Immunology and Serology

1
st
Semester SY 2013-2014
MWF 10:30-11:30 am


Submitted by:



Submitted to:



2013


EXTRACELLULAR BACTERIA
Bacillus anthracis
B anthracis has a tendency to form very long chains of rods and in culture is nonmotile
and nonhemolytic; colonies are characterized by a rough, uneven surface with multiple curled
extensions at the edge resembling a "Medusa head." B anthracis has a D-glutamic
acid polypeptide capsule of a single antigenic type that has antiphagocytic properties. B
anthracis endospores are extremely hardy and have been shown to survive in the environment
for decades. The organism also produces a potent exotoxin complex, which consists of two
enzymes, edema factor (EF) and lethal factor (LF) together with a receptor recognition protein
called protective antigen (PA).
Anthrax may present in three different forms: inhalational, cutaneous, and GI.
Inhalational anthrax is extremely rare, and any case should raise the suspicion of a biological
attack. The specific mechanisms of immunity against B anthracis are not known. Experimental
evidence favors antibody directed against the toxin complex, but the relative role of the
components of the toxin is not clear. The capsular glutamic acid is immunogenic, but antibody
against it is not protective.
Smears show large, gram-positive rods in chains. Spores are usually not seen in smears of
exudate because spores form when nutrients are insufficient, and nutrients are plentiful in
infected tissue. Nonhemolytic colonies form on blood agar aerobically. In case of a bioterror
attack, rapid diagnosis can be performed in special laboratories using polymerase chain reaction
(PCR)based assays. Another rapid diagnostic procedure is the direct fluorescent antibody test
that detects antigens of the organism in the lesion. Serologic tests, such as an ELISA test for
antibodies, require acute and convalescent serum samples and can only be used to make a
diagnosis retrospectively.

Bordatella pertusis
B. pertussis is a small, coccobacillary, encapsulated gram-negative rod.
B. pertussis causes whooping cough (pertussis). B. pertussis, a pathogen only for humans, is
transmitted by airborne droplets produced during the severe coughing episodes. The organisms
attach to the ciliated epithelium of the upper respiratory tract but do not invade the underlying
tissue. Decreased cilia activity followed by death of the ciliated epithelial cells are important
aspects of pathogenesis.
The surface exhibits a rod-like protein called the filamentous hemagglutinin (FHA)
because of its ability to bind to and agglutinate erythrocytes. FHA has strong adherence qualities,
based on domains in its structure that interact with anamino acid sequence (arginine, glycine,
aspartic acid) present in host integrins, epithelial cells, and macrophages. In addition to its
adherence functions, FHA also stimulates cytokine release and interferes with T
H
1 immune
responses. The organism surface also contains other adhesive structures includingpili and an
outer membrane protein called pertactin.
A direct immunofluorescent antibody (DFA) technique has been successfully applied to
nasopharyngeal smears for rapid diagnosis of pertussis. DFA is particularly helpful
in pertussis because of the many days required for culture results. Because the sensitivity and
specificity of DFA can vary with the quality of the reagents, these results should always be
confirmed by culture, if possible. Nucleic acid amplification tests have been developed, which
show potential for being highly sensitive but are not yet practical for most laboratories. Serologic
tests are widely used for epidemiologic studies but not for diagnosis of individual clinical cases.

Clostridium tetani
C tetani is a slim, Gram-positive rod, which forms spores readily in nature and in culture,
yielding a round terminal spore that gives the organism a drumstick appearance. C
tetani requires strict anaerobic conditions. Its identity is suggested by cultural and biochemical
characteristics, but definite identification depends on demonstrating the neurotoxic exotoxin. C
tetani spores remain viable in soil for many years and are resistant to most disinfectants and to
boiling for several minutes. Spores germinate under anaerobic conditions in the wound.
Organism produces exotoxin, which blocks release of inhibitory neurotransmitters (glycine and
GABA) from spinal neurons. Excitatory neurons are unopposed, and extreme muscle spasm
(tetanus, spastic paralysis) results. "Lock-jaw" and "risus sardonicus" are two examples of the
muscle spasms.
C. tetani produces two exotoxins: tetanolysin and tetanospasmin. Tetanolysin, which is
related to the clostridial toxins and streptolysin, plays no role in the pathogenesis of the disease.
Tetanospasmin, generally referred to as "tetanus toxin," is the neurotoxin that causes the
manifestations of disease.
There is no microbiologic or serologic diagnosis. Organisms are rarely isolated from the
wound site. C. tetani produces a terminal spore (i.e., a spore at the end of the rod). This gives the
organism the characteristic appearance of a tennis racket."

