, mycelial mass can form a visible fungal ball
(aspergilloma) within a cavity.

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An acute pneumonia may occur in severely immuno compromised patients,

mainly phagocyte defects or depressed neutrophil counts due to immuno suppressive
drugs.

Diagnosis

For invasive disease, lung aspiration, biopsy or serologic methods are useful to
detect aspergillus antibodies.

Treatment

Amphotericin B and itraconazole are used for invasive aspergillosis. In case of

pulmonary structural abnormalities and fungal balls, chemotherapy has little effect.
Surgical removal of localized lesion is also done for invasive disease.

Life Cycle of Plasmodium (5 Mark)

The plasmodia are sporozoa in which the sexual and asexual cycles of
reproduction are completed in different host species. The sexual reproducing occurs
within the gut of mosquitoes. Mosquitoes transmit the parasite when feeding humans
within human RBCS, plasmodia reproduce asexually; plasmodia burst RBC; and invade
other RBCS. This event produces periodic fever and anemia in the host and this
disease process known as malaria. Plasmodium vivax, P.ovale, p.malariae and
P.falciparum are the species of plasmodia infect humans.

Morphology

 The stained erythrocytic parasites have three charactertic features: Red Nuclear
chromatin; blue cytoplasm; and brownish – black malarial pigment or hemozoin
contain haemoglobin degradation product ferriprotoporphyrin IX.

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 There are changes in the shape of the cytoplasm and the division of the chromatin at
different stages of parasite development.
 Gametocytes are large in size and lack of nuclear division.
 Infected erythrocytes develop membrane invaginations or pink schuffner’s dots or
granules.
 P.Vivax and P.Ovale infectedn erythrocyte appear as pale, enlarged and contains
number schuffner dots. Asexual stages (trophozoite, schizont and merozoite) seen
simultaneously.
 Cells infected by P.ovale and elongated and frequently irregular or fimbriated in
appearance.
 In P.Malariae infections, the RBC is not enlarged and contains no granules. The
trophozoite appears as band forms and the merozoite are arranged in rosettes
around a clump of central pigments.
 In P.falciparum infections, the rings are very small and contain two chromatin dots.
More than are contain two chromatin dots. More than are parasites found per cell.
Intracytoplasmic granules known as Maurer’s dots and fewers in number. Schizonts
are merozoites are not present in the peripheral blood. Gametocytes are large and
banana shaped.

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Life Cycle

Sporogony or the Sexual Cycle

It begins when a female mosquito of the genus Aropheles ingests circulating male
and female gametocytes when feeing infected human.

Gametocyte enters into mosquito and reaches gut of the mosquito, the
gametocytes nature and undergo fertilization.

Resulting zygote penetrates the mosquito’s gut wall, lodges beneath the
basement membrane and vacuolates to form an oocyst.

Within Oocyst structure, thousands of sporozoites are formed.

Enlarged cyst ruptures, release sporozoites into the body cavity of the mosquito.

Sporozoites penetrate into salivary glands and mosquito become infections for
humans.

Completion of the sexual cycle in mosquitoes varies from 1 to 3 weeks depending

on the species of insect and parasite as well as on the temperature and humidity.

Asexual cycle – schizogony

It occurs in human and begins when the infected Anopheles takes a blood meal
from another individual.

Sporozoites from the mosquito’s salivary glands are injected into the human’s
subcutaneous capillaries and circulate in the peripheral blood.

Within hours sporozoite attaches to and invades liver cells (hepatocytes) by

sporozoite’s outer protein coat.

(In P.Vivax and P.Ovale infections, some of the sporozoite enters a dormant state
immediately after cell invasion)

Remaining sporozoites intiate exoerythrocytic schizogony, each sporozoite

produces about 2000 to 4000 daughter cells or merozoites.

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One to two weeks later, the infected liver cells rupture releasing merozoites into
the blood circulation.

Erythrocytic phase

It starts when released hepatic merozoites attach to a specific receptor on the

RBC surface. After attachment. Merozoite invaginates cell membrane and enters into
RBC.

Parasites appear as a ring – shaped trophozoite which enlarges and becomes

more active and irregular.

Within few hours, trophozoite multiplies in RBC producing multinucleated

schizont.

Cytoplasm condenses around each nucleus of the schizont to form new

merozoites.

About 48 – 72 hours after invasion, infected erythrocytes rupture and release

merozoites (daughter cells). (Produce first clinical manifestations of disease).

Newly relased merozoite invades other RBCs.

Later, few daughter cells (merozoites) transform into sexual forms or

gametocytes.

These cells continue to circulate in the peripheral vasculature until ingested by an

mosquito.

Occurrence

Malaria has a world wide distribution. P.vivax is the most widely distributed
species and P.malariae is found primarily in temperate and subtropical areas.
P.falciparum is found in the tropical region. P.ovale is rare and found mostly in Africa.

The malarial transmission depends on the density and feeding habits of mosquito
vectors and the prevalence of infected humans (parasite reservoirs).

Pathogenesis

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The fever, anemia circulatory changes and immunopathogenic charactertic of

malaria is due to the erythrocytic invasion by the plasmodia. Fever results when RBC
rupture that leads to release merozoites. Parasitized erythrocytes are removed by a
stimulated reticuloendothelial system causes anemia. When hemolysis is massive,
hemoglobin-nuria develops resulting in the production of dark urine. This process
with malaria is known as blackwater fever. Circulatory changes occur due to high
fever, resulting vasodilatation. Vasodilatation leads to a decrease in the circulating
blood volume and hypotension which is by changes in the small vessels and
capillaries.

Clinical features

The clinical features vary with the species of plasmodia. Generally, it includes
chills, fever, splenomegaly and anemia. Malarial paroxysm begins with cold stage
which persists for 20-60 minutes. During this time the patient experiences continuous
rigors and feels cold. It is followed by increase in body temperature, the rigors ceases
and vasodilatation starts. The temperature continues to rise for 3 to 8 hours reaching
a maximum of 40 to 41.7oC. The wet stage consists of a decrease in fever and profuse
sweating.

In falciparum malaria, capillary blockage can lead to several serious

complications. Few symptoms are vomiting, abdominal pain, and diarrhea and in
serve cases develop derlirium, convulsions, coma and death.

Diagnosis

Generally, stained smears of the peripheral blood are used to observe malarial
parasites in all symptomatic patients. To prepare thin and thick smears, capillary or
venous blood is used. These smears are stained with wright is used. These smears are
stained with wright or Giemsa stain to examine presence of erythrocytic parasites. For
thick smears, erythrocytes are lysed with water and stained to allow detection of very
mild parasitemia. Para sight F test is used to detect a protein (HRP2) excreted by
P.falciparum within minutes. Another test – Optimal detects parasite lactate
dehydrogenase and it can able to distinguish between p.falciparum and p.vivax.

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Treatment

Important task in treating malaria is destruction of three parasitic forms: the

erythrocytic schizont, the hepatic schizont and the erythrocytic gametocyte.
Chloroquine, Quinine, antifolates, Sulfonamide is used against erythrocytic schizont;
chloroquine and artemisinin are as helpful against erythrocyte gametocyte and for
hepatic schizont, primaquine are mostly used.

Life Cycle of Entamoeba histolytica (5 Mark)

Morphology

E.histoloytica has both trophozoite and cyst forms. The trophozoites are
microaerophilic found in the human or wall of the colon. Usually feed on bacteria and
tissue cells. It can multiply rapidly in the anaerobic environment of the gut.
Trophozoite mostly detected by their size (12 to 20mm in diameter); motility;
granular vacuolated endoplasm, and ectoplasm with finger – like pseudopods. In few
invasive strains, trophozoite in larger and may contain ingested erythrocytes within
their cytoplasm, 3.5 mm nucleus with central karyosome or nucleolus and finen
regular granules evenly distributed around the nuclear membrane.

Trophozoite encyst before leaving the gut. A cyst contains a single nucleus, a
glycogen vacuole and one or more ribosomal clusters known as chromatoid bodies.
Nature cysts can survive environmental temperatures up to 55 degree celcius,
chlorine wnc, and normal level of gastric acid.

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Life cycle

Humans are the main hosts and reservoirs of E.histolytica.

Transmission from person to person occurs when a parasite passed in the stool of
one host is ingested by another host through various ways.

Since Trophozoite die rapidly in the external environment, cyst can only able to
pass to the host. Human hosts may pass upto 45 million cysts daily.

Once cyst ability to enter into the body through ingestion, it passage through the
stomach and reaches the distal small bowel.

In Bowel, cyst disintegrates and releases the quadrinucleate parasite which

divides to form eight small trophozoites that reach the colon.

Colonization observed mostly in areas of fecal stasis such as the cecum and
rectosigmoid but also found throughout the large bowel.

Occurrence and transmission

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It’s infection rate are higher in warm climates particularly areas with low
sanitation. Symptomatic amebiasis caused as the result of direct person – to – person
fecal – oral spread under conditions of poor personal hygiene. Venereal transmission
is seen in male homosexuals as the result of oral – anal sexual contact. Food and
water are other modes of transmission.

Pathogenesis

A number of virulent factors are responsible for pathogenesis. For invasiveness,

the presence of a galactose – specific lectin capable of attachment (adhere) of the
organism to colonic mucosa and capacity to lyse host cells.

After adherence, the ameba release a pore forming protein that polymerizes in
the target cell membrane forming large tubular lesions and followed by cycolysis.

In most infections, tissue damage is less, thus the host remains without
symptoms. Few host to invasion such as protein malnutrition high-carbohydrate diets,
corticosteroid administration. Child hood and pregnancy make the host more
susceptible to invasion. Few coloric bacteria appear to enhance to enhance
invasiveness by providing more favourable conditions for survival and multiplication
or facilitate the adherence of the parasite to colonic mucosa.

Pathology

When amebas contact and lyse colonic epithelial cells small mucosal ulcerations
and little inflammatory response. Trophozoites are present in large numbers at the
junction between necrotic tissue and viable tissue. When the lesion is penetrates
below the superficial epithelium and a spreads in the submucosa producing lesions.
Extensive Ulcerations lead to secondary bacterial infection forming of granulation
tissue and fibrotic thickening of the colon. In few cases, the granulation tissue is
organized into large, humor-like masses known as amebomas. Amebomas occur in
ceecum, ascending colon, rectum, sigmoid, appendix and terminal ileum. Amebas
may also enter the portal circulation and move to the liver.

Clinical features

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In most cases, particularly temperature zones, E.histolytica is avirulent, living in

the towel as normal commensal inhabitant.

As the result of infections, Diarrhea, flatulence and cramping abdominal pain are
the common symptoms found. In fulminant disease, the stool consists of watery, foul
– smelling passages that contain mucus and blood. Fulminating amebic dysentery
occur in pregnant individuals and symptoms like high fever, severe abdominal cramps
and profuse diarrhea is produced. Amebic liver abscess may extend into surrounding
tissue produce pneumonia, empyema and peritoritis.

Diagnosis

For intestinal amebiasis, microscopic examination is done to detect organisms in

stool or sigmoidoscopic aspirates. Stools specimens or aspirates are used to identify
trophozoites and cysts by stained or wet preparations. Entamoeba species are
differentiated based on important charactertics. For example, E.histolytica ingests
erythrocytes; but E.dispar do not ingest.

Enzyme immunoassay and other methods can detect antigen in stool. The
diagnosis of extraintestinal amebiasis is more difficult because the parasite cannot be
recovered from stool and tissues. Serologic tests are employed to detect parasites
because intestinal disease and hepatic abscess have high levels of antiamebic
antibodies.

Treatment

Metronidazole is the drug is used and effective against all forms of amebiasis.
Along with metronidazole, diloxanide is used to improve the cure rates in intestinal
disease and diminish the chance of recrudescent disease in hepatic amebiasis.

Prevention

Efforts are needed for proper sanitary disposal of human feces and improvement
in personal hygienic practices.

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Trichomonas Vaginalis (5 Mark)

Trichomoniasis

Trichomoniasis is a sexually transmitted disease caused by Trichomonas vaginalis.

This disease produces a vaginitis with pain, discharge and dysuria. Men are usually
asymptomatic but may have urethritis and prostatitis.

Charactertics

T.Vaginalis is an pathogen infecting humans, The T.vaginalis trophozoite is oval

and size is about 7 by 15 mm. In asymptomatic patients, it’s size is doubled. In stained
preparations, a single, elongated nucleus and a small cytostome are found in anterior
portion. Five flagella are found, in which four outside the cell. The fifth bends back
and runs posteriorly along the outer edge of an undulating membrane. A microtubule
containing a supporting rod or axostyle bisects the trophozoite longitudinally and
protrudes through its posterior end. This axostyle is useful for attachment and
responsible for tissue damage.

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Trophozoite of Trichomonas Vaginalis

Culture charactertics

The organism can be grown on artifical media under anaerobic conditions at pH

5.5 to 6.0. Organisms can absorb soluble nutrients across the cell membrane.
Moreover, material such as bacteria, leukocytes and erythrocytes may be ingested
through any area of cell surface. T.Vaginalis lacks a cyst form but trophozoite able to
survive outside of the human host for 1 to 2 hours on moist surfaces. Moist
conditions like urine, semen and water it is viable for 24 hours.

Occurrence and Transmission

Trichomoniasis is a cosmopolitan disease usually transmitted by sexual

intercourse. About 25% of sexually active women become infected and 30 of to 70%
of their male sexual partners are also parasitized. Infection is rare in adult virgins but
rates as high as 70% are seen among sexual partners of infected patients. In women,
the peak incidence is between 16 and 35 years of age but high prevalence in the 30 to
50 year age group. Female neonates are occasionally to harbox T.Vaginalis, acquiring

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it during passage through the birth canal. When maternal estrogen is high then there
is transient decrease in the vaginal pH of the child making it more susceptible to
colonization. When estrogen levels drops, the parasite is eliminated in the vagina.

Pathogenesis

T.Vaginalis has direct contact with the squamous epithelium of the genitourinary
tract results in destruction of the epithelial cells and development of a neutrophillic
inflammatory reaction and petechial hemorrhages. The severity of the pathologic
changes is modulated by the changes in the microbial, hormonal and pH environment
of the vagina.

Clinical features

As the result of T.Vaginalis infection, persistent vaginitis produced in the women.

Generally most cases are asymptomatic at the time of diagnosis; most develop clinical
manifestations within 6 months. Approximately 75% of women develop a discharge
which is accompanied by vulvar itching or burning, and a disagreeable odor. In
symptomatic patients, physical examination reveals reddened vaginal and
endocervical mucosa. In severe cases, Petechial hemorrhages and extensive erosions
are present Trichomoniasis conditions results in increase the risk of in preterm birth
and enhance susceptibility to human immuno deficiency virus (HIV) infection.

In men, the urethra and prostrate are the usual sites of infection; few occasions
the seminal vesicles and epididymis may be involved. These infections are usually
asymptomatic because the organisms are removed from the urogenital tract by
voided urine. In symptomatic men, recurrent dysuria and scant, non purulent
discharge is found and acute purulent urethritis has been reported rarely.

Diagnosis

The diagnosis of trichomoniasis, detection and morphologic identification of the

organism in the genital tract is needed. In women, a drop of vaginal discharge is used
as specimen; in men, urethral exudate or urine sediment after prostrate massage may
be used. Identification is done by examining a wet mount preparation for the

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presence of motile organisms. Giemsa and papanicolaou stained smears are used for
examination. Serologic method include direct immuno-fluorescent antibody staining
has a sensitivity of 70 to 90%.

Treatment

Oral metronidazole is effective in curing more than 95% of all infections. It may
be given as a single does or over 7 days. When single-does therapy is used,
simultaneous treatment of sexual partners is needed to minimize recurrent
infections. During treatment, alcoholic consumption should be suspended. The drug
should not be used during the first trimester of pregnancy because of its potential
teratogenic activity. Drug can be used in the last two trimesters but should be used
for patients whose symptoms cannot be controlled by local therapies.

Giardia lamblia (5 Mark)

Giardiasis

Giardiasis is an intestinal disease caused by Giardia lamblia. Infection acquired

from untreated water sources but mostly disease is asymptomatic. When disease
occurs, it is in the form of a diarrhea lasting up to 4 weeks with foul – smelling, greasy
stools. Abdominal pain, nausea and vomiting are also present.

Charactertics

Giardia contains both a trophozoite and a cyst form. Trophozoite is a

sting-ray-shaped, 9-21 mm in length, 5-15 mm in width and 2 to 4 mm in thickness.

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Organism contains two nuclei and central parabasal bodies. Four pairs of flagella
found at anterior, lateral, ventral and posterior region. Cyst forms are oval and
smaller than trophozoites. Cyst possesses quadrinucleus (four nucleus), two sucking
discs, four parabasal bodies and eight axonemes. The nature cysts are the infective
form of the parasite, may survive in cold water for more than 2 months and resistant
to concentrations of chlorine. Cysts are transmitted from host to host by the fecal –
oral route. After transmission in the duodenum of a new host, cytoplasm divides to
produce two binucleate trophozoite.

Trophozoite present in the duodenum and jejunum, where they survive in the
alkaline environment and absorb nutrients from the intestinal tract. These
trophozoite move to mucous layer at the base of the microvilli with a peculiar
tumbling or falling leaf motility and with the help of a large ventral sucker, it attach to
the brush border of the intestinal epithelium. Unattached organisms may be carried
by the fecal stream to the large intestine. In the descending colon, the flagella are
withdrawn into cytoplasmic sheaths and a smooth, clear cyst is secreted. Thus, cystic
forms developed in the colon.

The genus Giardia is among the most widely distributed of intestinal protozoa;
they are found in fish, amphibians, reptiles, birds and mammals. On the basis of
central parabasal body morphology, three morphologically distinct groups of Giardia
have been described.

Transmission

Giardiasis has a cosmopolitan distribution; it is prevalent in areas with poor

sanitation and the population without adequate person hygiene. Infection is
transmitted by the fecal-oral route from person to person (families, childrens
between homosexuals) or in food and drinking water. The sources of infection include
untreated pond or stream water, sewage contaminated municipal water supplies and
domestic cats and dogs are prevalent of G.lamblia and may also acts as reservoirs for
human infection.

Pathogenesis

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Giardiasis disease may cause intestinal malabsorption (mainly fat and

cartohydrates) and inflammation as well as morphological changes in the small
intestine. Although, precise pathologenetic mechanisms responsible for these
changes remain poorly understood. Mechanical blockade of the intestinal mucosa by
large numbers of Giardia, damage to the brush border of the microvilli by the
parasite’s sucking disc, organism induced deconjugation of bile salts, altered intestinal
mobility have all been suggested for these changes in the intestine.

Clinical features

In endemic situations, over two third of infected patients are asymptomatic in

symptomatic patients, symptoms begin 1 to 3 weeks after exposure; they include
diarrhea, which is sudden and explosive in character. The stool is foul smelling, greasy
in appearance and floats in on water. Stool found with blood or mucus. Upper
abdominal cramping is common. Large quantities of intestinal gas produce abdominal
distention, sulfuric erucations and abundant flatus. Sulfuric erucations and abundant
flatus. Nausea, Vomiting and low-grade fever may be present. The acute illness
generally resolves in 1 to 4 weeks; in children, it may persist for months, leading to
significant malabsorption, weight loss and malnutrition.

In adults, the acute phase is often followed by a subacute or chronic phase

characterized by intermittent bouts of mushy stools, flatulence, and heart burn and
weight loss persists for weeks or months. In most individuals, symptoms and
organisms eventually disappear spontaneously. Lactose intolerance persists even
after the eradications of the organisms.

Diagnosis

The diagnosis involve mainly in identification of cyst in formed stool or the

trophozoite in diarrheal stools, duodenal secretions or jejunal biopsy specimens. In
acutely symptomatic patients, the parasites are identified by examining one to three
stool specimens with appropriate concentration and staining procedures are used. In
chronic cases, patients can be diagnosed by examining specimens taken at weekly
intervals over 4 to 5 weeks. Apart from stool specimens, duodenal secretions can be

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collected and examined for trophozoites in trichone or Giemsa 0 stained

preparations. Serological methods are reliable and commercially available. E.g.
enzyme immunoassays (EIA) are used for the direct detection of parasite antigen of in
stool. EIA method is sensitive and specific as microscopic examinations. The
organisms can be cultured in the laboratory but the methods are not currently used
for routine diagnostic work.

Treatment

The drugs such as quinacrine hydrochloride, metronidazole, furazolidone and

paramomycin are used for the treatment of giardiasis. Quinacrine and metronidazole
are more effective (70 to 95%) and preferred for patients capable of ingesting tablets.
Furazolidone is given to children as it is available as a liquid suspension but it has the
lowest cure rate. These three drugs require 5 to 7 days of therapy. Tinidazole is an
oral agent and it is safe and effective in single-dose treatment. In pregnant women,
these agents should not be used because of their potential teratogenicity. In this
circumstance, paromomycin is used but it is less effective agent.

Prevention

People should avoid ingestion of untreated surface water because of the

possibility of contamination by feces of infected animals. Use halogen tablets for
adequate disinfection of water and also safety is maintained by filtration procedures.

Leishmania (5 Mark)

Charactertics

Leishmania is the genera of Leishmania reside and reproduce within the gut of
specific insect hosts. When these vectors feed on a susceptible mammal, the parasite

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penetrates the feeding site, invades the blood and/or tissue of the new host and
multiplies to produce disease. The life cycle of leishmania is completed when a
second insect ingests the infected mammalian blood or tissue fluid. During the course
of passage through insect and vertebrate host, Leishmania undergo developmental
change. Within the gut of the insect, organism, assumes the promastigote form.
These protozoa are motile, fusiform and have a blunt posterior end and a pointed
anterior from which a single flagellum projects. It measure 15 to 30 mm in length and
1.5 to 4.0 mm in width. In the promastigote, the kinetoplast is located in the anterior
end. In the mammalian host, Leishmania appear as amastigote. The amastigote form
is found intracellular and is round or oval, measures 1.5 to 5.0 mm in diameter. It
contains a nucleus with a central karyosome, kinetoplast and an axoneme, But flagella
is absent.

Leishmania strain is classified into four major groups based on their serologic,
biochemical, cultural, nosologic and behavioral charactertics.

Transmisison

Leishmaniasis is the disease caused by leishmania. Leishmania tropica and

L.Mexicana produce a localized cutaneous lesion or ulcer. It known as oriental sore
and chicero ulcer. L.braziliensis is the cause of American mucocutaneous
leishmaniasis and L.donovani is the causative agent for kala azar disease.

These leishmania species are transmitted by phlebotomine sandflies. Sandflies

are small, delicate, short lived insects found throughout the tropics and subtropics
environment.

Life cycle of Leishmania

Insects feed on infected mammalian host (humans)

Amastigotes form ingested by the insects.

Amastigotes undergo developmental change to flagellated promastigote form,

multiply within the gut and migrate to the buccal cavity in the insect.

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When insect feed on human or animal host, the buccal promastigotes are
injected into the skin along with salivary peptides capable of inactivating host
macrophages.

Following phagocytosis, the promatigotes lose their flagella and multiply as the
rounded amastigote form within the phagolysosome.

(Macrophage killing mechanisms is inhibited).

Amastigote undergo Multiplication leads to the rupture of the phagocyte and

release of the daughter cells.

Sandfly ingest daughter cells (Amastigote) and get infected.

In insects, Amastigote transformed into promastigote form and undergo

multiplication.

When insect feed on another human host, life cycle continued.

Disease

Localized cutaneous Leishmaniasis Transmission

The disease is a zoonotic infection of tropical and subtropical rodents. Humans

are also infected when they are bitten by infected sandflies. Rodents serve as the
reservoir hosts of L.tropica and human infections occurs when they come in close
contact with animals. Although, sandflies may also transmit L.tropica directly from
human to human.

Clinical features

Within weeks to months after the bite of the sandfly, lesions appear on the
extremities or face. Lesions first appear as pruritic papules and followed by regional
lymphadenopathy. In a few months the papules ulcerate producing painless craters
with erythematous edges, sharp walls and granwlating base. Satellite lesions may
form around the edge of the primary sore. Multiple primary lesions are seen in some
patients. Spontaneous healing occurs in 3 to 12 months; make a flat, depigmented

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scar. For patients with acquired immunodeficiency syndrome (AIDS), multiple,

disseminated non healing lesions may be seen.

Diagnosis and Treatment

For diagnosis, materials are collected by biopsy, curettage or aspiration and it is

smeared, sectioned, stained and examined microscopically to Leishman – Donovan
bodies. Tissue biopsy material can also be cultured in liquid media. Leishmania also be
detected in tissue by the polymerase chair reaction (PCR) and these test allow direct,
rapid and specific diagnosis of all leishmanial infections.

To treat patients with more consequential lesions, pentavalent antimonial agents

and liposomal amphotericin B can be used. Moreover, ketoconazole and itraconazole
can be used alone or in combination with the previously mentioned agents for
cutaneous leishmaniasis. Preventive measures include the control of the sandfly
vector by use of insect repellants.

Life Cycle of Ascaris lumbricoides (5 Mark)

Charactertics

A.lumbricoides is the largest and most common of the intestinal helminthes. It

has short life span about 6 to 18 months and measure 15 to 40 cm in length. It is firm,
creamy cuticle and pointed ends. The male is slightly smaller than the female and
possesses a curved tail with copulatory spicules. The female worm passes measure 33
by 55mm; and has a rough mamillated, albuminous coat over their chitinous shells.
They are highly resistant to environmental conditions and may remain viable for 6
years in mild climates.

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Life cycle

The adult ascarids live in the small intestine. The eggs are deposited into the
intestinal lumen and passed in the feces. The eggs are embryonated in soil for a
minimum of 3 weeks and later become infections. The host ingests eggs and after
hatching, the larvae penetrate the intestinal mucosa and invade the portal venules
they move to the liver where they squeeze through that organ capillaries and exit in
the hepatic vein. They are carried to the right side of the heart and pump out to the
lung. In the course of this migration, the larvae increase in size. By the time they reach
the pulmonary capillaries but difficult to pass through to the left side of heart. They
rupture into the alveolar spaces and are loughed up and swallowed. Finally, they
access to the upper intestine, where they complete their naturation and mate.

Occurrence and Transmission

A lumbricoides causes a scanriasis. Its occuruence and transmission are similar to

that of Trichuris Ascariasis is a disease of warm climates and poor sanitation. The
parasite may acquire through ingestion of egg-contaminated food by the host; in dry,
windy climates, eggs may become airborne and be inhaled and swallowed by the
host. In tropical areas, the entire population may be involved.

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Pathogenesis

Ascariasis induces a protective immune response in the host. The severe

pulmonary damage induced by the migration of larvae through the lung; this damage
is due to a part of immediate hypersensitivity reaction to larval antigen.

Clinical features

Clinical features may result from either the migration of the larvae through the
lung or the presence of the adult worms in the intestinal lumen. Due to
hypersensitivity, there are severe symptoms such as fever, lough, wheezing and
shortness of breath can occur. Death may occur as the result of respiratory failure. If
the worm load is small, infections with adult worms may be asymptomatic. Clinical
attention is needed when the parasite is vomited up or passed in the stool.

Diagnosis

Stool examination, reveals charactertic and helpful in the identification. The

pulmonary phase of ascariasis is diagnosed by the finding of larvae and eosinophils in
the sputum.

Treatment and prevention

Albendazole, mebendazole and pyrantel pamoate are highly effective in treating

ascariasis. Preventive measure requires adequate sanitation facilities.

Taenia Saginata (Beef Tapeworm) (5 Mark)

Habitat

T.Saginata inhabits the human jejunum, where it may live for up to 25 years and
grow to maximum length of 10 mm.

Structure and life cycle

T.Saginata’s scolex (1mm) lacks hooklets but possesses the four sucking discs. The
creamy white strobila consists of 1000 to 2000 individual proglottids. The terminal

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segments are longer (20mm), wide (5mm) and contain a large uterus with 15 to 20
lateral branches. When fully gravid, string of 6 to 9 terminal proglottids, each
containing approximately 100,000 eggs, break free pass through the anal canal or
passed with stool when proglottids reaches soil, they disintegrate and releases eggs.
These eggs are 30 to 40 mm in diameter, spherical and possess a thick shell. In
appropriate environments, the hexacanth embryo may survive for months. When
these eggs are released, penetrates the intestinal wall, and embryo is transformed
into a white, ovoid cysticercus. Humans are infected when they ingest improperly
cooked meat containing these larval forms.

Beef Tapeworm Disease

Transmission

Beef Tapeworm diseases caused by Taenia saginata. T.Saginata is highly prevent

in less sanitation areas and under cooked beef is eaten.

Clinical manifestation

Most infected patients are asymptomatic. Some patients found with epigastric
discomfort, nausea, irritability, diarrhea and weight 10ss. In few occasions, the
proglottids may obstruct the appendix, biliary duct or pancreatic dust.

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Diagnosis

The diagnosis is done by identifying eggs or proglottids in the stool. Eggs may also
be distributed on the perianal area which recovered by adhesive cellophane tape
techniques. With this technique, 85 to 95% infections are detected.

Treatment and prevention

Praziquantel and niclo-samide are the agents are used for treatment. Both agents
highly effective in single-dose oral preparations. These drugs act directly on the
worm. Control measures include proper sanitary disposal of human feces; meat
inspection is needed because cysticerci are visible and thorough cooking – Internal
temper – atures of 56oC or more for 5 minutes or longer to destroy the cysticerci.
Salting or freezing for week at -15oC or less is also effective.

Taenia Solium (pork tapeworm)

Habitat

Like beef tapeworm. T.Solium inhabits the human jejunum where it may survive
for decades.

Structure and life cycle

T.Solium possesses a nostellum armed with row of hooklets. The strobile is

smaller than the T.Sagniata. It contains gravid segments measure 6 by 12 mm and the
uterus has only eight to twelve lateral branches. The eggs appear morphologically
identical to those of T.Saginata. T.Saginata infects only swine. Both pigs and humans
become intermediate host. When they ingest food contaminated with viable eggs.
Autoinfection is possible when the transport of the eggs from the perianal area to the
mouth by contaminated fingers.

When egg reaches stomach of intermediate host where they hatch and release
the hexacanth embryo. The embryo penetrates the intestinal wall and may be carried
by the lymphohamatogenous system to any of the tissues of the body. In tissues, it
develops into a 1 cm white, opalescent cysticercus within 3 to 4 months. The

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cysticercus may remain viable for up to 5 years and infecting humans when they
ingest under looked flesh. The scolex attaches to the mucosa and development into a
new adult worm.

Pork tapeworm Disease

Occurrence

This infection is widely distributed throughout the world, mainly in south and
Southeast Asia, Africa, Latin America and Eastern Europe.

Clinical manifestations

The signs and symptoms of infection with the adult worm are similar to those of
saginata. Clinical features differ because humans are serving as intermediate hosts.
Cysticerci develop in the subcutaneous tissues, muscles, hearts, lungs, liver, brain and
eye. When there is small number of cysticerci, it remain viable, tissue reaction is
moderate and the patients are asymptomatic the death of the larva stimulates a
marked inflammatory reaction, fever, muscle pains and eosinophilia.

The central nervous system found with lesions resulting cysticercosis. During the
acute invasive stage, patients experience Lever, headache and eosinophilia. Invasion
of CNS results Meningoencephalitic syndrome with CSF eosinophilic pleocytosis may
be present. Cysts can be found in the cerebrum, ventricles, subarachnoid spaces,
spinal cord and eye. Cerebral cysts are small, measuring 2cm or less in diameter.
These infectins can changes, intellectual impairments and seizures. Subarachnoid
lesions and cysticerci located within the fourth ventricle may obstruct the flow of CSF,
producing increased intracranial pressure with headache, vomiting, visual
disturbances or psychiatric abnormalities. Spinal lesions produce cord compression or
meningeal inflammation. Eye lesions give pain and visual disturbances.

Diagnosis

Adult worm is identified by procedunes used for T.Saginata cysticercosis is

suspected when an individual presents with neurdogic manifestations or
subcutaneous nodules. Roentgenograms of the soft tissues reveal dead, calcified

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cysticerci. Viable lesions may be detected by computed tomography (CT) or magnetic

nesonance imaging (MRI). For subarachnoid lesions, the diagnosis sample of a
subcutaneous nodule or specified antibodies in the circulating blood serum and CSF is
examined detect specific anticystivercal antibodies using enzyme immune assay and
western blot method.

Treatment and prevention

Like T.Saginata, same treatment followed patients with parenchymal lesions

usually respond to prolonged treatment with praziquantel or albendazole.
Corticosteroid administration helps minimize the inflammatory response to dying
cysticerci. Surgery and corticosteroids may help to ameliorate symptoms.

POLIO VIRUSES (5 Mark)

Enteroviruses are major subgroup of picornaviruses. Enteroviruses include the

polioviruses, coxsackieviruses, and echoviruses. Polioviruses have three serotypes,
namely type 1, 2 and 3.

Epidemiology

The virus is spread from man to man by fecal – oral route. The virus is also spread
by droplets.

Pathogenesis

After infection, the virus multiplies in the regional lumph nodes lead to a
viracmia, thus virus become disseminated throughout the body reaching spinal cord
and brain. Motor newrons are particularly Vulnerable to infection and neuronal
destruction occurs.

Manifestation

Most infections are either subclinical or mild. Three types of disease can be seen.
Abortive potomyelitis is a nonspecific illness of 2 to 3 day duration. Aseptic meningitis
(nonparalytic poliomyelitis) is characterized by signs of meningeal irritation (stiff neck,

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pain, and shiffness in the back) in addition to the signs of abortive poliomyelitis;
recovery is rapid and complete within a few days. Paralytic poliomyelitis is the major
infection and is often preceded by a period of minor illness; there is asymmetric
flaccid paralysis, with no significant sensory loss. All four limbs may be completely
paralyzed or the brainstem may be attacked with paralysis of the cranial nerves and
muscle of respiration. The maximum extent of involvement is evident within the a few
days of first paralysis. Later, it found as temporarily damaged neurons. Regain their
function, recovery begins and may continue for as long as 6 months; paralysis
persisting after this time is permanent.

Diagnosis

Virus can be isolated from feces and from throat swabs and cultivated on monkey
kidney, human amnion cell cultures. The identification of serotype is carried out by
neutralization tests.

Prevention

Inactivated polio vaccine (Salk) and live attenuated oral polio vaccine (sabin) are
available, for the prevention. Inactivated polio vaccine (IPV) contains formalin
inactivated strains of the three serotype of virus. The vaccine is given by deep
subcustaneous or intramuscular injection. Three doses of IPV is given (two doses 6-8
weeks apart and the third 8-12 months later). IPV produces long – lasting immunity
for all poliovirus types.

Oral polio vaccine (OPV) is composed of live, attenuated viruses grown in cultures
of monkey kidney cells and human diploid cells. The vaccine is given orally and
produces antibodies to all serotypes; these antibodies persist foro several years.
Booster doses are recommended to maintain adequate antibody levels. Continous
immunization program are needed to prevent the spread of this disease.

HEPATITIS VIRUSES (10 Mark)

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Hepatitis caused by viruses, bacteria and protozoa as well as drugs and toxins
(e.g.isoniazid, carbon tetrachloride and ethanol). Eventhough the causative agent is
different, clinical symptoms and course of acute virus hepatitis are similar. Hepatitis is
caused by five different viruses.

Hepatitis A

Hepatitis A Disease

Hepatitis is the causative agent. This virus is spread by the fecal – oral route and
major outbreaks results, when Hepatitis A associated with contaminated food or
water. The illness is usually subclinical. When symptomatic, there is fever and
jaundice fatal disease may occur, chronic heptatitis a narely occur.

Hepatitis A structure

Hepatitis A virus is an unenveloped, single – stranded RNA virus with cubic

symmetry andadimeter of 27 mm. The virus resists inactivation and is stable at – 20oC
with low pH. This virus comes under separate genus of Picornaviruses as a
hepatovirus. There is only one serotype of hepatitis A virus and these viruses can be
cultivated in primary liver cell cultures and in fetal rhesus monkey kidney cell cultures.

Hepatitis Virus

Features A B D C E

Type Single – Double Single – RNA RNA

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Strand RNA Strand DNA Strand RNA

Incubation
15 – 45 7 – 160 28 – 45 15 – 160 ?
Period

Onset of Usually
Slow Variable Insidious ?
infection Sudden

Infecting Children,
All ages All ages All ages Young adult
individual young adults

Transmission

Fecal – Oral +++   - +++

Sexual + ++ ++ + +?

Transfusion - ++ +++ +++ -

Severity Mild Moderate Often Severe Mild Variable

Chronic
Condition None 10 50 – 70 750% None
(%)

Carrier State None Yes Yes Yes ?

+, - indicate relative frequencies; Hepatitis C virus similar to Hepatitis G virus.

Epidemiology

Humans are major natural hosts of hepatitis A.Virus. Mode of transmission of

hepatitis A is fecal – oral route; Inoculation of infectious material intramuscularly and
transmission through blood transfusion. The disease is common in croded conditions
such as metanl hospitals, schools for the netarted and day – care centers have high
frequency. Frequent presence of virus in nature depends on sporadic subclinical
infections and person – to – person transmission. Outbreak of hepatitis A is possible
when people ingest under cooked seafood, usually shellfish from water contaminated
with human feces. Common – source of outbreaks are Vegetables as well as

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contaminating drinking water. The risk of clinically evident disease is much higher in
infected adults than in children.

Pathogenesis

After entry, the virus multiplies initially in the enteric mucosa. Virus is found in
the feces before onset on disease. In most patients with symptoms of the disease,
virus is not found in fecal specimens. After multiplication in the intestine, the virus
enters into the blood stream and viremia results, finally spread to the liver.
Replication in the liver induces host response such as lymphoid cell infilteration,
necrosis of liver parenchymal cells and proliferation of Kupffer cells. The extent of
necrosis depends upon severity of the disease. A variable degree of biliary statsis may
be present. IgE antibody persists in the serum in detectable levels and patients with
antihepatits A virus antibodies are immune to reinfection.

Manifestations

In hepatitis A virus infection, an incubation period of 10 to 50 days is usually

followed by the onset of fever; anorexia (poor apepitate); pausea; pain in the right
abdominal quadrant; and finally jaundice within several days. Dark Urine and Clay –
colored stools may be noticed by the patient 1 to 5 days before the onset of clinical
jaundice. The liver is enlarged and bilirubin levels are elevated as a resul of hepatic
inflammation and damage. Recovery occurs in days to weeks.

Many people with serologic evidence of acute hepatitis A are asymptomatic or

mildly ill, without jaundice. The infection – to – disease depends on age; it may be
high in children and older adults. Chronic hepatitis is none. In rare cases, fulminant
fatal hepatitis associated with extensive liver recrosis may occur.

Diagnosis

During early illness, Antibody to hepatitis A virus can be detected. Most patients
with symptoms or signs of acute heptatis A already have detectable antibody in
serum. Early antibody responses are predominantly IgM, which can be detected for
several weeks or months. During acute infection, high titers of virus – specific IgM

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antibody in serum is found which denotes the acute phase of illness. During
convalescence, IgG antibody predominates.

Treatment and prevention

There is no specific treatment for patients with acute hepatitis. Disease

supportive measures include adequate nutrition and rest. Control measures include
avoidance of exposure to contaminated food or water to reduce the risk of hepatitis A
infection.

Passive immunization – Immune serum globulin (ISG) manufactured from pools

of plasma from general population. ISG provides temporary protection it given before
or during the incubation period of the disease. ISG should be administered to
household contacts of hepatitis A patients and those known to have eaten uncooked
foods prepared or handled by infected individuals.