Enterotoxigenic Escherichia coli
Most strains of E coli ferment lactose rapidly and produce indole. These and other
biochemical reactions are sufficient to separate it from the other species.
Important virulence factors characterizing ETEC are heat-labile toxin (LT) and a heat-
stable toxin (STa). LT closely resembles cholera toxin, stimulating cyclic adenosine
monophosphate production, resulting in intestinal crypt chloride and water secretion and
reduced sodium chloride absorption by enterocytes. STa stimulates cyclic guanosine
monophosphate production, which, much like LT, causes enhanced chloride and water secretion
and impaired sodium chloride absorption by the small intestine.
The major clinical manifestation is diarrhea, which ranges from just a few loose stools
lasting less than a day to severe, watery diarrhea that may persist for several days to a week and
result in significant dehydration. Fever is uncommon and, when present, low grade. Nausea and
abdominal cramps may accompany the diarrhea, but vomiting is uncommon.
Stool is used for the detection of toxin-producing E. coli; test performed by state public
health laboratories. Stool does not contain WBCs, mucus, or RBCs.

Haemophilus influenzae
H. influenzae are short (1.5 micrometers) coccoid bacilli, sometimes occurring in pairs or
short chains. In cultures, the morphology depends both on the length of incubation and on the
medium. At 68 hours in rich medium, the small coccobacillary forms predominate. Later there
are longer rods, lysed bacteria, and very pleomorphic forms.
H influenzae produces no exotoxin. The nonencapsulated organism is a regular member
of the normal respiratory microbiota of humans. The capsule is antiphagocytic in the absence of
specific anticapsular antibodies. The polyribose phosphate capsule of type b H influenzae is the
major virulence factor. Type bH influenzae causes meningitis, pneumonia and empyema,
epiglottitis, cellulitis, septic arthritis, and occasionally other forms of invasive infection.
Nontypeable H influenzae tends to cause chronic bronchitis, otitis media, sinusitis, and
conjunctivitis after breakdown of normal host defense mechanisms.
The diagnostic tests used for H. influenza are commercial kits that are available for
immunologic detection of H influenzae antigens in spinal fluid. A positive test result indicates
that the fluid contains high concentrations of specific polysaccharide from H influenzae type b.
Next is by culture where specimens are grown on IsoVitaleX-enriched chocolate agar until
typical colonies appear. H influenzae is differentiated from related gram-negative bacilli by its
requirements for X and V factors and by its lack of hemolysis on blood agar.