Active Immunization

Live attenuated vaccines have poor immunogenicity and have not been effective
when given orally. Formalin – killed vaccines induce antibody theres and are almost
100% protective. This vaccine is preferred for those with prolonged or repeated
exposure to hepatitis.

Hepatitis - B

Structure

Hepatitis B Virus is an enveloped DNA virus belonging to the family

hepadnaviridae. The complete virion is a spherical particule (42nm that consists of an
envelope around core (27nm). The core comprises a nucleocapsid that contains the
DNA genome.

Hepatitis B Disease

Hepatitis B Virus was formerly known as serum hepatitis. This virus causes
hepatitis B disease. This form of hepatitis is transmitted by needle use or blood
transfusion. Hepatitis B is usually an asymptomatic or mild illness with fever and

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jaundice for days to weeks. It becomes chronic in 10% of patients and may lead to
cirrhosis or hepatocellular carcinoma.

Epidemiology

Hepatitis B infection is found worldwide but prevalence rates vary between

countries. Chronic carriers constitute the main reservoir of infection and about 10%
of patients with HIV infection are chronic carriers of hepatitis B. In some Far East
Countries, 5-15% of all persons has the virus and most are asymptomatic. Virus
transmitted sexually and it is prevalent in male homosexuals, patient undergoes
hemodialysis or immunosuppressive drugs, patients with Down’s syndrome and
injection drug users. Needle stick injuries more maximum exposure to hepatitis
viruses as there is chance for direct contact with blood or other body fluids.

Injants acquired infection during the birth process by swallowing of infected

blood or fluids or through abrasions. Hepatocellular carcinoma has strong association
with persistent hepatitis B virus. In many parts of Africa and Asia, primary liver cancer
accounts for 20% to 30% of all types of malignancies. The persons with chronic
hepatitis B have increased risk of developing malignancy.

Pathogenesis

The major mode of acquisition of Virus is through close personal contact with
body fluids of infected individuals, HBsAg has been found in most body fluids
including saliva, semen, blood and cervical Secretions. Most transmission of infections
is possible through inadequately sterilized hypodermic needles or instruments used
in tattooing and ear piercing.

Symptoms like Rash or arthritis are found and jaundice appears to related to
circulating immune complexes that activate the complement system. Antibodies are
produced against HBsAg, and are protective to recover from disease patients with
depressed T-lymphocyte function have a high frequency of chronic infection with the

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hepatitis virus. In chronic carriers, there is persistant hepatitis B virion production and
antibody to HBcAg does not appear to the protective.

In chronics active hepatitis B, the continued presence of inflammatory to is of

infection results in necrosis of hepatocytes, collapse of the reticular framework of the
liver and progressive necrosis. The increasing fibrosis can result in the syndrome of
post necrotic hepatic necrosis.

Hepatitis B viral DNA get integrated into host cell DNA, may results in
hepatocellular carcinomas. This virus may well activate a cellular oncogene. It is also
possible that the virus does not play such molecular role in oncogenicity because the
natural history of chronic hepatitis B infection involves cycles of damage or death of
liver cells interspersed with periods of intense regenerative hyperplasia. This
significantly increases the opportunity for spontaneous mutational changes that may
activate cellular oncogenes.

Manifestations

For hepatitis B, the incubation period may be as brief as 7 days or as long as 160
days. Acute hepatitis B is usually manifested by the gradual onset of fatigue, loss of
appetite, nausea and pain. Early in the course of disease, pain and Swelling of the
joints and occasionally arthritis may occur with increasing Involvement of liver there is
increasing cholestasis and hence, clay- colored stools, darkening of urine and
jaundice. Symptoms may persist for months.

In general, the symptoms associated with acute hepatitis B are more severe and
more prolonged than those of hepatitis A. In fulminant hepatitis, it leads to extensive
liver necrosis and death. Chronic hepatitis occur in approximately 10% of all patient
with hepatitis B infection, but the risk is much higher for newborn, children and the
immunocompromised Chronic hepatitis may lead to cirrtosis liver failure or
hepatocellular carcinoma in 25% patients.

Diagnosis

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During the acute phases of disease , when there is active viral replication,
large amounts of HBsAg and hepatitis B virus can be detected in the serum , when
fully developed virions, there is high levels of DNA polymerase and HBcAg. Although
HBcAg is also present, antibody against it invariable occurs and prevents its detection.
With resolution of acute hepatotos B, HBsAg and HBcAg disappear from serum with
the development of antibodies (anti-HBs and ant-HBe) against them. The
development of anti-HBs indicates elimination of infection and protection against
re-infection. Anti-HBc is detected early in the course of disease and persisits in serum
for year. Anti-HBc is not protective but epidemiologic marker of infection. The
laboratory diagnosis of acute hepatitis B is made by detecting Igm antibody in serum.
HBsAg may also be detected in serum, past infection with hepatitis B is determined by
detecting IgG anti-HBc, anti-HBs or both.

In patients with chronic hepatitis B, evidence of viral persistence can be found in

serum. HBsAg can be detected throughout the active disease process and anti-HBs
does not develop, which accounts for the chronicity of the disease. However, HBsAg is
detected. Two types of chronic hepatitis can be distinguished. In this HBsAg is
detected but not HBeAg: these patients usully show minimal evidence of liver dys
function. In the other, both antigens are found: the process is more active with
continued hepatic damage that may result in cirrhosis.

Treatment

There is no specific treatment for acute hepatitis B. A high calorie diet is

desirable. For chronic hepatitis B diseases, interferon alpha provides long-term
benefit in minority of patients. Lamivudine (3TC), a potent inhibitor of HIV, may use
against hepatitis B virus, but resistance to this agent developed in about 25% of
patients after 12 months of therapy. Adefovir, a nucleotide analog of adenosine
monophosptate is used for treatment. This treatment is considered for patients
exhibiting chronic hepatitis B for more than 6 months with detectable serum levels of
HBsAg, HBcAg and hepatitis B- DNA.

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Prevention

Safe sex practices and avoidance of needle is needed. Stick injuries or injection
drug use are approaches needed to reduce the risk of hepatitis B infection. Both
active prophylasis and passive prophylaxis can be done. Post exposure prophylaxis
with (HBIG) Hepatitis B immunoglobulin) should be followed by active immunization
with vaccine. Recombinant vaccine can be recommended for medical personnel such
as laboratory workers and injection drug users who come into contact with blood or
other potentially infected materials, pre-exposure prophylaxis with Recombinant
Vaccine can recommended for all children.

A combination of active and passive immunization is the most effective approach

to prevent neonatal acquisition and the development of carriage in the neonate.
Infants born to those who are positive should receive HBIG in the delivery room
followed by three does of hepatitis B vaccine beginning 24 hours after birth. A similar
combination of passive and active immunization is used for unimmunized persons
who have been exposed by needle stick or similar injuries.

Hepatitis D (Delta hepatitis)

Delta – hepatitis disease

Delta hepatitis is most prevalent in groups at high risk of hepatitis B. Injection

drug users are those at greatest risk and other risk include dialysis, nonparenteral and
vertical transmission.

Structure and Transmission

Delta hepatitis is caused by the hepatitis D virus. It has small single-stranded RNA
virus. This virus requires the presence of hepatitis B surface antigens for its
transmission and hepatitis D is found only in persons with acute or chronic hepatitis B
infection. Along with RNA , proteins are found that constituted the delta antigen. This
protein RNA complex is surrounded by HBsAg.

Manifestations

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Two major types of delta infection have been noted : 1. Simultaneous delta and
hepatitis B infection , 2. Delta superinfection with chronic hepatitis B Simultaneous
infection with both delta and hepatitis B results in clinical hepatitis that is mostly a
similar to acute hepatitis A or B: persons with chronic hepatitis B who acquires
infection with hepatitis D suffer relapses of jaundice and have chances to develops
chronic cirrhosis . Delta infection is occurs in populations with a high incidence of
chronic hepatitis B and have resulted in rapidly progressive liver disease causing death
in 20% infected persons.

Hepatitis-E

Hepatitis E is the cause of hepatitis. It is spread by the fecal-oral route. Hepatitis E

virus is an RNA virus, Spherical, 27 to 34 nm in size and unenveloped and found spikes
on their surface. This virus is frequently subclinical when symptomatic; it causes only
acute disease that may fulminate, especially in pregnant women. In endemic are as, it
has the high rate in young adults and infections is usually associated with
contaminated drinking water. The incutation period is approximately 40 days. The
diagnosis is done by detecting the presence of specific IgM antibody. No treatment is
available. Liver transplant may be only recourse in seriously ill patients.

Hepatitis-G

Hapatitis G is a RNA virus similar to hepatitis C. It comes under the flavivirus

family. An antibody assay can detect past infection but not present infection.
Detection of acute infection requires a PCR assay method for viral RNA in Serum. It is
closely associated with hepatitis C, the majority of patients infected by hepatitis c are
also infected by hepatitis G. Hepatitis G is a blood born virus. Patients infected with
both viruses do not appear to have worse disease than those do not appear to have
worse disease than those infected by hepatitis C alone. Currently, there is no usefully
serologic test and no therapy is establisted.

HIV-HUMAN IMMUNODEFICIENCY VIRUS (AIDS) (10 Mark)

Structure

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The HIV Virion is about 100nm in diameter it contains two copies of

single-stranded RNA.The RNA are coated with the nucleocapsid protein (NC) and the
RNA-protein complexes are enclosed in a capsid. Around the capsid, envelope is
surrounded, which contain two viral glycoprotein. On the surface of envelope, gp120
(surface glycoprotein) is present. Transmembrane or gp41 is found in the envelope.
Between the capsid and the envelope is the Matrix protein is present. The virion core
contains three-specific proteins that are essential for viral replication:reverse
transcriptase (RT), protease (PR) and, integrase (IN).

Life cycle

Viral Entry

The virions attach to cellular membrane receptors and enter the cell by
direct fusion with the plasma membrane. For HIV-1, the virion attatchment protein is
the surface glycoprotein (gp120) which attach to cellular receptor, CD4 moecule,
CXC R4 or CCR5 acting as coreceptors. These receptors occur primarily in the plasma
membrane of CD4+ T lymphocytes, cells of the monocyte-Macrophyage Series and
some other target cells. Early in the infection, virus infect macrophage by using the
CCR5 receptor Later, Virus become Syncytia-forming variants, they infect T-cell by
using CXCR4 corecoptor, thus virus has rapid advancement to AIDS. The HIV-I
transmembrane proteing gp41 is responsible of jusion of the viral and cell membrane,
leading to entry of the virion core complex into the cytoplasm of the cell.

HIV-I can also infect cells such as fibroblasts and certain brain cells that lack the
CD4 surface molecule. With the help of transmembrane protein gp41, HIV sufficient
to promote entry into the cells. Fusion activity may also play an important role in
amplification of the effects of the virus infection mainly during the later stages of
infection because infected cells expressing viral glycoproteins in their membranes
readily fuse with uninfected CD4+ T-Lymphocytes to form large syncytia. This fusion
provides direct cell-to-cell transmission of the virus that bypasses the usual
extracellular phase and may contribute to the overall depletion of CD4+ lymphocytes
in an infected individual.

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Viral Replication

After entry of the viral core into the cytoplasm of the infected cell, the RNA is
copied into double-stranded DNA by viral reverse transcriptase. This process is known
as reverse transcription. Linear DNA molecules are enter to the nucleus and
integrates into a host cell chromosome. The viral genes called the provirus are
replicated and inherited as long as the infected cell continues to divide. HIV-1
produce envelope proteins, and series of viral regulatory and accessory proteins. In
HIV-1 each time the viral RNA is reverse transcribed , one to two new mutations are
introduced into the resulting DNA. Thus, it produce new viral genome (mutant), these
genome accumulate rapidly over the course of the infection. There is a variability in
the surface envelope protein gp120. This is one of the reasons for the failure of the
immune system to control the infection and also the increases in viral virulence that
appear to occur during the course of the infection.

(AIDS) Acquired Immunodeficiency Syndrome

The primary infection in AIDS ranges from asymptomatic to an infectious illness

with up to a few weeks of fever, malaise, arthralgias and rash. After long
asymptomatic period, AIDS disease emerges. The effect o the virus on the immure
system causes an viral, bacterial, fungal and parasitic oppuntunistic infections.

Epidemiology

The AIDS Syndrome was first recognized in the United States in 1981, when they
found unusual member of rare skin cancers (kapost’s sarcoma) and opportunistic
infections were occurring among male homosexuals. In 1985, HIV-2 was found to be
endemic in parts of West Africa and cause AIDS.

Transmission

The HIV virus is transmitted between humans in three ways: Sexually perinatally
and by exposure to contaminated blood or body fluids. The majority of HIV infection
result form sexual contact. The virus has been found high titers in semen and cervical
secretions. Infection is facilitated by breaks epithelial surfaces which provide direct

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access to the tissues or bloodstream. The fragility of the rectal mucosa, together with
large number of sexual contacts is probable contributing factors to the predominance
of the disease among male homosexuals. The risk of perinatal transmission form an
infected mother to her child has been estimated to range from 15 to 40%.

Transmission of infection by blood is largely associated with staring of needles

and Syringes by injecting drug users are at extremely high risk. Hetrosexual
transmission could occur and that the injection could be transmitted from mother to
infant either by intravterine spread or during the birth process. It was also found that
the disease had its greatest prevalence in parts of Africa, where the spread was
predominantly heterosexual.

Transmission of infection to health care workers after accidental sticks with

potentially contaminated needles is very rare (less than 1%), because the amount of
injections virus in the blood infected cases is small and larger volumes or repeated
exposures are needed for a sigrificant chance of infection. Transmission does not
occur through day, to-day nonsexual contact with infected individuals or through
insect vectors because the virus need for direct mucosal or blood contact. Bread milk
contains virus infect breastfeeding infants.

Pathogenesis
the pathogenesis of HIV infection is very complex.

Infection

The intial target of HIV is CD4 molecules, particularly on the surface of

CD4+helper T lymphocytes, monocytes and macrophages. The virus can also infect
other human cells with CD4 and a wide range of CD 4 negative cells including renal
and gastrointestinal epithelium and brain astrocytes. The virus replicates in
macrophages and these cells serve as areservoir for continued expansion of the
infections to other cell types by cell-to-cell fusion, which allows the virus to spread
without being exposed to neutralizing antibody. Infected macrophages may
participate in breakdown of the blood-brain barrier, allowing exposure of the central
nervous system.

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Kinetic Studies shows that more that 50% of the viral load measured on any given
day has been produced in the past 24 hours. Because 99% of the viral load is
produced by cells that were infected with in the past 48 to 72 hours. When kinetic
studies are performed on changes in CD4 cell counts, it is estimated that up to 1
billion CD 4 cells are produced per day in response to the injection and that the
half-life of these cells is only 1.6 days.

Latent State

The long asymptomatic period following HIV infection occurs despite active virus
replication in the host. Several factors can terminate the long latent periods of HIV.
Mutations occur during viral replication that appears to enhance induction of virulent
forms of the virus, infect different cell types. Thus mutated forms of HIV isolated from
the later stages of disease infect broad range of cell types and grow rapidly than those
isolated in asymptomatic period. In latent period, little or no replication because of
intense immunological reactions found within the lymphoid tissue. This implies that
the immune system is capable of controlling the virus in the early course f disease but
it lost as the disease progresses over time.

Studies shows that individuals with early-state disease have less than 10
infectious virions/ML of plasma, whereas those in late-stage disease have about
between 100 and 1000 infectious virions/ML of plasma. These studies implies that
either viral replication was increasing during later stages of disease due to more
virulent mutations and/or the immune system had lost its ability to clear free virus as
the disease progresses.

Immune Deficiency

The primary immune defect in AIDS results from the reduction in the numbers
and effectiveness of CD 4+ helper inducer T lymphocytes. This is due to direct killing
of CD4+ T lymphocytes, by the virus. There are also functional defects in CD4+
lymphocytes affecting Lymphokine production and leading to inhibition of some
macrophage function.

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At the end, there is a disturbance of immune balance that can give rise to
malignancies as well as the susceptibility of AIDS patients to a range of opportunistic
viral, fungal and bacterial infections.

Manifestations

The intial infection with HIV is usually asymptomatic, although in some cases
illness develops 2 to 4 weeks after infection and lasts about 2 to week. The illness
includes fever, malaise, lymphadenopathy, hepatosplenomegaly, arthralgias and rash.
The infection is lifelong, as the virus persists and integrates into the host cells DNA.

The initial infection is followed by asymptomatic period , in most cases continues

for years before the disease becomes clinically apparent. During this time virus can be
isolated from blood, semen, and the cervix approximately 50% of infected individuals
develop significant disease within 10 years of infection.

As the disease progresses, the number of CD4+T lymphocytes decline. There is

increasing immunodeficiency and opportunistic infections become more frequent,
severe and difficult to treat. As the result of opportunistic infections, patients found
with oral thrush, meningitis, CMV are found.

Common opportunistic infection in AIDS patients.

Protozoan

Toxoplasmosis and cryptosporidiosis.

Fungal

Cryptococcosis, Candidiasis, Histoplasmosis.

Mycobacterial

Disseminated tuberculosis, Mycobacterium avium , intracellulare complex infections.

Viral

Persistent mucocutaneous herpes simplex,

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Cytomegalo virus retinitis, gastrointestinal or disseminated infection,

Varicell-Zoster, persistent or disseminated

Progressive multifocal leuko encephalopathy.

As the duration of survival of AIDS patients become longer, due to therapy with
drugs, an increased number developed neurologic manifestations of the disease and
lymphoid neoplasms , HIV is a virus and can be isolated from cerebrospinal fluid
of 50 to 70% of patients, CNS involvement may be asymptomatic, but many patients
develop a subacute neurologic , illness that produce clinical symptoms varying from
mild cognitive dysfunction to severe dementia. Loss of complex cogritive function is
usually the first sign of illness progression to severe memory loss, depression, seizures
and coma may result.

Diagnosis

The diagnosis of AIDS is most commonly confirmed by detecting antibody to the

virus or its components.

Screening Test

It is performed using whole viral lysates as the target antigens in enzyme

immunoassay (EIA) tests. These tests have a high level of sensitivity, but false positive
results may also occur, so all positive EIA tests must be confirmed.

Confirmative Test

Western blot analysis is used, which detects antibodies to specific viral proteins.
In this procedure, viral proteins are separated by electrophoresis, transferred to
nitrocellulose paper and incubated with patient sera; antibody bound to the
individual proteins is detected by enzyme-labelled anti-human globulin sera. Sera
from infected patients have antibodies that react with the envelope glycoprotein,
core proteins or both.

The combination of EIA and western blot tests gives a high degree of specificity to
test results, but antibody is not detectable by these procedures in the first 2 to 4

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weeks after injection. During this period, the individual can still transmit the injection
to others by sexual contact or blood donation.

More practical approaches include nucleic acid-based assays such as the

polymerase chain reaction (PCR) for plasma HIV RNA or DNA and the branched chain
DNA (6 DNA) assay. These are also useful in assessing the benefits of antiviral therapy,
as well as in determining if infants born to seropositive mothers are infected or simply
demonstrating passively transmitted transplacental antibody.

Treatment

Various combinations of drugs are used in treatment. Few anti HIV drugs listed in
the chapter viruses.

Prevention

Education is the cornerstone of prevention. Educated people about the means of

transmission, Detection and treatment of HIV-injected pregnant woman have also
been shown to be effective in reducing perinatal injection. The avoidance of breast
feeding is by HIV positive mothers to prevent transmission.

STAPHYLOCOCCUS (10 Mark)

Staphylococcus comes under the family Micrococcaceae. They form clusters,

some single cells, pairs, short chains are also seen. They are nonflagellate, nonmotile
and on-spore forming, Staphylococci are facultative anaerobes, and produce catalase
enzyme. There is around 20 species found in man of which s.aureus is coagulase
positive and the rest coagulase-negative. Major Medical important orgarisms are
S.aureus, S.epidermidis and S.Saprophyticus.