Pseudomonas aeruginosa
P aeruginosa is motile and rod shaped, measuring about 0.6 x 2micrometers. It is gram
negative and occurs as single bacteria, in pairs, and occasionally in short chains. P
aeruginosa grows well at 3742C; its growth at 42C helps differentiate it from
otherPseudomonas species in the fluorescent group. It is oxidase positive. It does not ferment
carbohydrates, but many strains oxidize glucose. Identification is usually based on colonial
morphology, oxidase positivity, the presence of characteristic pigments, and growth at 42C.
Differentiation of P aeruginosa from other pseudomonads on the basis of biochemical activity
requires testing with a large battery of substrates. The bacterium attaches to and colonizes the
mucous membranes or skin, invades locally, and produces systemic disease. These processes are
promoted by the pili, enzymes, and toxins. Lipopolysaccharide plays a direct role in causing
fever, shock, oliguria, leukocytosis and leukopenia, disseminated intravascular coagulation, and
adult respiratory distress syndrome.
Pili (fimbriae) extend from the cell surface and promote attachment to host epithelial
cells. The exopolysaccharide is responsible for the mucoid colonies seen in cultures from
patients with CF. The lipopolysaccharide, which exists in multiple immunotypes, is responsible
for many of the endotoxic properties of the organism. P aeruginosa can be typed by
lipopolysaccharide immunotype and by pyocin (bacteriocin) susceptibility. Most P
aeruginosa isolates from clinical infections produce extracellular enzymes, including elastases,
proteases, and two hemolysins (a heat-labile phospholipase C and a heat-stable glycolipid).
Many strains of P aeruginosa produce exotoxin A, which causes tissue necrosis and is
lethal for animals when injected in purified form. The toxin blocks protein synthesis by a
mechanism of action identical to that of diphtheria toxin, although the structures of the two
toxins are not identical. Antitoxins to exotoxin A are found in some human sera, including those
of patients who have recovered from serious P aeruginosa infections.
There are no specified serologic tests for P. aeruginosa but the most common diagnostic
tests performed are smears and culture. In smears, Gram-negative rods are often seen. No
specific morphologic characteristics differentiate pseudomonads in specimens from enteric or
other gram-negative rods; and in culture Specimens are plated on blood agar and the differential
media commonly used to grow the enteric gram-negative rods. Pseudomonads grow readily on
most of these media, but they may grow more slowly than the enterics. P aeruginosa does not
ferment lactose and is easily differentiated from the lactose-fermenting bacteria. Culture is the
specific test for diagnosis of P aeruginosa infection.

Staphylococcus aureus
Staphylococci are spherical gram-positive cocci arranged in irregular grapelike clusters.
All staphylococci produce catalase, whereas no streptococci do (catalase degrades H
2
O
2
into
O
2
and H
2
O). Catalase is an important virulence factor because H
2
O
2
is microbicidal and its
degradation limits the ability of neutrophils to kill. Coagulase is an enzyme that causes plasma
to clot by activating prothrombin to form thrombin. Thrombin then catalyzes the activation of
fibrinogen to form the fibrin clot.
Protein A is the major protein in the cell wall. It is an important virulence factor because
it binds to the Fc portion of IgG at the complement-binding site, thereby preventing the
activation of complement. As a consequence, no C3b is produced, and the opsonization and
phagocytosis of the organisms are greatly reduced. Protein A is used in certain tests in the
clinical laboratory because it binds to IgG and forms a coagglutinate" with antigenantibody
complexes. The coagulase-negative staphylococci do not produce protein A.
Teichoic acids are polymers of ribitol phosphate. They mediate adherence of the
staphylococci to mucosal cells. Lipoteichoic acids play a role in the induction of septic shock by
inducing cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) from
macrophages.
Polysaccharide capsule is also an important virulence factor. Surface receptors for
specific staphylococcal bacteriophages permit the phage typing" of strains for epidemiologic
purposes. Teichoic acids make up part of these receptors. The peptidoglycan of Sta. aureus has
endotoxin-like properties (i.e., it can stimulate macrophages to produce cytokines and can
activate the complement and coagulation cascades). This explains the ability of Sta. aureus to
cause the clinical findings of septic shock yet not possess endotoxin.
Laboratory diagnosis include smears from staphylococcal lesions that reveal gram-
positive cocci in grapelike clusters. Cultures of Sta. aureus typically yield golden-yellow
colonies that are usually beta hemolytic. Sta. aureusis coagulase-positive. Mannitol-salt agar is a
commonly used screening device for Sta. aureus. Cultures of coagulase-negative staphylococci
typically yield white colonies that are nonhemolytic.