Staphylococcus Aureus

Morphology

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They are spherical cocci arranged in clusters. S.aueus is Gram-positive in growing

cultures. In old cultures, in resolving lesions and the presence of antibiotics, they lose
their gram positivity.

Cultural Charactertics

They are aerobes and facultative anerobes, optimum temperature for growth is
370 and optimum PH is 7.5.

Blood Agar

After overnight incubation on blood agar, S.aureus produce white colonies tend
to turn golden color (aureus) with time. Most strains of S.aureus show zone of
B.hemolysis surround the colony.

Mac Conkey agar

S.aureus produces pink colonies due to lactose fermentation.

Virulence factor

Exofoliatin

It causes splitting of the epidermis between the stratum spinosum and stratum
granulosum, probably by disruption of intercellular junction. Two antigenic variants of
exofoliatin are antigenic in humans.

Pyrogenic Toxin Superantigens

The pyrogenic toxin superantigens are a family of secreted proteins able to

stimulated systemic effects due to absorption for the site where they are produced by
staphylococci. This toxins stimulates both T cells and macrophages to release massive
amounts of cytokine particularly tumor necrosis factor-x and interleukin-1 other
activities of these toxins are pynogenicity and enhanced susceptibility to the lethal
effects endotoxin.

Staphylococcal Enterotoxin

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Enterotoxins stimulate gastro intestinal symptoms (primarily vomiting) in humans

and animals. There are several antigenically district proteins in this class: enterotoxin
A, B, C toxins are formed, they are quite stable, retaining activity after exposure to
gastric and jejunal enzymes. In addition to superantigen mediated actions, they
appear to act directly on neural receptors in the upper gastrointestinal tract leading
to stimulation of the vomiting center in the brain.

Toxin Shock Syndrome Toxin (TSST-1)

TSST-1 is the major cause for staphyiococcal toxic syndrome. It can stimulated
the release of cytokines through the superantigen mechanisms but also have direct
toxic effects endothelial cells. Toxic effects on endothelial cells may lead to capillary
leakage, hypotension and shock.

Coagulase

It activates a coagulase- reacting factor (CRF) present in plasma, causing the

plasma to clot by conversion of fibrinogen to fibrin. There are several antigenic types
of coagulase e.g.A, B, C, D, E, F, G and H. Coagulase may also act to coat the bacterial
cells with fibrin, rendering them resistant to opsonization and phagocytosis.

Staphylo kinase (Fibrinolysin)

Many Strains of S.aureus produce straphylokinase. It is antigenically and

enzymatically distinct from streptoRinase produced by streptococcus. Fibrinolysin
may breakdown fibrin clots and allows spread of infection to contifuous tissues.

Hyaluronidase

It hydrolyzes hyaluronic acid present in the intercellullin ground substance of

connective tissue, thus facilitating the spread of the organisms to adjacent areas.

Epidemiology

The basic human habitat of S.aureus is the anterior nares. Some nasal carriers are
individuals with colonization at other sites such as the perineum may disseminate the

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organisms with desquamated epithelial cells, thus make source of infections to

others.

Pathogenesis

Primary Infection

The intial stages of colonization by S.aureus are mediated by a numbr of surface

proteins , each protein bind to host elements in or covering tissues, body fluids or
foreign bodies such catheters.

One beyond the mucosal or skin barrier, any mechanism that protects the
organisms from phagocytosis may allow multiplication to continue and alpha-toxin to
intiate local injury. A Factor protein A interferes with phagocytosis, thus effectively
diminishing opsonization. Production of coagulase can retard migration of phagocytes
to the site of infection and even phagocytosed S.aurenus may resist lysosomal killing.
The acute inflammatory response continues and lesions are localized due to the
fibrotic reaction to the alpha-toxin mediated injury to host cells.

In the skin, spontaneous resolution of the boil is by granulation and fibrosis. In

the lung, kidney, bone and other organs, the process may continue to spread with
statellite foci. The actions of the cytotoxins are highly destructive creating cavities and
massive necrosis. Circulating staphylococci may also shed cell wall peptidoglycan
producing massive complement activation, leucopenia, thrombocytopenia and septic
shock.

Toxins

Enterotoxins - In staphylococcal food poisoning the contaminating bacteria

produce pyrogenic exotoxin in the food that can initiate its enterotoxic action on the
intestine within hours of its digestion. Thus results in vomiting and diarrhea within 1
to 5 hours. Diarrhea is due to the inhibition of water absorption from the lumen of
the intestine and increased transmucosal fluid flux into the lumen.

Exofoliative Toxin

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Exofoliative toxin cause blisterlike separation of the epidermis by their action on

intercellular juctions, which is localized to the site of skin injection. In Staphylococcal
scalded skin syndrome , absorbed toxin causes extensive epithelial desquamation at
site remote from the primary injection.

Tsst-1

In staphylococcal TSS, the pyrogenic exotoxin TSSt-1 is produced during the

staptylococcal infection with systemic disease as a result of absorption of toxin from
the local site.

In some cases, women carry S.aureus in Vaginal flora have the potential to
produce TSST-1. In presence of strain, the combination of menstruation and
high-absorbency tampon usage appear to provide growth conditions that enhance
the production of TSST-1, Toxin absorbed from the Vagina can circulate to produce
superantigen mediated cytokine release and direct effect on the vasculture.

Manifestations

Primary infection.

Furuncle and Carbuncle

The furuncle or boil is a superficial skin infection that develops in a hair follicle ,
sebaceous glands or sweat gland. Furunculosis is often complication of acne vugaris.
Infection at the base of the eyelash gives rise to the common stye. This infection is
usually benign and the infection resolves upon spontaneous drainage of pus. Infection
can spread from a furuncle with the development of one or more abscesses in
adjacent cutaneous tissues. This lesions known as a carbuncle occurs most often on
the back of the neck. Carbuncles are serious lesions that may result in blood stream
invasion.

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Chronic Furnculosis

Some individuals have repeated attacks of boils caused by the same strain of
S.aureus causes chronic furunculosis. Delayed type hypersensitivity to staphylococcal
products appears responsible for inflammation and necrosis.

Impetigo

Strains of S.aureus that produce exfoliatin cause bullous impetigo characterized

by large blisters containing many staphylococcus in the superficial layers of the skin.

Deep Lesions

The infection of deep tissues is by bacteremic spread from a skin lesion. These
include infection of bones, joints deep organs and soft tissues,. In children acute
osteomyelitis are caused by S.aureus. Lesions are produce destruction.

Scalded Skin Syndrome

It results from the production of exfoliatin in a staphylococcal lesion, e.g.

conjuctiveitis Erythema and intraepidermal desquamation takes place at remote sites.
The face, axilla and groin tend to be affected first but the Erythema, bullous formation
and desquamation of epithelial sheets can spread to desquamation of epithelial
sheets can spread to all parts of the body.

Toxic Shock Syndrome

The disease is characterized by high fever, vomiting, diarrhea, sore throat and
muscle pain. With in 48 hours, it may progress to severe shock with evidence of renal
and hepatic damage. A skin rash may develop followed by desquamation at a deeper
level.

Diagnosis

Most acute, untreated lesions contain large number of Gram-positive

staphylococci grow overnight on blood agar incubated aerobically. Catalase and
coagulase tests are used for identification of colonies.

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For deep staphylococcal infections, lesions, aspiration is need for diagnosis blood
cultures are necessary for deep infections.

Treatment

Penicillin and Cephalosporins are used against S.aureus. Methcillin, nafcillin and
oxacillin is used against resistant strains. Vancomycin, clindamycin or erythromycin is
also against resistant strains.

Prevention

Clothes and bedding should be washed at a sufficiently high temperature (70o C

or higher ) to destroy staphylococci or dry- cleaned. In adults, the use of chlorhexidine
or hexacholorphene soaps increases the bactericidal activity of the skin. Methicillin or
vancomycin given during and shortly after surgery may reduce the chance for intra
operative infection.

STREPTOCOCCI (10 Mark)

The genus streptococci are Gram-positive cocci arranged in chains that form large
portion of the indigenous microflora of the oropharynx. Streptococci include harmless
species and also contain three important pathogens of humans. One is S.pyogenes,
causes strep throat, which can lead to rheumatic fever and heart disease, some
strains ability to cause deep tissue infections, usually called flesh-eating bacteria.
Second is S.agalactiae the most frequent cause of sepsis in newborns. Last is
S.pneumonia causes pneumonia and meningitis.

Morphology

Streptococci are coccal cells that are generally smaller and ovoid in shape. They
are usually arranged in chains. Length may differ from a single pair to continuous
chains, depending on the species and growth conditions. Mostly streptococci are not

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acid fast, do not form spores and non motile. Few species form capsules composed of
polysaccharide or hyaluronic acid Majority of streptococci are aerobes and facultative
anaerobes but few are obligate anaerobes.

Culture and Biochemical Characteristics

Streptococci grow in enriched media under aerobic and anaerobic conditions.

Growth is enhanced by the presence of carbon dioxide. Usually, blood agar is used
because it contains most growth requirement and also serves as an indicator for
patterns of hemolysis. The colonies are small, 2mm in diameter and they may be
surrounded by a zone where the erythrocytes blood suspended in the agar have been
hemolyzed. When the zone around the colonies is clear, this is called B-hemolysis.
When incomplete hemolysis found with a green discoloration of the agar, is called
alpha-hemolysis. Last, is no hemolysis is found, and then these organisms are termed
as gamma hemolytic streptococci. Streptococci are catalase negative and on glucose
formentation, it yields lactic acid.

Group A Streptococci (Streptococcus Pyogenes)

Morphology

It appear in purulent lesions or broth cultures as spherical or ovoid cells in chains

of short to medium length. On blood agar plates, colories are compact, small and
surrounded by a 2 to 3mm zone of B hemoloysis. B hemolysis is caused by either of
two hemolysins, streptolysin S are B hemolytic only under anaerobic conditions,
because the remaining streptolysin O is not active in the presence of oxygen.

Pathogenesis

Acute infections

Group A streptococcus which adhere to epithelial cells of the nasopharynx and

skin. M protein, LTA and protein F are helpful adherence. In the naspharynx, these
three involved in attachment to glycoprotein fibronectin lowering the epithelial cell
surface. The group A Streptococci has the capacity to be highly invasive. The events
are following attachment that trigger invasion. M protein, protein F and other

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fibronectin-binding proteins are required for invasion of phagocytes. After the intial
events f attachment and invasion, activity of the M protein, immunoglobulin binding
proteins and the C5a peptidase play the key role in allowing the streptococcal
infection to continue. M protein plays as essential role in group A streptococcal
resistant to phogocytosis.

The combined effect of streptokinase DNAase, and hyaluronidase may prevent

effective localization of the infection, while the streptolysins produce tissue injury and
are toxic to phagocytic cells.

In streptococcal toxic shock syndrome (STSS), there is massive cytokine release

stimulated by the superantigenicity of the SPEs causes shock, renal impairment and
diarrhea.

Acute Rheumatic Fever (ARF)

Acute Rheumatic fever is an autoimmune state induced by streptococcal

infection. Streptococcal pharyngitis patients who develop ARF have higher levels of
antistrepcococcal and autoreactive antibodies and T cells. Some of these react with
both heart tissue and Streptococcal antigens. Antibodies to the group A carbohydrate
may play a role in injury to the valvular endothelium. A cellular reaction pattern
consisting of lymphocytes and macrophages aggregated around fibrinoid deposits is
found in human hearts. This lesion called the Aschoff body is characteristic of
rheumatic carditis.

Acute Glomerulonephritis

The renal injury of acute glomerulonephritis is caused by deposition in the

glomerulus of antigen-antibody complexes with complement activation and
consequent inflammation.

Epidemiology

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Pharyngitis

Pharyngitis usually found in children between 5 to 15 years of age streptococcal

transmission is by person to person from the large droplets produced infected
persons during coughing, sneezing or even conversation. Asymptomatic carrier are
the major source, they colonized in the nose as well as the throat. Group A
streptococci can survive for some time in dried secretions, environmental sources and
fonites.

Impetigo

Its occurs when transient skin colonization with group A streptococci is combined
with minor trauma such as insect bites. The tiny skin pustules are spread locally by
scratching and to others by direct contact or shared fomites such as towels. Impetigo
is most common in summer months when insects are biting and when the hygiene is
low.

Wound and puerperal infections

Transmission from patient to patient is by the hands of physicians or other

medical attendants who fail to follow hand washing procedures. Other ways by
transferring organisms from another patients or come from the health care workers.

Manifestations

Streptococcal pharyngitis

It is most frequent between the ages of 5 and 15 years. The illness includes acute
sore throat, malaise, fever and headace. Infection involves the fonsillar pillars, uvula
and soft palate which become red, swollen, and covered with yellow-white exudates.
Usually fever is gone by third to fifth day and other signs subside within week.
Occasionally the infection may spread locally to produce pertonsillar or
retinopharyngeal abscesses, otitis media, suppurative cervical adenitis and acute
sinusitis. Rarely , extensive spread occurs, producing meningitis, preumonia or
bacteremia with metastatic infection in distant organs.

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Impetigo

The primary lesion of Streptococcal impetigo is a small (1cm) vesicle surrounded

by an area of erythema. The vesicles enlarge over a period of days, become pustular
and break to form a yellow crust. The lesions appear in 20 to 5 year old children on
body surfaces, typically faces and lower extremities. Multiple lesions many coalesce
to form deeper ulcerated areas.

Erysipelas

Erysipelas is a distinct form of streptococcal infection of the skin and

subcutaneous tissues, primarily affecting the dermis. It is characterized by a spreading
area of erythema and edema with pain and systemic manifestations including fever
and lymphadenopathy.

Puerperal Infection

Infection of the endometrium at or near delivery is a life threatening form of

group. A streptococcal infection.

Scarlet Fever

In scarlet fever, the buccal mucosa, temples and cheeks are deep red. Punctate
hemorrhages appear on the hard and soft palates and the tongue becomes covered
with yellow-white exudates through which the red papillae are prominent (strawberry
tongue). A diffuse red rash appears on the second day of illness, spreading from the
upper chest to the trunk and extremities.

Streptococcal Toxic Shock Syndrome (STSS)

STSS may begin at the site of any group A streptococcal infection. The systemic
illness starts with vague myalgia, chills and severe pain at the infected site. In the skin
and soft tissues a it leads to necrosis fascitis and myonecrosis. STSS continues with
nausea, Vomiting and diarrhea followed by hypotension, shock and organ failure.

Acute Rhematic Fever (ARF)

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It is a nonsuppurative inflammatory disease characterized by fever, carditis,

subcutaneous nodules, chorea and migratory polyarthritis. Cardiac enlargement,
Valvular murmurs and effusions are seen and reflect endocardial, myocardial and
epicardial damage, which can lead to heart failure.

Acute Glomerulonephritis

Post streptococcal glomerulonephritis is primarily a disease of childhood that

begins 1 to 4 weeks after streptococcal pharyngitis and 3 to 6 weeks after skin
infection. It is characterized by edema, hypertension, hematuria, proteniuria,
occasionally progressive course of infection leads to renal failure and death.

Diagnosis

For streptococcal pharyngitis, culture of the posterior pharynx and tonsils is

required for diagnosis. Blood agar plates incubated anaerobically give the
identification of B-hemolysis B-hemolytic colonies are identified by Lancefield
grouping using immunofluorescence or agglutination methods. Serologic methods are
used to detect rheumatic fever.

Treatment

Pencillin G, cephalosporins, tetracyclines, and macrolides are used against Group

A Streptococci. Impetigo is treated with erythromycin to cover the prospect of
S.arueus involvement. Adequate treatment of Streptococcal pharyngitis within 10
days of onset prevents rheumatic fever. Treatment does not prevent the
development of acute glomerulorephritis.

Prevention

Penicillin prophylaxis is used to prevent recurrences of acute rheumatic fever

during the most susceptible ages (5 to 15 years)

Group B Streptococci (Streptococcus Agalatiae)

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Group B Streptococci produce short chains and diplococcal pairs of spherical

or ovoid Gram-positive cells. Colonies are larger and B-hemolysis is less distinct or
may be absent. It has lancefield B antigen and produce polysaccharide capsules.

Epidemiology

The organism is resident in the gastrointestinal tract and spread to other sites,
mainly Vagina. Group B Streptococci can be found in the vaginal flora of 10 to 30% of
women and during pregnancy and delivery; these organisms may again access to the
amniotic fluid or colonize the newborn as it passes through the birth canal. The risk is
higher when organisms are present that decrease the infant’s innate resistance or
increase the chances of transmission (ruptured amniotic membrane).

Pathogenesis

The capsule is the major factor to infection. The sialic acid capsule bind serum
factor H, which in turn accelerates degradation of C3b. Thus make
opsonophagoaytosis ineffective.

Manifestations

Respiratory distress, fever, lethargy, irritability, aprea and hypotension are

common. Pneumonia is common and meningitis is present in 5 to 10% of cases. The
onset of infection is typically in the first few days of life, and signs cases. Adult GBS
infections may be serious; they may be fatal if patients are immunocompromised.

Diagnosis

Diagnosis is by culture of blood, cerebrospinal fluid or other specimen. Serologic

methods used to determine the lancefield.

Treatment

Penicillin is the treatment of choice. For neonatal infections are often initially treated
with combinations of penicillin and an aminoglycoside.

Prevention
Attempt is needed to reduce contact of the infant with the organism.

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Antimicrbical prophylaxis with penicillin or ampicillin has been shown to reduce

transmission and disease in high-risk populations.

STREPTOCOCCUS PNEUMONIAE

Morphology and Structure

S.pneumoniae (Pneumococci) are Gram-positive, oval cocci typically arranged

end to end in pairs (diplococcus) giving the cells a bullet shape. Pneumococcus
contains capsule. All virulent strains have capsules, composed of polysaccharides
oligosaccharides, and other components. They are antigenic and have more than 90
Serotypes.

Pneumococcal cell wall structure is similar to other streptococci. Teichoic acid

and phosphocholine are rooted in the peptidoglycan extending outside into the
capsule where they provide binding domains for a variety of surface proteins. For
example, choline binding protein is able to bind to both preumococcal cell wall
cholines and carbohydrates present on the surface f epithelial cells.

Epidemiology

S.Pneumonia is a leading cause of pneumonia, acute purulent meningitis,

bacteremia , and other invasive infections. World wide, more than 5 million children
die every year from pneumococcal disease. S.Preumoniae is also the most frequent
cause of otitis media in children. Pneumococcal infections are most lommon in the
very goining child (less than 2 years) and in the old (above 60 years). Alcoholism,
diabetes mellitus, chronic renal disease, asplenia and some malignancies are all
associated with more frequent and serious pneumococcal infection.

Infections results when pneumococci colonizes nasopharynx, when can be found

in 5 to 40% of healthy persons depending on age, season and other factors. The
highest rates are among children in the winter. Respiratory secretions containing
pneumococci may be transmitted form person to person by direct contact or from
the microaerosols created by coughing and sneezing. These microaersols

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transmissible possible in crowded living conditions, particularly when colonized

persons are mixed with susceptible ones (child care centers, prisons). About 23 o the
90 pneumococcal serotypes produce disease.

Pathogenesis

Pneumococcal adherence to nasopharyrgeal cells involves multiple factors. The

choline binding proteins of pneumococci bind to carbohydrates covering or exposed
on the surface of host epithelial cells. This binding is helped by the by the exposure of
additional receptors by neuraminidase digestion stimulated cytokine activation of
host cells. Aspiration of respiratory secretions containing these pneumococci is the
intial event leading to pneumonia. Normally, aspirated organisms are cleared rapidly
by the defense mechanisms of the lower respiratory tract, including the cough and
epiglottic relexes; the mucociliary blanket and phagocytosis by alveolar macrophages.
When host factors impair the clearance mechanisms can also pneumococci to reach
the alveoli and multiply there. Host factors include chronic pulmonary disease;
damage to bronchial epithelium from smoking or air pollution; and respiratory
dysfunction from alcoholic intoxication, narcotics, anesthesia and trauma.

When organisms reach the alveolus, there is an involvement of pneumococcal

virulence factors in two stages. The first stage is occurring in early stage of infection,
when the surface capsule of pneumococci acts to block phagocytosis by complement
inhibition. This allows the organisms to multiply and spread. The second stage occurs
when organisms begin to disintegrate and release a number of factors, thus causing
injury. These factors include pneumolysin, autolysin and components of the cell wall.

Pneumococcal Disease

The most common form of infection with S.pneumoniae is pneumonia, which

begins with fever and a shaking chill followed by signs that localize the disease to the
lung. These include difficulty breathing and cough with production f purulent sputum,
sometimes with blood.

Manifestations

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Pneumococcal Pneumonia

Clinically, pneumococcal pneumonia begin abruptly with a shaking chill and high
fever. Cough with production of sputum pink to rusty in color and pleurstic chest pain
are common. Without therapy, sustained fever, pleuristic pain and productive cough
continue for 5 to 10 days after onset of the disease. Although infection may occur at
any age, but the incidence and mortality of pneumococcal pneumonia increase
sharply after 50 years.

Pneumococcal Meningitis

Acute purulent meningitis may follow pneumococcal pneumonia, infection at

another site, or appear with no apparent infection. It may also develop after trauma
involving the skull. The mortality is slightly higher with pneumococcal meningitis than
with others of pyogenic meningitis.

Diagnosis

Sputum collection may be difficult because specimens contaminated with

respiratory flora. Lower respiratory specimen may be used for diagnosis. When
specimen is gram stained, pneumococcal infection typically show Gram positive,
lancet-shaped diplococci.

S.Pneumonia grows well over right on blood agar medium and is usually
distinguished from viridans streptococci by susceptibility to the synthetic chemical or
by bile solubility. Blood cultures are valuable to cultures of local fluids or exudates
because Bacteremia is common in pneumococcal pnuemoria and meningitis.

Treatment

Pencillins is still the choice for susceptible strains but resistance rates not exceed.
Penicillin-resistant strains may be treated with erythromycin, vancomycin or
quinolones, if susceptible. High doses of third generation cephalosporins have also
been used in situations such as meningitis, which may over resistance.

Prevention

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A vaccines prepared from capsular polysaccharide extracted from the 23 most

common serotypes of S.Pneumoniae is used. This vaccine is presently recommended
for patients who are particularly susceptible to pneumococcal infection because of
advanced age protein conjugate vaccine is recommended for children.

Treponema Pallidium (Syphilis) (10 Mark)

T.Pallidium

Morphology

T.Pallidium is a slim spiroctute 5 to 15 micro metre long with regular spirals but
resembles like corkscrews. The organisms are seen only by immunofluorescence, dark
field microscopy, or silver impregnation histologic techniques. T.Pallidium cells shows
slow, rotating motility.

Growth Charactertics

T.Pallidium has been grown in cultured mammalian cells. It prefers low oxygen
tensions, but it is not a strict anaerote. With controlled oxygen tension and pH, the
organism can multiply through several generations in primary cell culture, but it is
difficult to subculture. Growth is generally slow with a mean generation time about
30 hours. In vivo growth is usually achieved by injection into rabbit testes.

T.Pallidium is extremely susceptible to changes in physiologic conditions. It dies

rapidly on drying and is readily killed by a wide range of detergents and disinfectants.

Syphilis Disease

Syphilis is the disease acquired by the direct contact of mucous membranes

during sexual intercource. Syphilis is caused by Treponema pallidum. The Syphilis
disease begins with a lesion at the pint of entry, usually a genital ulcer. After healing
of the ulcer, the organisms spread systemically and few weeks later, disease found as
generalized muculopapular rash called secondary syphilis. The disease then enters to
Latency phase. The Latent infection may be cleared by the immune system or

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reappear as tertiary syphilis years or decade’s later Tertiary syphilis is characterized by

focal lesions. Isolated foci lesions found in bone or liver may be unnoticed, but
infection of the cardiovascular or nervous systems can cause severe progressive
dementia or a ruptured aortic aneurysm is lead to death, if syphilis is untreated.

Antigenic Structure

T.Pallidium lacks proteins and other exposed antigens on their surface. The
outer membrane of T.Pallidium contains antigenic transmembrane proteins and
lipoproteins but quantity is very less.

Epidemiology

T.Pallidium is a human pathogen under natural conditions. In most cases,

infection is acquired from direct sexual contact with individual who has an active
primary or secondary syphilitic lesion. The studies suggest that transmission occurs in
over 50% of sexual contacts where a lesion is present. The disease may also spread by
nongenital contact with a lesion (e.g. of the lip), sharing of needles by intravenous
drug users or transplacental transmission to the fetus within approximately the first 3
years of the maternal infection transfusion is also a source of infection.

Pathogenesis

Strains of T.Pallidium are inoculated into the spirochetes reach the subepithelial
tissues through in apparent breaks in the skin or by passage between the epithelial
cells of mucous passage between the epithelial cells of mucous membranes, where it
multiplies slowly with little intial tissue reaction. Slow multiplication of the organisms
produces endarteritis. The small arterioles show swelling and proliferataion of their
endothelial cells. This reduces or obstructs local blood supply, probably forming
necrotic ulceration of the primary lesion and subsequent destruction at other sites.
Dense, granulomatous cuffs of lymphocytes, monocytes and plasma cells surround
the vessels. Although the primary lesion heals spontaneously the bacteria disseminate
to other organs by way of local lymph nodes and the blood stream.

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Syphilis is then silent until the disseminated secondary stage develops and then
silent again with entry into latency. During latent periods, organisms bind to host
proteins, immunoglobulins and complement to its surface, but without change in
viability or motility of the organisms.

The inflammatory response to immune complexes, spirochetal lipoproteins and

complement in arteriolar walls accounts for some of the injury in syphilitic lesions. In
late syphilis, the granulomatous nature of the lesions with injury is caused by
delayed-type hypersensitivity responses prolonged by persistence of the spirochetes.

Manifestations

Primary Syphilis

The primary syphilitic lesion is a papule which evolves to an ulcer at the site of
infection. This is found in the external genitalia or cervix but could be in the anal or
oral area depending on the nature of sexual contact. The lesion becomes indurated
and ulcerates but remains painless although sensitive to touch. The fully developed
ulcer with a firm base and raised margins is called chanchre, firm, non suppurative
painless enlargement of the regional lymph nodes usually develops within 1 week of
the primary lesion and may persist for months. The median incubation period from
sexual contact until appearance of the primary lesion is about 3 weeks. It heals
spontaneously after 4 to 6 weeks.

Secondary Syphilis

Secondary or disseminated syphilis develops 2 to 8 weeks after the appearance of

the chanchre. The primary lesion has usually heated but may still be present. This
form of syphilis is characterized by a symmetric mucocutaneous maculopapular rash
and generalized non tender lymph node enlargement with fever, malaise and other
manifestation of systemic infection. Skin lesion is distributed on the trunk and
extremites, including the palm, sole and face. Some patients develop painless
mucosal warty erosions called condylomatalata. These erosions usually develop in

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warm, moist sites such as the genitals and perineum. Only one third of patients, they
recover spontaneously after a few days to many weeks. In the remaining two third of
patients, the illness enter the latent stage.

Latent Syphilis

Latent syphilis is a form where there is no clinical manifestation but continuing

infection is evidenced by serologic tests. In the first few years latency may be found
with progressively less sever relapses of secondary syphilis. In late latent syphilis
(more than 4 years) relapses stop and patients become resistant to re-infection.
Transmission to other is possible from relapsing secondary lesions and by transfusion
or other contact with blood products. Mothers may transmit T.pallidium to their
foetus throughout latency.

Tertiary Syphilis

Patients with untreated syphilis develop tertiary syphilis. The manifestations may
appear as early as 5 years after infection but charactertistically occur after 15 to 20
years. The manifestation depend on the body sites involved and most important
involved are the nervous and cardiovascular system

Neurosyphilis is due to the damage produced by a mixture of meningovasculitis

and degenerative parenchymal changes in any part of the nervous system. The
chronic meningitis is evolved with fever, headache, focal neurologic findings and
increased cell and protein in the cerebrospinal fluid (CSF). Cortical degeneration of
the brain causes mental changes ranging from decreased memory to hallucinations or
frank psychosis. In the spinal cord demyelination of the posterior columns, dorsal
roots and dorsal root ganglia produce a syndrome called tabes dorsalis which includes
at axia, wide based gait, and loss of sensation. The most advanced central nervous
system (CNS) has a combination of neurologic deficits and behavioral disturbances
called paresis (Personality effect, reflexes, eyes, sentorium, intellect, speech).

Cardiovascular syphilis is due to arteritic involving the vasa vasorum of the aorta
causing a medical necrosis and loss of elastic fibers. This results in dilatation of aorta
and aortic valve ring seen. This leads to aneurysms of the ascending and transverse

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segments of the aorta and or artic valve incompetence. The expanding aneurysm can
produce pressure necrosis of adjacent structures or even rupture. A localized,
granulamatous reaction to T.Pallidum infection called a gumma may be found to skin,
bones, joints or other organ. Clinical manifestations are related to the local
destruction as with other mass-producing lesions such as tumors.

Congenital Syphilis

Fetuses are susceptible to Syphilis only after the fourth month of gestation and
adequate treatment of infected mothers before that time prevents tetal damage.
Untreated maternal infection may result in fetal loss or congenital syphilis, which is
analogous to secondary syphilis in the adult. The most common finding is rhinitis and
a maculopapular rash. Bone involvement produce charactertic changes in the
architecture of the entire skeletal system. Anemia, thrombocytopenia and liver failure
are events are found at the end.

Diagnosis

Microscopy

T.Pallidium can be seen by dark field microscopy in primary and secondary

lesions. The suspect lesion must be cleaned and abraded to produce a serous
transudate from below the surface of the ulcer base. This material can be collected in
a capillary tube or placed directly on a microscope slip for dark field observation. For
diagnosis, corkscrew morphology and charactertic motility is noticed oral and anal
lesions is not used for dark field microscopy because of the risk of misinterpretation
of other spirochetes present in the normal flora.

Serologic Tests

Using Serologic tests, syphilis can be diagnosed. These tests detect either lipid or
specific treponemal antigens. The former are called non-tremporemal tests and the
latter are referred to as treponemal tests. They used in screening, diagnosis and
therapeutic evaluation of syphilis.

Nontreponemal Test

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It is used for screening, but they must be confirmed by one of the more
specific treponemal test. Nontreponemal are valuable for treatment because the
height of the antibody is directly related to activity of disease.

Treponemal Tests

This test detect antibody specific to T.Pallidium such as an indirect

immunofluorescent procedure called fluorescent treponemal antibody (FTA-ABS)
which uses spirochetes fixed to slides. The “ABS” refers to an absorption step that
removes non-specific antispirochetal antibody found in normal serum.

Another method, micro (MHA-TP) hemagglutination test uses antigens attached

to the surface of erythrocytes which then agglutinate in the presence of specific
antibody.

Treponemal tests are specific, so its main role in diagnosis is to confirm positive
RPR and VDRL results obtained in the evaluation of a patient suspect for syphilis or in
screening programs. They are not useful for screening or following therapy because
once positive, they usually remain so for life.

IgM antibodies is used to diagnose congenital syphilis but use of serologic tests in
congenital syphilis is complicated because of the presence of IgG antibodies in infants,
who acquires it transplacentally from their mothers.

Treatment and Prevention

Penicillin is used against T.Pallidium and penicillin is the preferred treatment in all
stages because of T.Pallidium sensitiveness. In primary, secondary or latent syphilis
person hypersensitive to penicillin may be treated with tetracyclines, erythromycin or
cephalosporins. Safe sex practices are as effective for prevention of syphilis.

MYCOBACTERIA (10 Mark)

Mycobacteria

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Mycobacterium is a genus of Gram-positive bacilli that demonstrate the straining

of charactertic of acid-fastness. It’s species, Mycobacterium tuberculosis is the
causative agent of tuberculosis. A second important species Mycobacterium leprae, is
the causative agent to leprosy. A large number of less pathogenic species collectively
referred as atypical mycobacteria or nontuberculous mycobacteria are cause disease
agents in immunocompromised patients, mainly those with AIDS.

General Charactertics

Morphology and Structure

The mycobacteria are slim, Gram-positive bacilli (0.2-0.4X2.10mm). They are

nonmotile, obligate aerobes that do not spore spores. The cell wall contains
peptidoglycan similar to other Gram-positive organisms, but it contains
N-glycolylmuramic acid. Peptidoglycan similar to other Gram-Positive organism, but it
contains N-glycolylmuramic acid. Peptidoglycan is attached to myriad of branched
chain polysaccharides proteins and lipids. The important charactertics are long-chain
fatty acids called mycolic acids. The mycolic acids are present, so it is names as
mycobacteria

Growth Charactertics

M.Tuberculosis show enhanced growth in 10% carbon dioxide and at a PH of

about 6.5 to 6.8. Nutritional requirement vary among species. Some nonpathogens
can multiply on the washes of water faucets to the strict intracellular parasite
M.Leprae, which does not grow in artificial media or cell culture. Mycobacteria grow
more slowly than most human pathogenic bacteria because of their hydrophobic cell
surface, which cause them to clump and inhibit permeability of nutrients into the cell.
When surfactant (Tween 80) is added to cultures of M.Tuberculosis wets the surface
and lead to disperse and more rapid growth.

Mycobacterial Disease

Mycobacteria include a wide range of species pathogenic for humans and

animals. M.Tuberculosis occurs only in humans under natural conditions.

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M.Intracellulare can infect various host including humans but also exits in the
free-living state. Disease is caused by mycobacteria usually develop slowly, follow a
chronic course, and elicit a granulamatous response.

Mycobacteria do not produce exotoxins or endotoxins. Diseases are result of two

related host response. 1. Delayed-type hyper sensitivity reaction to mycobacterial
proteins, results in the destruction of non-activated macrophages containing
multiplying organisms. It is detected by intradermal injections of purified proteins
from the mycobacteria. 2. call-mediated immunity activates macrophages enabling
them to destroy mycobacteria contained within their cytoplasm.

Mycobacterium Tuberculosis

Charactertics

M.Tuberculosis is a slim, strongly acid-alcohol fast rod. It usually show irregular

beading in its staining, appear as connected series of acid-fast granules. It grows at
370C but not at room temperature and requires enriched or complex media for
primary growth. Growth is enhanced by 5 to 10% carbon dioxide but has slow mean
generation time of 12 to 24 hours Lowenstation Jensen medium. It contains
homogenized egg in nutrient base with dyes to inhibit the growth of
nonmycobacterial contaminants. The dry, rough, buff-colored colories usually appear
after 3 to 6 weeks of incubation.

Tuberculosis Disease

Tuberculosis is a systemic infection manifested only by evidence of an immune

response in most exposed individuals. In some infected persons, the disease either
progresses or commonly, reactivates after an asymptomatic period (years). The most
common reactivation form is a chronic pneumonia with fever, cough , bloody sputum,
and weight loss. Disease may spread outside of the lung and become devastating
when it reaches the central nervous system.

Epidemiology

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The great majority of tuberculous infections are contracted by inhalation of

droplet nuclei carrying the causative organism. Humans may also be infected through
the gastrointestinal tract following the ingestion of milk from tubercurlous lows or
rarely through abraded skin. It has been estimated that a single cough can generates
as may as 3000 infected droplet nuclei and that less than 10 bacilli may intiake a
pulmonary infection in a susceptible individual.

Pathogenesis

Primary Infection

Primary tuberculosis is the response to the intial infection an individual not

previously infected and sensitized to tuberculoprotein. Inhaled droplet nucleic
containing small numbers of tubercle bacilli are deposited in the peripheral
respiratory alveoli; mostly in well ventilated middle and lower tobes. Here , they are
engulfed by non-specifically activated alveolar macrophages. These cells destroy the
ingested organisms depends on their interent microbicidal capacity. If the alveolar
macrophages are unable to destroy ingested mycobacteria, they continue to multiply
until the macrophage bursts. The released organisms are quickly ingested by
inactivated blood macrophages, with cells are attracted to the lung.

The ingested mycobacteria continue to multiply intracellulary without damage to

their host cell. Some of the bacterial containing macrophages, are transported
through lymphatic channels to the hilar lymph nodes. From here, they may
disseminate through blood and lymphatic systems to a number of tissues, including
the liver, spleen, kidney, bone, brain, meninges and apics of the lungs. The
inflammatory in tissue is minor, and the sign and symptoms are absent. However, the
primary site of infection and some enlarged hilar lymph nodes can be detected
radiologically. In infants and immunocompromised adults, hematogenous
dissemination of organisms may occasionly produce life threating meningitis.

Reactivation Tuberculosis

Reactivation usually occurs in body areas relatively high oxygen tension and low
lymphatic drainage, mostly in apex of the lung. The lesion spread slowly, coalescing

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tubercles with numerous tubercule bacilli and large areas of caseous necrosis.
Necrosis mostly found in the wall of a small bronchus from which the necrotic
material is discharged, resulting in a pulmonary cavity and bronchial spread. Small
blood vessels are also eroded. The chronic fever and weight loss may be mediated in
part by macrophage-derived tumor necrosis factor.

Manifestations

Primary Tuberculosis

It is either asymptomatic or manifest only by fever and malaise. Radiographs

show infiltrates in the mid-zones of the lung and enlarging draining lymph nodes
found around the hilum. Primary tuberculosis may reactivate or organisms
disseminate to many organs to produce active military tuberculosis. The later may
result from a necrotic tubercle eroding into a small blood vessel.

Reactivation Tuberculosis

Reactivation tuberculosis is most common in old men. Generally Reactivation are

associated with a period of immunosuppression malnutrition, alcoholism, diabetes,
old age are the factors to induce immunosuppression. Recently reactivatin and
progressive primary tuberculosis among young adults have increased as a
complication of AIDS.

Symptoms include cough, as disease progresses sputum is produced, which later is

mixed with blood (hemoptysis). Fever, malaise, tatigue, disease continues. Untreated,
the progressive cough, fever and weight loss of pulmonary tuberculosis, may result in
death after 2 to 5 years. The course in AIDS and CMI compromised patients is more
rapid.

Diagnosis

Tuberculin Test

The tuberculin skin test measures (DTH) delayed type hypersensitivity to

tuberculoprotein. Purified protein derivated (PPD) is randamized and its activity

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expressed in tuberculin units (TU). Most intial skin test employs 5 TS (intermediate
strength). When and high degree of hypersensitivity or eye or skin tuberculosis is
suspected m then 1 to (first strength) or less is used initially to avoid the risk of an
excessive reaction locally or at the site of a mycobacterial lesion.

The test is performed by inter dermal injection that is read 48 to 72 hours later.
Around the area, induration of 10mm or more accompanied by erythema constitutes
a positive reaction, although smaller areas of induration and erythema indicate a
lesser degree of sensitization to mycobacterial proteins. No induration indicates
negative reaction. A positive tuberculin test indicates that the individual has been
infected at some time with M.Tuberculosis or with a strongly cross reacting
mycobacterium of another species.

Treatment

Isoniazid, ethambutol, rifampin, pyrazinamide, streptomycin and the combination

of these agents constitute the primary drugs of choice for treatment of tuberculosis.
Second line of drugs is aminosalicyclic acid, Ethionamide, Cycloserine,
fluoroquinolones and kanamycin, etc. is used for treatment.

Prevention

At present, the bacillus (almette-Guerin (BCG) vaccine is the only available

vaccine. It is used for prophylaxis and administers intadermally.