Streptococcus pyogenes
Individual cocci are spherical or ovoid and are arranged in chains The cocci divide in a
plane perpendicular to the long axis of the chain. The members of the chain often have a striking
diplococcal appearance, and rodlike forms are occasionally seen. Streptococci are gram positive;
however, as a culture ages and the bacteria die, they lose their gram positivity and can appear to
be gram negative; for some streptococci, this can occur after overnight incubation.
M protein is a major virulence factor of S pyogenes. m protein appears as hairlike
projections of the streptococcal cell wall. unlike m protein, t substance is acid labile and heat
labile. It is obtained from streptococci by proteolytic digestion, which rapidly destroys m
proteins. t substance permits differentiation of certain types of streptococci by agglutination with
specific antisera, but other types share the same t substance. Yet another surface antigen has been
called r protein. Other antigenic structures include streptokinase (fibrinolysin), streptococcal
deoxyribonucleases a, b, c, and d degrade dna (DNAses), hyaluronidase, pyrogenic exotoxins
(erythrogenic toxin), and hemolysins such as streptolysin O and streptolysin S.
For the serologic tests, a rise in the titer of antibodies to many group A streptococcal
antigens can be estimated. Such antibodies include ASO, particularly in respiratory disease; anti-
DNase B and antihyaluronidase, particularly in skin infections; antistreptokinase; anti-M type-
specific antibodies; and others. Of these, the anti-ASO titer is most widely used.

Treponema pallidum
T pallidum are slender spirals measuring about 0.2 micrometers in width and 5
15 micrometers in length. The spiral coils are regularly spaced at a distance of 1 micrometers
from one another. The organisms are actively motile, rotating steadily around their endoflagella
even after attaching to cells by their tapered ends. The long axis of the spiral is ordinarily straight
but may sometimes bend so that the organism forms a complete circle for moments at a time,
returning then to its normal straight position.
The outer membrane surrounds the periplasmic space and the peptidoglycancytoplasmic
membrane complex. Membrane proteins are present that contain covalently bound lipids at their
amino terminals. The lipids appear to anchor the proteins to the cytoplasmic or outer membranes
and keep the proteins inaccessible to antibodies. The endoflagella are in the periplasmic space. T
pallidum subspecies pallidum has hyaluronidase that breaks down the hyaluronic acid in the
ground substance of tissue and presumably enhances the invasiveness of the organism. The
protein profiles of T pallidum (all the subspecies) are indistinguishable; more than 100 protein
antigens have been noted. The endoflagella are composed of three core proteins that are
homologous to other bacterial flagellin proteins plus an unrelated sheath protein. Cardiolipin is
an important component of the treponemal antigens.
Serologic tests for syphilis include either nontreponemal or treponemal antigens.
Nontreponemal tests: The antigens in these tests contain measured amounts of
cardiolipin, cholesterol, and purified lecithin in quantities sufficient to yield a standardized
amount of reactivity. Historically, the cardiolipin was extracted from beef heart or liver with
added lecithin and cholesterol to enhance reaction with syphilitic "reagin" antibodies. Reagin is a
mixture of IgM and IgG antibodies reactive with the cardiolipincholesterollecithin complex.
All of the tests are based on the fact that the particles of the lipid antigen remain dispersed in
normal serum but flocculate when combining with reagin. The VDRL and unheated serum
reagin (USR) tests require microscopic examination to detect flocculation. The rapid plasma
reagin (RPR) test and toluidine red unheated serum test (TRUST) have colored particles that
become caught in the mesh of the antigenantibody complex, allowing the tests to be read
without microscopic magnification. Results develop within a few minutes, particularly if the
suspension is agitated.
Treponemal Antibody Tests: The treponemal tests measure antibodies against T
pallidum antigens. The tests are used to determine if a positive result from a nontreponemal test
is truly positive or falsely positive. A positive result of a treponemal test on a serum specimen
that is also positive on a nontreponemal test is a strong indication of T pallidum infection. The
traditional treponemal tests are less useful as screening tests because once positive after initial
syphilitic infection the tests remain positive for life independent of therapy for syphilis. Serial
dilutions of serum are not done in the treponemal tests, and results are reported as reactive or
nonreactive (or occasionally inconclusive). T pallidumparticle agglutination (TP-PA), T
pallidum hemagglutination (TPHA), microhemagglutination T pallidum(MHA-TP) , and
fluorescent treponemal antibody absorbed (FTA-ABS) are examples of treponemal antibody
tests.