Mycobacterium Leprae

Mycobacterium leprae causes leprosy. M.Leprae is an acid-fast bacillus that is not

grown in artificial medium or tissue culture beyond a few generations. It can be
grown in the footpads of normal mice, in thymectomized irradiated mice and in the
armadillo. Its growth in animals is very slow, with a doubling time of 12 to 14 days.
The structure and cell wall components appear to be similar to other mycobacteria.
M.Leprae produce mycoside, phenolic glycolipid 1 (PGL-1).

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Leprosy Disease

Leprosy is a chronic granulomatous disease of the peripheral nerves and

superficial tissues, particularly the nasal mucosa. Disease ranges from slowly resolving
anesthetic skin lesions to the disjguring facial lesions.

Epidemiology

Transmission is by small droplets from the nasal secretions from cases of

lepramatous leprosy. Other possible transmission is by traumatic inoculation through
minor skin lesions. The infectivity of M.Leprae is low. Most cases have had prolonged
close contact with an infected individuals. Biting insects may also be involved.

Pathogenesis

M.Leprae is an obligate intracellular parasite that must multiply in host cells to

persist. In humans, they found in the macrophages. PGL-1 and LAM have been helpful
in the ability to survive and multiply in these cells. The organisms may invade
peripheral sensory nerves, resulting in patchy anesthesia. Few lepra are seen in
tuberculoid lesions, which are granulomatous with extensive epitheloid cells, giant
cells and lymphocytic infiltration. In Lepromatous Leprosy, Cell mediated immunity is
deficient thus growth of M.Leprae is not stopped.

Manifestations

Two major forms of disease are tuberculoid and lepromatorus.

Tuberculoid Leprosy

In lepromatous leprosy cell mediated immunity is deficient and patients are

anergic to lepromin. Growth of M.Leprae is not stopped. Skin lesions are extensive
symmetric and diffuse, particularly on the face, with thickening of the looser skin of
the lips, forehead and ears. Damage may be severe, with loss of nasal bones and
septum, digits and with testicular atrophy in men.

Diagnosis

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It involves preparation of acid-fast stained scrapings of infected tissue,

particularly nasal mucosa or ear lobes. By this staining large numbers of acid-fast
bacilli is detected. Skin biopsies used for the diagnosis of tuberculoid leprosy by
histological methods. In serodiagnosis, PGL-1 based serologic tests are done.

Treatment and Prevention

Sulfones such as dapsone are used for the treatment. When dapsone combined
with rifampin can control or uses tuberculoid leprosy, when given for 6 months.
Prevention involves recognition and treatment of infectious patients and early
diagnosis of the disease in close contacts.

Clostridium (10 Mark)

These bacteria require anaerobic condition for growth. Most of the organisms
produce endogenous infections adjacent to the mucosal surfaces, where they are
members of the normal flora. The clostridia form spores that aid to produce diseases,
such as tetanus and botulism.

Anaerobes Characteristics

Anaerobes survive under anaerobic conditions, to initiate and sustain growth.

Usually, anaerobes fail to grow in the presence of 10% oxygen, but some are sensitive
to oxygen concentrations as low as 0.5% and are killed by even brief exposures to air.
Most of the pathogenic species can survive in 2 to 8% oxygen, they are described as
aerotolerant that require the culture medium to be prepared and stored under
anaerobic conditions.

Anaerobes lack the cytochromes required to use oxygen as a terminal electron

acceptor in energy-yielding reactions and thus generate energy solely by
fermentation.

Clostridium Perfringes

Charactertics

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It is a large, Gram-positive, nonmotile rod with square ends. It grows on blood

agar medium under anaerobic conditions, producing colonies surrounded by a double
zone of hemolysis. In broth contain carbohydrate, as the result of growth
C.perfringens, there is production of large amount of hydrogen and carbon dioxide
gas which produced in necrotic tissues termed as gas gangnene.

Exotoxins

It produces multiple exotoxins and classified into five types (A to E). Type A
exotoxin is the most important in humans and is found consistently in the colon and
often in soil. The most important exotoxin is the toxin, a phospholipase that hydrolyze
lecithin and sphingomyelin, thus disrupting the cell membrane of various host cells
including erythrocytes lenkocytes and muscle cells. The o-toxin is a pore forming toxin
which is closely related to streptolysin O. This toxin alters capillary permeability and is
toxic to heart muscle.

Epidemiology

Gas Gangrene

When traumatic wounds with muscle damage are contaminated with

C.pertringens results in gas gangrene. The clostridia often found in patients own
intestinal flora or spores in the environment. Compound fractures, wounds or other
trauma allows entry of the between the injury and wound management allows
multiplication of the organisms.

Clostridial Food Poisoning

When large numbers of an enterotoxin producing C.perfringens are ingesting ,

that cause food poisoning. Out breaks probably results, when food source is get
contaminated with the strains.

Pathogenesis

Gas Gangrene

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If the oxidation-reduction potential in a wound is low, C.perfringens spores

germinate and can multiply, producing X-toxin. Thus, process passes along the muscle
bundles, producing rapidly spreading edema and necrosis that are more favourable
growth of the bacteria. As the disease progresses, increased vascular permeability
and systemic absorption of the toxin and inflammatory mediators leads to shock toxin
and oxygens results due to the metabolic activities of C.perfringens.

Clostridia Food Poisoning

The spore of some C. perfringens strains are often particularly heat resistant and
can withstand temperatures of 100o c for an hours or more. Thus, spores that survive
initial cooking can convert to the vegetative form and multiply if food is not
refringerated or rewarmed. After ingestion, the enterotoxin is released into the upper
gastrointestinal tract, causing a fluid outpouring in which the ileum is most severely
involved.

Manifestations

Gas Gangrene

Gas gangrene usually beings 1 to 4 days after the injury but sometime may start
within 10 hours. Earlier, there is severe pain at the site of the wound accompanied by
a sense of heaviness or pressure. The disease progresses with edema, tenderness and
pallor, which is followed by discoloration and hemorrhagic bullae. Late sign includes
gas, which appear as crepitance in the tissue. Systemic infection present as shock with
intravascular hemolysis, hypotension ans renal failure leading to coma and death.

Food Poisoning

The incubation period of 8 to 24 hours is followed by nausea, abdominal pain

and diarrhea. There is no fever and vomiting is rare. Spontaneous recovery usually
occurs with in 24 hours.

Diagnosis

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Diagnosis is based mainly on clinicial observations. It is quite common to isolated

C.perfringens from contaminated wounds of patients. Occasionally, C.perfringens is
even isolated form blood cultures of patients who do not develop serious clostridial
infection. In clostridial food poisoning isolation of more than 10 5 C.perfringens per
gram of ingested food in the absence of any other cause is usually sufficient to
confirm the causative agent.

Treatment and Prevention

Treatment of gas gangrene and endometritis must be initiated immediately

because these conditions are almost fatal if untreated. Excision of all devitalized
tissue is of most importance, because it denies the organism the anaerobic conditions
required for further multiplication and toxin production. Administration of massive
doses of penicillin is used for the treatment.

Clostridium Tetani

Charactertics

C.tetani is a slim, Gram-positive rod, which may lose gram positivity to very young
or old cultures. It forms spores readily in nature and in culture, yields a typical in
round terminal spore that gives the organism a drumstrick appearance before the
residual a vegetative cell disintegrates. C.tetani requires strict anaerobic conditions
and, is flagellate and motile.

Tetanospasmin

C.tetani produces neurotoxic exotoxin called tetanospasmin or tetanus toxin, a

metalloproteinase that enzymatically degrades a protein required for docking of
neurotransmitter vesicles at the appropriate site on presynaptic membranes. Loss of
this function prevents release of neurotransmitters used by inhibitory afferent motor
neurons. Thus the effect produces spasms. The toxin is heat labile, antigenic, readily
neutralized by antitoxin and rapidly destroyed by intestinal proteases. When toxin
treat with formaldehyde yields a nontoxic product or toxoid that retains the
antigenecity of toxin and thus stimulates production of antitoxin.

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Tetanus

The feature of tetanus is severe muscle spasms (or “lockjaw” when the jaw
muscles are involved). Muscle spasms occurs minimal or no inflammation at the
primary site of infection, which may be unnoticed even though the outcome is fatal.

Epidemiology

The spores of C.tetani found in soil, expecially those that have been treated with
manure and the organism is sometimes found in the lower intestinal tract of humans
and animals. The spores are entering into wounds when wound contaminated with
soil or foreign bodies. In some countries, the majority of tetanus occurs in recently
delivered infants when the umbilical cord is severed or bandaged in a nonsterile
manner. Similarly, tetanus may follow after an unskilled abortion, female circumcision
and even surgery performed with nonsterile instruments or dressings.

Pathogenesis

The predisposing factor for tentanus is an area of very low oxidation- reduction
potential in which tetanus spores can germinate in a large splinter after introduction
of soil, or necrosis after infection of contamination illicit drugs. Injection with
facultative or other anaerobic organisms can contribute to the development of an
appropriate nidus for spore germination. Generally, tetanus bacilli multiply locally and
neither damage nor invade adjacent tissues. Tetanospasmin is produced at the site of
infection and enters the presynaptic terminals of lower motor neurons, reaching the
central nevous system (CNS) manily by exploiting the retrograde axonal transport
system in the nerves. In the spinal cord, tetanospasmin acts at the lever of the
anterior horn cells, where its blockage of post synaptic inhibition of spinal motor
reflexes produces antagonist muscles. This process takes place initially in the area of
the causative lesion but ay extend up and down the spinal cord.

Manifestations

The incubation period of the disease is from 4 days to several weeks. The shortest
incubation period is usually found when wounds in areas supplied by the cranial

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motor nerves, which helpful in the short transmission route for the toxin to the CNS.
In general, shortest incubation period are associated with more severe disease.

Treatment

Treatment of tetanus involves neutralization of any unbound toxin with large

does of human tetanus immune globulin (HTIG), which is derived from the blood of
volunteers, hypermmunized with toxoid. Supportive measures includes maintenance
of a quiet dark environment, sedation and adequate airway, these are required until
axons regenerated. Benzodiazepines are also used to indirectely antagonized the
effects of toxin.

Prevention

Active immunization with tetanus toxoid, combined with diphtheria toxiod and
pertussis vaccine (DTap) is used for primary immunization in childhood and DT for
adults. This usage can completely prevent the disease the immunization efforts have
been focused on pregnant women, because transplacental transfer of antibodies to
the fetus also prevents the highly lethal neonatal tetanus.

Unimmunized persons with tetanus-prone wounds should be given passive

immunity with a prophylactic does of HTIG as soon as possible. This immunization
provides immediate protection. If immunization is incomplete or the wound has been
neglected and poses a serious risk of disease, HTIG is appropriate to use in this
condtion.

Clostridium Botulinum

Charactertics

C.botulinum is a large Gram-positive rod like the other clostridia. It produce

spores which resist boiling for long periods, and moist heat at 21OC is required for

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certain destruction. Germination of spores and growth of C.botulinum can occur in a

variety of alkaline or nentral foodstuffs when conditions are sufficiently anaerobic.

Toxin

C.botulinum grows under anaerobic conditions, produce family of neurotoxin of

extra toxicity. Botulinum toxin is the most potent toxin, with an estimated lethal dose
for human of less than 1 Mg. botulinum toxin is also a metalloproteinase that acts on
the presynaptic membranes at neuromuscular junction. Thus, toxin cleaves proteins
involved in the release of acetycholine at the synapse. The blockage of acetylcholine
release result in paralysis of the motor system, but it also causes dysfunction of the
autonomic nervous system. C.botulinum is classified into multiple types (A to G)
based on the antigenic specificity of the neurotoxins. All toxins are heat labile and
destroy rapidly at 100oC but are resistant to the enzymes of the gastrointestinal tract.
If unheated toxin is ingested, it is readily absored and distributed in the bloodstream.

Botulism Disease

It begins with cranial nerve palsies and develops into descending symmetrical
motor paralysis, which may involve the respiratory muscles. The time course depends
on the amount of toxin present and whether it was ingested preformed in food or
produced endogenously in the intestinal tract or a wound.

Epidemiology

Spores of C.botulinum usually are found in soil, pond and lake sediments. It
spores contaminate food; they may convert to the vegetative state, multiply and
produce toxin in storage under proper conditions. This may occur with no change in
food waste, color or odor. The alkaline conditions provided by vegetables, such as
green beans and mushrooms and fish support the growth of C.botulinum and the
acidic conditions provided by foods such as canned fruit do not support the growth of
the bacterium. Botulism often occurs after ingestion of food that have not been
heated at temperatures sufficient to kill. C.botulinum spores. Because the toxin is

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heat labile, food must be ingested uncooked or after insufficient cooking to cause
botulism. Infant and wound botulism results when the toxin is produced
endogenously (inside), spores enter from environment, by either ingest or
contaminate wounds.

Pathogenensis

Food-borne botulism is an intoxication, not an infection. The ingested preformed

toxin is absorbed in the intestinal tract and reaches its neuromuscular junction target
via the blood stream. After bound, its inhibition of acetylcholine release causes
paralysis due to lack of neuromuscular transmission. The specific disease
manifestations depend on the specific nerves to which the circulating toxin binds.
Cardiac arrhythmias and blood pressure instability are due to effects of the toxin on
the autonomic nervous system.

Manifestation

Food-borne botulism usually starts 12 to 36 hours after ingestion of the toxin.

The first signs are nausea, dry mouth and diarrhea. Cranial nerve signs include blurred
vision, papillary dilatation and nystagmus occurs. Symmetrical paralysis begins with
the ocular laryngeal and respiratory muscles and spread to the trunk and extremities.
The most serious condition is complete respiratory paralysis. Mortality is about 10 to
20%.

Infant Botulism

It occurs in infants between the ages of 3 weeks and 8 months is the commonly
diagnosed form of botulism. The organisms are introduced on weaning or with dietary
supplements, and multiply in the infant’s colon, with absorption of small amounts of
toxin. The infants shows constipation,poor muscle tone, lethargy and feeding
problems and have ophthalmic and other paralyses similar to those in adult botulism.

Wound Botulism

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Usually, wounds infected with other organisms rarely allow C.botulinum to grow.
Contaminated wounds of drungs users are sites of toxin production. Diseases are
similar to food poisoning or it may be with weakness localized to the injured
extremity.

Diagnosis

The toxin can be detected in blood, intestinal contents or remaining food.

C.botulinum may also be isolated from stool or from food stuffs susprected of
responsibility for botulism.

Treatment and Prevention

The intensive supportive measures, particularly mechanical ventilation is neede,

with proper ventilatory support, mortaliy should be less then 10%. The administration
of large doses of horse C.botulism antitoxin is thought to useful in neutralizing free
toxin. Frequent hupersensitivity reactions related to the equine orign of this
preparation makes it unsuitable for use in infants. Antimicrobial agents are given only
to patients with wound botulism.

Adequate pressure looking or autoclaving can kills spores and heating food at
100o C for 10 minutes before eating destroys the toxin. Food from damaged cans
should be not even being tasted because of the extreme toxicity of the C.botulinum
toxin.

NEISSERIA (10 Mark)

Neisseria

Neisseria are Gram-negative diplococci. The genus contains two pathogenic and
many commensal species; most of them are inhabitants of the upper respiratory and
alimentary tracts. The pathogenic species are neisseria meningitides

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(meningococcus), major cause of meningitis and bacteremia and nesseria gonorr

hoeae (gonococcus), and the cause of gonorrhea.

General Charactertics

Neisseria are Gram-negative cocci that typically appear in pairs with the opposing
sides flattened, giving a “kidney bean” appearance. They are nonmotile, non-spore
forming and on-acid fast bacteria. Their cell walls are Gram-negative bacteria, with a
peptidoglycan layer and an outer membrane containing endotoxic glycolipid
complexed with protein. Both N.meningitidis and N.gonorrhoeae are similar in
structure except that the meningococcus has a polysaccharide capsule external to the
cell wall.

Neisseria Meningitidis

Charactertics

Meaningococci produce smooth colonies on blood agar plates after overnight

incubation. Based on the antigenic specificity of a polysachharide capsule, organism
can be divided into twelve serogroups. The most important disease-producing
serogroups are A,B, C,W-135 and Y. in addition to this N.meningitidis strains may
contain two distinct classes of pili and multiple classes of OMPS. Some OMPS, porins
and adherence proteins have structural and functional similarities to those found in
gonococci.

Epidemiology

Meninogococci are found in the nasopharyngeal flora of approximately 10 % of

healthy individuals. Organisms transmitted into the body by inhalation of aerosolized
respiratory droplets. Close, prolonged contact such as occurs in families and closed
populations promotes transmission.

Meningococcal infection mostly found in children less than 6 years of age. They
occur isolated cases, as sporadic small epidemics, or in small family or
closed-population outbreaks. B, C and Y are the most common serogroups involved in

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infection. Group a meningococci have the capability to cause wide spread epidemics
among communities.

Meningococcal Disease

Meningococci may produce fulminant infection of the blood stream and central
nervous system (CNS). The major disease is acute, purulent meningitis with fever,
headache, seizures, and mental signs to inflammation and increased intracranial
pressue. N.meningitidis infections found rash, purpura, thrombocytopenia and other
manifestations associated with endotoxemia. The bacterium causes infections in
which patients may progress from normal health to death in less than a day.

Pathogenesis

The meningococcus is a human pathogen, which exist as a normal flora or

produce acute disease. Some individuals, the carrier state is assocated with
acquisition of protective antibodies, but for other, spread from the nasopharynx to
bacteremia, endotoxemia and meningitis takes place quaickly. After inhalation,
meningococci use pili for initial attachment to the microvilli of the nonciliated
naspharyngeal epithelium to invade the body. In the invasion process, the microvilli
come in close contact with the bacteria, which enter the cells in membrane bound
vesicles. One inside meningococci quickly pass through the cytoplasm; enter into the
submucosa on the other side. During the process, they damage the ciliated cells by
direct release of endotoxin.

After entry into the mucosa, meningococci ability to produce disease is enhanced
by several factors that allow and evade the host immune response. N.meningitids
contains proteins which are able to acquire iron from the human iron transport
protein transferin. The bacteria contain prolysaccharide capsule enables
meningococci to resist complement mediated bactericidal activity and neutrophil
phagocytosis. Meningococcal LPS also has features that facilitate evasion of host
immune responses. Menigogococcal LPS chemical structure resembles sphingolipids
found in the human brain enough for them to be recognized as self by the immune
system. The LOS and sialic acid interferes with complement deposition.

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The most serious manifestations of meningococcal disease caused when

organisms spread to the blood stream, namely meninges, Bacteremia occurs in the
choroids plexus with high rate of blood flow. After CNS invasion, an intense
subarachroid space inflammatory responsse is generated; induce by the release of
wall peptidoglycan fragments, LPS and other virulence factors causing the release of
inflammatory cytokines.

The meningococcal infection cause potent, endotoxic activity, in culture

organisms release endotoxin-containing blets of its outer membrane from the cell
surface.

Manifestations

The most form of meningococcal infection is acute purulent meningitis. A

prominent feature of meningococcal meningitis is the appearance of scattered skin
petechiae, which evolve into ecchymoses or a diffuse petechial rash. Disseminated
intravascular coagulation (DIC) Syndrome results when endotoxic shock brought on by
meningococcal bacteremia (meningococcemia) and rash (cutaneous manitestations).
Meningococcemia occurs without meningitis and may progress to fulminant DIC and
shock with bilateral hemorrhagic destruction of adrenal glands
(watehouse-friderichsen syndrome). In few occasions, the disease is not always
fulminant and some patients have only low-grade fever, arthritis and skin lesions that
develop slowly over a period of days or weeks. Meningococci rarely cause pneumonia.

Diagnosis

Examination of cerebrospinal fluid (CSF) reveals typics bean-shaped,

Gram-negative diplococci in gram stain smears. Definitive diagnosis is by culture of
CSF, blood or skin lesions. Growth is maximum on blood of chocolate agar after 18
hours of incubation. Species are differentiated by carbohydrate degradation patterns
or immunological tests. Serogrouping is done by slide agglutination.

Treatment

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Penicillin is the treatment of choice for meningococcal infections because of its

antimeningococcal actitivy and good CSF penertration. Resistance mediated by
B.lactamase and altered pencillin binding proteins has been seen. Third generation
cephalosporins such as celotaxime are effective alternative to penicillin.

Prevention

Rifampin and ciprofloxacin were used as the primary means of preventing spread
of meningococcal infections. Close contact and small age groups are severely
infected. Most important prophylaxis is to avoid close contact with affected
individuals.

Purified polysaccharide meningococcal vaccines have been shown to prevent

group A and C disease in high risk populatins. A, C, Y and W-135 polysaccharides
vaccines are also used. Routine immunization of children is not recommended.

Neisseria Gonorrhoeae

General Charactertics

N.gonorrhoeae grows well on chocolate agar and on specialized medium

enriched to ensure its growth. For growth, it requires carbon dioxide
supplementation. Small, smooth non pigmented colonies appear after 18 to 24 hours
and are well developed (2 to 4mm) after 48 hours. Gonococci possesss numerous pili,
these structures are associated with virulence and may mediate initial attachment to
epithelial surfaces.

Gonorrhea Disease

Gonorrhea disease is primarily localized to mucosal surfaces with infrequent

spread to the blood stream or deeptissues. Gonorrhea infection is transmitted
sexually by direct genital contact, and the primary manifestation is pain and purulent
discharge at the infected site. In men, the infected site is the urethra, and in women,
the uterine cervix. If infection extends to the fallopian tubes produces fever and lower
abdominal pain, a syndrome called pelvic inflammatory disease (PID). For women
sterility or ectopic pregnancy can be long-term lonsequences of gonorrhea.

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Epidemiology

Infection rates among adolescents are very high and increasing year by year.
The major reservoir for continued spread to gonorrhea is the asymptomatic patient,
screening programs and case contact studies have shown that almost 50% of infected
women are asymptomatic or do not have symptoms usally associated with venereal
infection. But most men (95%) have acute symptoms with infection usually, persons
are treated become asymptomatic but remain infectious. Asymptomatic, male and
female patients can remain infectious for months. The attack rates are high in person
involving genital intercourse with an infected patient. The organism may also be
transmitted by oral-genital contact or by rectal intercourses. Non-sexual transmission
such as toilet seats found with gonococci, fomite transmission of purulent
vulvovaginitis is very rare.

Pathogenesis

Gonococci are not normal inhabitants of the human body. Organism introduced onto
a mucosal surface by sexual contact with infected individual adherence ligands such
as pili, opa proteins and Los allow initial attachment of the bacteria to receptors
(CD46, CD66) on nonciliated epithelial cells. Pili are the primary mediators of
adherence to urethral and vaginal epithelium, nonciliated fallopian tube cells, sperm
and neutrophils. Opa proteins are involved in cervical and urethral epithelia cell
adherence an in adhesion between gonococcal cells.

After attachment, gonococci invade epithelial cells. The microvilli surround the
bacteria and are helpful bacteria to enter into the host cell. This process is called
parasite-directed endocytosis.

Manifestations

Genital Gonorrhea

In men, the primary site of infection is the urethra. Symptoms begin 2 to 7 days
after infection and consist primarily of purulent corethral discharge and dysuria. A
local extension can lead to epididymitis or prostatitis. The endocervix is the primary

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site in women, in whom symptoms include incrased vaginal discharge, coronary

frequency, dysuria abdominal pain and menstrual abnormalities. Symptoms may be
mild or absent in either sex, particularly women.

Other Local Infections

Rectal gonorrhea occurs after rectal intercourse or in women, after

contamination with infected vaginal secretions. This condition is generally
asymptomatic but may cause discharge and rectal bleeding pharyngeal gonorrhea is
transmitted by oralgenital sex and is usually asymptomatic. Sore throat and cervical
adenitis may occur. Infection in Bartholin’s glands in women, may lead to abscess
formation.

Inoculation of gonococci into the conjunctiva produces a Severe, acute, purulent

conjunctivitis. These infections may oaccur at any age, the most serious form is
gonococcal ophthalmia neonatorum, a disease acquired by a newborn from an
infected mother. The diseas was a common cause of blindness, which can be
prevented by the use of prophylactic topical eye drops ox ointment (silver nitrate,
erythromycin or tetracycline) at birth.

Pelvic Inflammatory Disease (PID)

The local forms of gonorrhea and their extensions such as PID may lead to
bacteremia. In the bacteremic phase, the primary features are fever; migratory
polyarthralgia; and a petechial, maculopapular or pustular rash. Some of these
features may be immunologically mediated; gonococci are infrequently isolated from
the skin or joints at this stage despite. The bactermia may lead to metastatic infection
such as endocarditis and meningitis, but the most common is purulent arthritis. The
arthritis involves large joints such as elbows and knees. Gonococci are readily cultured
from pus.

Diagnosis

Staining

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The smear is from a genital site, shows the presence of multiple pairs of bean
shaped, gram-negative diplococci within neutrophils. The direct Gram smear is more
than 95% sensitive and specific in symptomatic men. Unfortunately, it is only 50 to 70
% senstivie in women.

Direct Detection

Direct detection is possible using immunoassay and nucleic acid bybridization

methods that detect gonococci in clinical specimens without culture. The serologic
test for gonorrhea has not yet achieved the needed sensitivity and specificity. A test
that would detect the disease in asymptomatic patients would be very useful in
control of this disease.

Treatment

Definitive treatment was easily accomplished with a single intramuscular

injection of penicillin G. due to resistant strains, there is shift in treatment of genital
gonorrhea to third-genereation cephal osporins. The recommended agents used as
single dose treatment either intramuscularly (ceftriaxone) or orally (cefixime). Other
agents used for primary treatment include flororoquinolones (ciprofloxacin or
ofloxacin) and azithromycin. Doxycycline is also effective but must be given orally for
7 days.

Prevention

Prevention methods used are condoms, which block direct mucosal contact or
vaginal foams, which also inhibit the gonococcus. These methods provide protection
against gonorrhea if used properly. The availability of a good serologic test would
greatly aid control. The development of gonococcal vaccine awaits further
understanding of immunity and its relationship to the shifting target provided by the
gonococcus.

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VIBRIO (10 Mark)

Vibrio

Virbios are curved, gram-negative rods commonly found in salt water. Cells may
be linked end to end, forming S shapes and spirals. They are highly motile with a
single polar flagellum, non-spore forming, oxidase positive and can grow under
aerobic or anaerobic conditions. The cell envelop structure is similar to that of other
gram-negative bacteria. Vibrio cholerae cause of a water-loss diarrhea called cholera.

Organism Features Epidemiology Disease

V.mimicus Related to Ingestion of raw Watery diarrhea

V.cholerae, Cholera seafood.
like entertoixin

V.parahoemolyticus Produces bowel, Costal seawater; Watery; diarrhea

inflammation, ingesting raw dysentery.
enterotoxin seafood

V.Vulnifus Produces powerful Coastal sea water; Fulminant

siderophores which ingesting raw bacteremia
scavengeiron from seafood or following ingestion
host transferring contamination of cellulites from
and lactoferrin wound with sea wound
water contamination

V.Alginolyticus Wounds Cellulitis

contaminated by
seawater

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Vibrio Cholerae

Facultative Fecal-oral, water Choler toxin (Ace, Watery diarrhea

borne, pandemics Zot) (cholera)

Vibrio Cholerae

Growth and Structure

V.cholerae grows under alkaline (pH 8.0 to 9.5) conditions that inhibit many other
gram-negative bacteria. V.cholerae is distinguished form other vibrios by it
biochemical reactions, lipopolysac charide (LPS) O antigenic structure, and production
of cholera toxin (CT). There are over 150 O antigen serotypes, only two o which (01
and O139) cause cholera. O139 strains possess a unique O antigen and have a
polysaccharide capsule. V.cholerae possesss long filamentous pili that form bundles
on the bacterial surface. The pili is chemical structure is similar to those of the
gonococcus. All strains capable of causing cholera produce a colonizing factor known
as the toxin-coregulated pilus (TCP).

Cholera Toxin

Cholera toxin is an A-B type ADP-ribosylating toxin. Cholera toxin molecule is an

aggregate of multiple polypeptide chains organized into two toxic subunits (A1,A22)
and five binding (B) units. The B units bind to a GM1-ganglioside receptor found on
the surface of many types of cells. After binding to the cell the A1 subunit is released
form the toxin molecule and enter the cell.

Cholera

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Cholera produces the watery diarrhea. Intestinal fluids pour out in voluminous
bowel movements; this leads to dehydration and electrolyte imbalance. These effects
are due to the action of cholera toxin secreted by V.cholerae in the bowel lumen.

Epidemiology

Epidemic cholera is spread primarily by contaminated water. These is due to the

condition of poor sanitation, particularly where sewage treatment is absent. It’s
incubation period is 2 days.

Cholera is endemic in India and Africa. The new serotype (O139) is fully in
persons whose immunity is due to exposure to the old serotype.

Pathogenesis

After enter into the body, V.cholerae reaches the small intestine in sufficient
numbers to multiply and colonize. In healthy people, ingestion of large numbers of
bacteria is required to pass the acid barrier of the stomach. V.Cholerae contain
surface pili which helps to adhere to the epithelia surface, thus organisms colonized
entire intestinal tract form the jejunum to the colon. The important feature of
V.cholerae pathogencity is the ability of virulent strains to secrete cholera toxin,
which is responsible for the disease cholera. The water and electrolyte shift from the
cell to the intestinal lumen is the fundamental cause of the watery diarrhea of
cholera.

Fluid Loss

The fluid loss that results from the adenylate cyclase stimulation of cells depends
on the balance between the amount of bacterial growth, toxin production, fluid
secretion and growth, toxin production, fluid secretin and fluid absorption in the
entire gastrointestional tract. The outpouring of fluid and electrolytes is greatest in
the small intestine, where the secretory capacity is high and absorptive capacity low.

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Small intestine loses liters of fluid, with same sodium content as plasma but two to
five times the potassium and bicarbonate concentrations. The results are dehydratin
(isotonic fluid loss), hypokalemic (Potassium loss) and metabolic acidosis (bicarbonate
loss). The intestinal muscosa remains unaltered except hyperemia because V.cholerae
does not invade or otherwise injure the enterocyte.

Manifestations

Cholera has a rapid onset, beginning with abdominal fullness and discomfort,
rushes of peristalsis and loose stools. Vomiting may also occur. The stool quickly
become watery, voluminous, odorless and contain mucus flecks, giving it an
appearance called rice-water stools. Clinical features of cholera result from the
extensive fluid loss and electrolyte imbalance, which can lead to extreme
dehydration, hypotesion and death within hours if untreated.

Diagnosis

The initial suspicion of cholera depends on recognition of the typical clinical

features. Diagnosis is accomplished by isolation of V.Cholerae from the Stool. The
organisms grows on blood agar and macconkey agar medium, but its isolation is
enhanced by the use of a selective medium (thiosulfate-citrate-bile-salt-sucrose agar).
After isolation, the organisms are readily indentified by biochemical reations.

Treatment

Balancing the diarrheal fluid and ionic losses with adequate fluid and electrolyte
replacement is needed for recovery. This is accomplished by oral and intravenous
administration of solutions of glucose with near physiologic concentrations of sodium
and chloride and higher than physiologic concentrations of potassium and
bicarbonate. Antimicorobial therapy plays a secondary role to fluid replacement.
Tetracyclines shorten the duration of diarrhea and magnitude of fluid loss.
Trimethoprim sulfamethoxazole and erythromycin are alternatives for use in children
and pregnant women.

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Prevention

Epidemic cholera, a disease caused due to poor sanitation. These disease does
not persist when treatment and disposal of human waste is adequate main preventive
measures are good sanitary conditions, boiling or chlorination of water. Vaccines have
been disappointing and protection is not long lasting.

Unit – 5

TYPES OF IMMUNITY (10 Mark)

Immunity is the resistance produced by the body against microorganisms and their
products. Immunity is the ability of the human body to tolerate the presence of
material indigenous to the body (“self”) and to eliminate foreign (“nonself”)
material. The discrimatory ability provides protection from infectious disease, since
most microbes are identified as foreign by the immune system.

Immunity to microbes is usually indicated by the presence of antibody to that

organism. Immunity is generally specific to a single organism or group of closely
related organisms.

There are two basic mechanism for acquiring immunity, they are active and passivity
immunity.

Active immunity is protection that is produced by the persons own immune system.
This type of immunity is usually permanent.

Passive immunity is protection by products produced by an animal or human and

transferred to human, usually by injection. Passive immunity often provides effective
protection, but this protection disappears with time, usually within a few weeks or
months.

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Immunity is classified into two types. They are innate immunity and acquired
immunity.

1. Innate immunity
 Species
 Racial
 Individual

2. Acquired immunity
 Active
a. Natural
b. Artificial
 Passive
a. Natural
b. Artificial

Innate immunity

The innate immunity system is the first line of defense against invading organisms.
Innate and adaptive (acquired) immune system has both cellular and humoral
components by which they carry our their protective function. In addition, the innate
immune system also has anatomical features that function as barriers to infection.
The elements of the innate (non-specific) immune system include anatomical barriers,
secretory molecules and cellular components. Mechanical anatomical barriers are the
skin and internal epithelial layers, the movement of the intestines and the oscillation
of bronchopulmonary cilia.

Innate immunity varies based on species, racial and individual.

Species

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It is a complete resistance to a pathogen by all members of species. E.g. all

humans resistant to plant pathogens.

Racial

There is difference in resistance within species. These difference are genetic in

origin. E.g. some people may be resistant to infection but other people susceptible to
infection.

Individual

There is difference in resistance within races. It is also genetic in origin. E.g.

homozygous twins show similar degree resistance or susceptible to leprosy and
tuberculosis.

Factors determining innate immunity

1. Age – there is difference in innate immunity between age groups. New borne child
susceptible to infection because of immature immune system. In old people, repair in
the immune system lead to susceptibility to infection. Adult people able to resist
infection because of well developed immune system.
2. Hormones – due to endocrine disorders, there is a chance of increased susceptibility
to infection. For example, glucocorticoids inhibit the ability of phagocytes to ingest
foreign particles. Pregnant women are susceptible to infection due to increased levels
of steroids during pregnancy.
3. Nutrition – as a result of malnutrition, there is a decline in immunity mainly
cell-mediate and antibody-mediated immunity. Protein malnutrition causes 1. Lowers
(C3 and factor B of the complement system. 2. Decreases the interferon response and
3. Inhibits neutrophil activity.

Mechanism of innate immunity

1. Skin and mucus membrane – skin and mucus membrane act as barrier to prevent
infections. Skin provide mechanical barrier and act as bactericidal activity. Sweat,
sebaceous secretions, fatty acids and soaps contribute for protection. Mucosa of
respiratory contains cilia which help in preventing the entry of dust particles. Cough
reflex is the main defense mechanism of respiratory tract. Conjunctiva of eyes is
constantly flushed with lacrimal secretion which helps to kill bacteria. Saliva inhibits
microbes. Gastric juice is of high acidic nature which can destroy microbes.
2. Phagocytosis – phagocytic cells present in blood and tissues engulf and kill microbes
and foreign molecules. Phagocytosis is carried out by neutrophils and macrophages.

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3. Inflammation – it is a non-specific defense mechanism. Inflammation occurs during

tissue injury caused by pathogens. During this mechanism, microbes are
phagocytosed and killed.
4. Fever – during infection, there is rise in temperature which helps in speed up the
physiological process and destruction of microbes.

Acquired immunity

When innate immunity breached, the acquired immunity come into action against
pathogens. The resistance that an individual acquires during his life time is known as
acquired immunity. Immunity is antigen-specific and may be antibody-mediated or
cell-mediated.

Acquired immunity is classified into two types

1. Active immunity
2. Passive immunity

Active immunity

It is produced when an individual’s own immune system produces specific immune

defensive cells or molecules called antibodies or immunoglobulins, upon expose to an
infectious agent or its toxins. In this immunity, long lasting memory cells are
developed. Active immunity can be acquired naturally or artificially.

Active immunity is classified into

1. Natural active immunity

2. Artificial active immunity

Natural active immunity

The host’s immune system plays an active role in preventing disease by producing
specific antibodies and T lymphocytes. It follows the recovery from a disease.
Immunity develops memory cells which are long lasting. Memory cells produced
either from a sub clinical or clinical infection. Thus, it provides long-lasting immunity
due to repeated sub clinical infections. E.g. diphtheria, whooping cough, measles
and mumps. Few cases like influenza virus give immunity for short period is due to the
ability of influenza virus to undergo antigenic variation.

Artificial active immunity

In artificial active immunity, the person’s immune system produces antibodies and
specific defense cells. These resistance developed by immune system which is
induced by vaccines (live or killed preparations). Vaccines initiate a mini infection

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without causing disease. Vaccines are given as single doses. Killed vaccines are less
immunogenic than live vaccines and protection lasts only for short period.

Passive immunity

Passive immunity is established when ready-made antibodies or defensive cells are

introduced into the body. This immunity is temporary because antibodies or cells
persist in the body only for a few weeks or months. No memory cells are developed
and B and T cells don’t clone.

Passive immunity can be acquired naturally or artificially. Passive immunity can be

classified into two types

1. Natural passive immunity

2. Artificial passive immunity

Natural passive immunity

Natural passive immunity is the immunity transferred from mother to foetus through
placenta. IgG antibodies can cross placental barrier to reach the foetus. These
antibodies protect foetus from infectious diseases.

Artificial passive immunity

It is the immunity transferred passively to a recipient by administration of antibodies.

Immunization is carried out by injection of serum of animals. These antibodies remain
effective for 10 days.

HYPERSENSITIVITIES (10 Mark)

Immune system protects the host against microbial invaders and cancer cells. In
human host, disorders (malfunctions) occur in the immune system. Immune disorders
can be categorized as hypersensitivities, autoimmune diseases, transplantation
(tissues) rejection and immunodeficiency.

Hypersensitivity is an exaggerated immune response that results in tissue

damage and is manifested in the individual on second or subsequent contact with an
antigen. Whenever the immune system acts excessively or inappropriately to an

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antigen and pathological changes occur, the person is said to be hypersensitive.

Hypersensitivity reactions can be classifies as either immediate or delayed. They
classified based on the relative amount for time for an immunological response to the
antigen. In 1963, Peter Gell and Robert Coombs developed a classification system for
reactions responsible for hypersensitivities. Thus hypersensitivity can be classified
into five types. They are

Type – I Anaphylactic

Type – II Cytotoxic.

Type – III Immune complex

Type – IV Cell mediated or delayed

Type - V Stimulatory or antireceptor.

Type – I , II , III, and V are known as immediate type reactions, because reactions
develop in less than 24 hours after re exposure to an antigen. Their reactions occur
during the interaction of antigen with humoral antibodies. Type IV is called delayed
hypersensitivity, reaction develop in 24- 48 hours. Type IV mediated by
T-lymphocytes.

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Type – I Hypersensitivity:

Allergic reactions occur when an individual who has produced IgE

antibody in response to an antigen (allergen) subsequently encounters the same
antigen. An allergy is one kind of type – I hypersensitivity reaction.

An antigenic substance that can trigger the allergic state is known as

allergen, it may be a protein or chemically complex low molecular weight substance.
Generally, most people do not respond to allergen but an allergic person is often
sensitive to several different allergen. Type-I hypersensitivity is an allergic reaction
occurring immediately following an individual’s second contact with the antigen ( the
allergen). As the result of contact, vasoactive amines and histamine are produced.
Hypersensitivity may be local or generalized, depending upon the amount of
histamine released, the site of its release and route of stimulating antigen. In local
anaphylaxis, small amount of antigen administered to mucous membrane or skin,
whereas generalized anaphylaxis, large amount of antigen administered systemically.