Vibrio cholera
Vibrios are curved, comma-shaped gram-negative rods. V. cholerae is divided into two
groups according to the nature of its O cell wall antigen. Members of the O1 group cause
epidemic disease, whereas non-O1 organisms either cause sporadic disease or are nonpathogens.
The O1 organisms have two biotypes, called El Tor and cholerae, and three serotypes, called
Ogawa, Inaba, and Hikojima. These features are used to characterize isolates in epidemiologic
investigations. Serogroup O139 organisms, which caused a major epidemic in 1992, are
identified by their reaction to antisera to the O139 polysaccharide antigens (O antigen).
V. cholerae is transmitted by fecal contamination of water and food, primarily from
human sources. Human carriers are frequently asymptomatic and include individuals who are
either in the incubation period or convalescing. The main animal reservoirs are marine shellfish,
such as shrimp and oysters. Ingestion of these without adequate cooking can transmit the disease.
Adherence to the cells of the brush border of the gut, which is a requirement for
colonization, is related to secretion of the bacterial enzyme mucinase, which dissolves the
protective glycoprotein coating over the intestinal cells. After adhering, the organism multiplies
and secretes an enterotoxin called choleragen (cholera toxin). This exotoxin can reproduce the
symptoms of cholera even in the absence of the Vibrio organisms.
For diagnosis of sporadic cases in this country, a culture of the diarrhea stool
containing V. choleraewill show colorless colonies on MacConkey's agar because lactose is
fermented slowly. The organism is oxidase-positive, which distinguishes it from members of the
Enterobacteriaceae. On TSI agar, an acid slant and an acid butt without gas or H
2
S are seen
because the organism ferments sucrose. A presumptive diagnosis of V. cholerae can be
confirmed by agglutination of the organism by polyvalent O1 or non-O1 antiserum. A
retrospective diagnosis can be made serologically by detecting a rise in antibody titer in acute-
and convalescent-phase sera.















INTRACELLULAR BACTERIA

Brucella spp (Brucella melitensis, Brucella sui, Brucella abortus, Brucella canis)
The appearance in young cultures varies from cocci to rods 1.2 microns in length, with
short coccobacillary forms predominating. They are gram-negative but often stain irregularly,
and they are aerobic, nonmotile, and non-spore-forming.
Brucella melitensis typically infects goats; Brucella suis, swine; Brucella abortus, cattle;
and Brucella canis, dogs. The pathogens can be transmitted to humans directly from diseased
animals or indirectly in food. They cause characteristic granulomas in the organs of the RES.
The primary clinical symptom is the undulant fever.
Serologic diagnosis is by means of pathogen identification or antibody assay using a
standardized agglutination reaction, blocking antibodies, and ELISA assay. To be reliable, serum
agglutination tests must be performed with standardized heat-killed, phenolized, smooth brucella
antigens. IgG agglutinin titers above 1:80 indicate active infection. Individuals injected with
cholera vaccine may develop agglutination titers to brucellae. If the serum agglutination test is
negative in patients with strong clinical evidence of brucella infection, tests must be made for the
presence of "blocking" antibodies. These can be detected by adding antihuman globulin to the
antigen-serum mixture. Brucellosis agglutinins are cross-reactive with tularemia agglutinins, and
tests for both diseases should be done on positive sera; usually, the titer for one disease will be
much higher than that for the other.
Blocking antidobies are IgA antibodies that interfere with agglutination by IgG and IgM
and cause a serologic test to be negative in low serum dilutions (prozone) although positive in
higher dilutions. These antibodies appear during the subacute stage of infection, tend to persist
for many years independently of activity of infection, and are detected by the Coombs
antiglobulin method.
IgG, IgA, and IgM antibodies may be detected using ELISA assays, which use
cytoplasmic proteins as antigens. These assays tend to be more sensitive and specific than the
agglutination test.

Chlamydia trachomatis
Obligate intracellular parasites. Not seen on Gram-stained smear. Exists as inactive
elementary body extracellularly and as metabolically active, dividing reticulate body
intracellularly. Habitat is the human genital tract and eyes. Transmission is by sexual contact and
during passage of neonate through birth canal. Transmission in trachoma is chiefly by hand-to-
eye contact.
The serologic test of choice is the microimmunofluorescence (MIF) test, in which high-
titer purified EBs mixed with embryonated chicken yolk sac material are affixed to a glass
microscope slide to which dilutions of sera are applied. After incubation and
washing, fluorescein-conjugated IgG or IgM antibody is applied. The test is read with an
epifluorescence microscope, with the highest dilution of serum producing visible fluorescence
designated as the titer. Although the complement fixation (CF) test can also be used, it employs
only lipopolysaccharide (LPS) as the antigen and therefore identifies the pathogen only to the
genus level. However, serologic testing is not recommended for diagnosis of uncomplicated
genital infections of the cervix, urethra, and lower genital tract or for C. trachomatis screening of
asymptomatic individuals.