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Hay fever and asthma is a good example of local anaphylaxis involving the upper
respiratory tract. Common examples of allergen’s that produce systematic
anaphylaxis include drugs (penicillin), antisera, peanut, and insect venom from the
stings or bites of wasps, hornets or bees.

Mechanism:

Initial exposure to a soluble allergen, B cells are stimulate to differentiate

into plasma cells and produce specific IgE molecules. IgE are called regain, IgE binds to
the Fc receptors of mast cells (basophils and eosinophils can also be activated) and
sensitizes these cells, making the individual allergic to the allergen. When a second
exposure of allergen occurs, the allergen attaches to the surface bound IgE on the
sensitized mast cells causing degranulation. Degranulation releases physiological
mediators such as histamine, leukotrienes, heparin, prostaglandins, PAF(
platelet-activation factor), ECF-A (eosinophil chemotactic factor of anaphylaxix) and
proteolytic enzymes. Mediators trigger smooth muscle contraction, vasodilation,
increased vascular permeability and mucous secretion. These responses are termed
anaphylaxis.

Skin Test:

Skin testing can used to identify the allergen responsible for allergies.
These tests involve inoculating small amounts of suspect allergen responsible for
allergies. These tests involve inoculating small amounts of suspect allergen into the
skin. Sensitivity to the antigen is shown by a rapid inflammatory reaction
characterized by redness, swelling, and itching at the site of inoculation. The affected
area in which the allergen-mast cell reaction takes place is called a wheal and flare
reaction site.

Once the responsible allergen has been identified, the individual should
avoid contact with it. Desensitization procedure consists of a series of allergen doses
injected beneath the skin to stimulate the production of IgG antibodies rather than
IgE antibodies. The circulating IgE antibodies can then act as blocking antibodies to

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intercept and neutralize allergens before they have time to react with mast
cell-bound IgE. Suppressor T-cell activity also may cause a decrease in IgE synthesis.

Bronchial Asthma:

It is an example of an atopic allergy (local anaphylaxis) involving the

lower respiratory tract. In bronchial asthma , the air sacs (alveoli) become over
distended and fill with fluid and mucus; the smooth muscle contacts and narrows the
walls of the bronchi, Bronchial constriction productes a wheezing or whistling sound
during exhalation. Relief is obtained from bronchodilators that help relax the
bronchial muscles and from expectorants and liquefacients that dissolve and expel
mucous plugs that accumulate.

Food Allergies:

Food such as eggs, peanuts, sea foods, citrus fruits and chocolate are
examples for food allergen. Allergens that enter the body through the digestive
system may cause food allergies. Hives (eruptions of the skin) are a good diagnostic
sign of a food allergy. Food allergies can be partially controlled with antihistamines or
avoiding food allergen.

Type II Hypersensitivity:

Type II hypersensivity is called a cytolytic or cytotoxic reaction because it results in

the destruction of host cells either by lysis or toxic mediators. In type II, IgG or IgM
are directed against cell surface or tissue associated antigens. Antibodies usually
stimulate the complement pathway and a variety of effector cells. The antibodies
interact with complement (C1q) and the effector cells through their Fc regions. The
damage mechanisms are a reflection of the normal physiological processes involved
in interaction of the immune system with pathogens. Example for Type II
hypersensivity is results when a person receives a transfusion with blood from a
donor with a different blood group.

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Haemolytic anemia:

Haemolytic anemia may be induced by administration of drugs. To treat hypertension,

drugs such a penicillin bind to the surface of red blood cell and form complex on the
surface of these cells. Complement-fixing antibodies are produced and react with the
RBC-bound drug activates the complement system, resulting RBC lysis and anemia.

Blood Transfusion:

When a person receives a transfusion with from a donor with a different

blood group, the antibodies present in recipient’s blood group, the antibodies present
in recipient’s serum agglutinate and lyse donor RBCs with liberation of free
haemoglobin into the plasma. This leads to jaundice, fever and kidney failure.

Type III hypersensivity:

Type III hypersensivity involves the formation of immune complexes

(Antigen-Antibody complexes). Normally, these complexes are removed effectively by
the fixed monocytes and macrophages. In the presence of excess amounts of some
soluble antigens, the antigen-antibody complexes may not be efficiently removed.
Accumulation of these complexes will activate complement that triggers a variety of
inflammatory processes. The inflammation causes damage, mainly of blood vessels,
kidney glomerular basement membranes (glomerulonephritis), joints (arthritis) and
skin.

Disease caused from type III reactions are come from three groups.

1. Persistent viral, bacterial or protozoan infection with a weak immune response,

leads to chronic immune complex formation and deposition of the complex in host
tissues.

2. during an autoimmune disease, the continued production of autoantibody to self

antigen lead to prolonged immune complex formation. This overloads the

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moncyte-macrophage system and tissue deposition of the complexes occur e.g. in the
disease of systemic lupus erythematous.

3. Immune complexes can form at body surfaces (lungs), due to rrpeated inhalation of
allergens from molds, plants or animals.

Example. Farmer’s lung disease, an individual repeatedly exposed to mold hay, as the
result circulating antibodies (IgG) produces. When the allergen (fungal spores) enters
the alveoli of the lungs, local immune complexes form, leading to inflammation.

Some group A Streptococal infections can produce an immunologically

mediated acute gllierulonephritis. Complexes of antibody and Streptococcal antigen
are deposited within the kidney glomeruli and generate a type III hypersensitivity
reaction.

Type IV Hypersensitivity:

Type IV hypersensivity involves delayed T-cell-mediated immune reactions. It is

also known as delayed hypersensitivity because T-cell appears in 24 – 48 hours after
the presensitized host encounters the antigen.

Type IV reactions occurs when antigens binding to tissue cells are

phagocytosed by macrophages and (antigen) then presented to receptors on the Th 1
cell surface in class I MHC. Contact between the antigen and Th 1 cell causes the cell
to proliferate and release cytokines. Cytokines attract lympocytes, macrophages and
basophils to the affected tissue. Extensive tissue damage may result. Examples of type
IV hypersensitivities include tuberculin hypersensitivity. (The TB skin test), allergic,
transplantation rejection and killing of cancer cells.

Tuberculin Hypersensitivity:

A partially purified protein called tuberculin, which is obtained from the cacillus that
causes tuberculosis. Tuberculin is injected into the skin of the forearm. The response
in a tuberculin-positive individual begins in about 8 hours and a reddened area
surrounding the injection site becomes indurated (firm and hard) within 12 to 24
hours. The Th1 cells that migrated into the injection site are responsibe for

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induration. The reaction reaches its peak in 48 hours and then subsides. The sixe of
the induration is directly relted to the amount of antigen that was introduced and to a
degree of hypersensitivity of the tested individual.

Other microbial products used in skin testing are histoplasmin for histopasmosis,
coccidioidin for Coccidioidomycosis, lepromin for leprosy and brucellergen for
brucellosis.

Allergic dermatitis:

Allergic contact dermatitis is caused by haptens that combine with proteins

in the skin to form the allergen that elicits the immune response. The haptens are the
antigenic deteminatnts, and the skin proteins are the carrier molecules for haptens.
Examples for hapten are cosmetics, plant material (poison), topical chemotherapeutic
agents, metals and jewelry.

Several important chronic diseases involve cell and tissue destruction by

type IV hypersensitivity reaction. In these chronic infections, macrophages and T cells
are continually stimulated. Example are leprosy, tuberculosis, leishmaniasis,
candidiasis, and herpes simplex infections.

Type V Hypersensitivity:

This is an antibody-mediated hypersensitivity. In this, antibodies react with key

surface components such as a hormone receptor and stimulate the cell. Example.
Graves’s disease, the thyroid stimulates autoantibody which activates thyroid
hormones.

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IgG (5 Mark)

Immunoglobulin G (IgG) is a type of antibody. Each IgG has two antigen binding sites.
Representing approximately 75% of serum antibodies in humans, IgG is the most
common type of antibody found in the circulation. IgG molecules are created and
released by plasma B cells. IgG is the main type of antibody found in blood
and extracellular fluid allowing it to control infection of body tissues. By binding many
kinds of pathogens such as viruses, bacteria, and fungi, IgG protects the body from
infection.
1. Immunoglobulin G is the most abundant immunoglobulin in the body.
2. It constitutes about 75% of the total Igs.
3. It is found in both intravascular and extravascular pools.
4. During primary response, low level of IgG produced but in the secondary response, it
is produce in high level.
5. It is a glycoprotein with molecular weight of 1, 50,000 daltons.
6. IgG present in normal serum is about 12 mg/ml.
7. IgG is the only class of Igs that can cross the placenta and responsible for infant
Protection. Breast milk also contains IgG along with IgA.

8. IgG has four subclasses named IgG 1, IgG 2, IgG 3 and Ig4. Each subclass possesses
distinct type of & chain.
9. IgG involves in Immunological reactions such as complement fixation, precipitation
and neutralization of toxins and viruses.

IgM (5 Mark)

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Immunoglobulin M, or IgM for short, is a basic antibody that is produced by B cells.

IgM is by far the physically largest antibody in the human circulatory system. It is the
first antibody to appear in response to initial exposure to an antigen. The spleen,
where plasmablasts responsible for antibody production reside, is the major site of
specific IgM production.
1. IgM is the largest antibody; it tends to remain in the blood and efficient in killing
bacteria.
2. IgM is a glycoprotein with molecular weight of about 9, 00,000 daltons.
3. Normal serum contains about 10% of IgM and its half life is about 5 days.
4. Its structure consists of pentamer with five immunoglobulins.
5. IgM found in serum but completely absent in body cavities or secretions.
6. During immune response, IgM is the first antibody to appear.
7. It is the first antibody to be synthesized by foetus by about 20 weeks of age.
8. IgM is usually short lived; hence their presence in the serum indicates recent
infection.

9. IgM may occur in monmeric form in high conantration.

10. In IgM, there is additional peptide called the joing (J) chain. J chain may be
responsible for the polymerization which occurs before the molecule is secreted by
plasma cell.
11. Generally, IgM can bind to antigen molecules but if antigens are large that bound to
one site, they prevent the binding of other antigen molecule to the adjacent site.

IgA (5 Mark)

1. Human IgA constitutes about 13% (2.1mg/ml) of the antibody in human serum.
2. The IgA is present in secretions (tears, saliva, nasal secretions, bronchial and digestive
tract mucus and mammary gland secretions) is secretory IgA.
3. Serum IgA has molecular weight of 1,60,000 daltons.
4. There are two classes of IgA: IgA 1 and IgA 2.

IgE (5 Mark)

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1. IgE is found in trace amounts in the blood.

2. It can triggers allergies.
3. Human IgE make up less than 0.003% (0.4µg/ml) of the antibody in serum.
4. It has molecular weight 1,90,000 daltons and half life is about two days.
5. It is heat labile and get inactivated by heating it at 56˚C for 30 minutes.
6. It is produced in the linings of the respiratory and intestinal tracts.
7. Increased level of IgE are seen in type I hypersensitivity.

IgD (5 Mark)

Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in

the plasma membranes of immature B-lymphocytes where it is usually coexpressed
with another cell surface antibody called IgM. IgD is also produced in a secreted form
that is found in very small amounts in blood serum, representing 0.25% of
immunoglobulins in serum. Relative molecular mass and half-life of sIgD is
185 kDa and 2.8 days, respectively. Secreted IgD is produced as
a monomeric antibody with two heavy chains of the delta (δ) class, and two Ig light
chains.
1. IgD is remain membrane bound and regulates the cell’s activation.
2. Its molecular weight is approximately 1, 75,000 daltons.
3. It is present on the surface of B lymphocytes.
4. IgD constitutes less than 1% (40µg/ml) of the antibody in human serum.

VDRL Test: (5 Mark)

VDRL is the simple and rapid test which requires only a small quantity
of serum. It is sensitive and specific test than other. IgM or IgG antibody present in
the patient serum caused a suspension of cardiolipin antigen to flocculate. This test is
done as a slide test in which inactivated patient serum is mixed with a freshly
prepared suspension of cardiolipin-lecithin-cholesterol antigen on a glass slide. The
mixture is rotated mechanically for 4 minutes after which the flocculation can be
detected under a low power objective microscope.

Procedure:

Preparation of antigen.

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1. Pipette 0.4 ml of buffered saline into a 30 ml round bottom screw cap bottle.

2. Add 0.5ml of VDRL antigen drop by drop while gently rotating bottle.

3. Add 4.1 ml of buffered saline more.

4. Replace the top of the bottle and all it to stand for 15-30 minutes.

Serum: Heat clear serum samples in a water bath at 56 degree celcius for 30 minutes.
Each 4 hours, the serum should be reheated for 10 minutes.

Qualitative test:

It is carried out on a glass slides.

1. Pipette 50 micro litre of inactivated patient serum into the paraffin ring on the glass
slide.

2. Pipette 50 micro litre each of positive and negative sera into other paraffin rings.

3. Add one drop of working antigen suspension to each of these paraffin rings.

4. Mix with wooden sticks and rotate slide for 4 minutes with hand on a flat surface in
a circular manner.

5. Observe the test results under a low power objective of a microscope.

Observation – the antigen particles are seen as small fusiform needles which remain
more or less evenly dispersed in case of non-reactive serum. In case of a weak
positive test, small clumps of antigen with little and in case of a positive test large
clumps of antigen are obtained. The control should be for validity of the results of the
tests. Specimen giving a weak positive or positive reaction should be tested
quantitatively. It is performed with serial dilutions of patient serum.

VDRL test is used as a screening test and positive resultys in

approximately 70% of primary and 99% of screening syphilis. Antibody becomes
detactable 7-10 days after the appearance of primary chancre. The titre of antibody
may rise to 8 or 16 during primary syphilis and 16-128 during secondary syphilis. False
negative reaction is possible due to prozone phenomenon when the patient serum

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contains high titre of antibody. VDRL test indicates active infection, therefore, it can
be used to monitor the efficacy of antibacterial therapy. The major disadvantages of
VDRL slide test are fresh antigen need to be prepared and use microscope to read the
results.

Tube test:

Flocculation test can be carried out in the tubes. Example is Kahn test for
syphilis and standardization of toxins and toxoids.

Widal test. (5 Mark)

It is an agglutination test which detects the presence of agglutinins in

patient serum against H and O suspensions of the enteric bacteria.e.g. Salmonella
typhi and S.typhi. Two types. Two types of antigens are used; they are flagellar (H)
antigen and somatic (O) antigen. H antigen is a formolised suspension of the
organisms which on combination with antibody, forms large, loose and fluffy clumps
resembles cotton wool. For H agglutination conical tuves are used. O antigen is
prepared by treating the bacterial suspension with alcohol. When combine with
antibody it forms fine granular deposit resembling chalk powder at the base of
round-bottomed (felix) tubes.

Principle:

Patient’s suffering from enteric fever would possess antibodies in therir sera which
can react and agglutinate serial doubling dilutions of killed, salmonella antigens in a
tube agglutination test.

Requirements:

Widal rack, round-bottomed Felix tubes, conical bottomed Dryer’s tubes, water bath,
doubly diluted patient serum, killed suspensions of S.typhi O antigen, S.typhi H
antigen, and S.paratyphi AH antigen.

Preparation of antigens.

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Salmonella typhi strain is used to prepare S.typhi O and S.typhi H antigens for
S. paratyphi A and S. paratyphi B are not taken as they cross react with S.typhi O
antigen. H antigen suspension is prepared by treating overnight broth culture.

Diluted serum = 0.1 ml saline + 0.9 ml of patient serum.

Patient serum is doubly diluted by mixing and transferring from 1: 10 to 1:640 in

three rows. First row usually compresies of Felix tubes , where somatic S.typhi O
antigen is added. For all the remaining rows, Dreyer’s tubes are taken; where
different flagellar antigen H antigens are added. Each tube must contain 0.5 ml of
diluted serum. A test tube with only saline is kept in each row as control. All the tubes
(including control) in a row are mixed with 0.5 ml of antigen suspension. The first row
is treated with S.typhi O antigen, the second row with S.typhi H antigen and the third
row with S. paratyphi AH antigen. The widal rack is placed in a water bath maintained
at 37 degre celcius for overnight incubation.

Results:

The control tubes must be examined first, where they should give no agglutination.
The agglutination of O antigen appears as matt or carpet at the bottom. Agglutination
of H antigens appear loose, wooly or cottony. The highest dilution of serum that
produces a positive agglutination is taken as titre.

Slide Widal test

This test is most popular and gives rapid results.

Qualitative test – one drop each of undiluted patient’s serum samples for the four
antigens are placed on the encircled card and one drop of each of the four Salmonella
antigens are added separately and gently rotate for one minute. Appearance of
agglutination gives qualitative results.

Interpretation of widal test:

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Timing is most important, as antibodies begin to arise during end of first week. The
titre increase during second, third and fourth week after which it gradually declines.
The test may be negative in early part of first week.

 Patients already treated with antibiotics may not show any rise in titre, instead there
may be fall in titre. Patients treated with antibiotics in the early stages may not give
positive results.
 Antigen suspensions with fimbrial antigens may sometimes give false positive
reactions due to sharing of fimbrial antigens by some Enterobacteriaceae members.
 Patients who have received vaccines against Salmonella give false positive reactions.
This can be differentiated from true infection by repeating test after a week. True
untreated infection results in rise in titre whereas vaccinated from individuals don’t
demonstrate any rise in titre.

Enzyme-linked Immunoassay (ELISA) (5 Mark)

Enzyme-linked immunoassay is an important method for detecting

and measuring antigens or antibodies. It looks similar like radioimmunoassay with
little difference. The difference is that for enzyme immunoassays the antigens or
antibody is conjugated to an enzyme rather than a radioactive isotope. The enzyme is
then detected by its ability to convert a colourless substance to a coloured one.
Enzyme immunoassays are highly sensitive, and easy to perform. Solid phase
immunoassays are most widely used are called enzyme linked immunosorbent assays
(ELISAs). The ELISA can be used to detect and determine concentrations of antigens
or antibody. The test done is polystyrene well (microtitre plate) or tube. Different
types of ELISA are available. Few types are presented here.

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Indirect ELISA:

Objective:

To detect the anti-HIV-1 and anti-HIV-2 antibodies in the patient serum.

Procedure:

1. The wells of the polystyrene microtitre plate are coated with purified HIV-1 and
HIV-2 antigens.

2. Diluted test serum or plasma sample is added to a well and incubated. If antibodies
specific for HIV-1 or HIV-2 are present in test sample they will form complexes with
antigens coated on the well.

3. Well is then washed and a conjugate of goat antihuman immunoglobulin

(antiglobulin), which has been labelled with the enzyme horse radish peroxidase is
added. If the antigen-antibody complex is present, the peroxidase conjugate will
bind to the complex and remains in the well. If the conjugate fraction remaining free
in the well is removed by washing.

4. Later, incubate in the presence of a colourless enzyme substrate (H2O2) and

chromogen. If anti-HIV-1 and anti-HIV-2 antibodies are present in the test sample,
then on incubation with enzyme substate produces a yellow-orange colour in the test

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well. If the sample contains no anti-HIV-1 and anti-HIV-2 then the antiglobulin tagged
enzyme cannot be found and no colour develops.

Results:

The test give yellow-orange colour in the well are termed as HIV-Positive and
if no colour found in the well are HIV-Negative.

Competitive ELISA:

Objective:

To detect the anti-HIV antibodies in the patient serum.

Procedure – The wells of the polystyrene microtitre plate are coated with HIV
antigens. The test sample and human anti-HIV, which has been labelled with the
enzyme horse-radish peroxidase, are incubated in a well incubated in a well. When
the sample contains no anti-HIV, solid-phase antigen-labelled antibody complex will
be formed. The incubation with enzyme substate produces a yellow-orange colour in
the test well. If anti-HIV is present in the test sample, it competes with the labelled
antibody for the available solid-phase antigen and no colour or reduced colour
develops.

Advantage – it takes less time to perform than indirect ELISA and no pre dilution of
test serum is required.

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