Francisella tularensis
Francisella tularensis, the causative agent of tularemia, is a small, nonmotile, facultative
aerobic, intracellular gram-negative coccobacilli. Tularemia grows in aerobic environments.
Despite its inability to form spores, it is nonetheless quite hardy and able to persist for several
weeks in water, soil, vegetation, or in animal products.
The diagnosis of tularemia is most frequently confirmed by agglutination testing.
Microagglutination and tube agglutination are the techniques most commonly used to detect
antibody to F. tularensis. In the standard tube agglutination test, a single titer of greater than or
equal to 1:160 is interpreted as a presumptive positive result. A fourfold increase in titer between
paired serum samples collected 23 weeks apart is considered diagnostic. False-negative
serologic responses are obtained early in infection; up to 30% of patients infected for 3 weeks
have sera that test negative. Late in infection, titers into the thousands are common, and titers of
1:201:80 may persist for years. Enzyme-linked immunosorbent assays have proved useful for
the detection of both antibodies and antigens. Serologic tests are used to diagnose infections
by C. psittaci and C. pneumoniae but are rarely helpful in diagnosing disease caused by C.
trachomatis because the frequency of infection is so high that many people already have
antibodies.

Legionella pneumophila
Legionella pneumophila is a weakly staining gram-negative bacillus that causes Pontiac
fever (acute influenza-like illness) and legionnaires disease (a pneumonia that may progress to a
severe multisystem illness). It does not grow on routine bacteriologic culture media. There are at
least 6 serogroups of L. pneumophila and at least 22 species of Legionella. Indirect
immunofluorescent assays for L. pneumophila serogroup 1 (IgM and/or IgG) and serogroups 16
(IgM and/or IgG) are both available.
Legionella Antibody Test (Serum) provides only a retrospective laboratory diagnosis
because it generally takes more than 3 weeks to mount a detectable antibody response. More than
a fourfold rise in titer to >1:128 in specimens gathered more than 3 weeks apart indicates recent
infection. A single titer of >1:256 is considered diagnostic. About 5060% of cases of
legionellosis may have a positive direct fluorescent antibody test. Culture can have a sensitivity
of 50%. All three methods may increase sensitivity to 90%. This test is species-specific.
Polyvalent antiserum is needed to test for all serogroups and species. Urine Legionella antigen
testing, in adjunct to cultures, may provide a rapid turnaround for results. The urine antigen test
is very specific, but the sensitivity ranges from 70% to 90% because it detects primarily
serogroup 1 infections.

Listeria monocytogenes
L. monocytogenes is a small gram-positive rod arranged in V- or L-shaped formations
similar to corynebacteria. The organism exhibits an unusual tumbling movement that
distinguishes it from the corynebacteria, which are nonmotile. Colonies on a blood agar plate
produce a narrow zone of -hemolysis that resembles the hemolysis of some streptococci.
Listeria grows well at cold temperatures, so storage of contaminated food in the refrigerator can
increase the risk of gastroenteritis. This paradoxical growth in the cold is called cold
enhancement."
Serological tests for the detection of antibodies have not been traditionally used for the
diagnosis of listeriosis. A number of formats have been tried and they have all been found to be
largely unreliable, lacking sensitivity and specificity. Experimental serological assays based on
the detection of anti-listeriolysin O have been used in some epidemiological investigations and
as support for the diagnosis of culture-negative central nervous system infections.
Immunohistochemical detection of L. monocytogenes antigens is a useful tool for the diagnosis
of the encephalitic form of the disease.

Mycobacterium tuberculosis
Aerobic, acid-fast rods. High lipid content of cell wall, which prevents dyes used in Gram
stain from staining organism. Lipids include mycolic acids and wax D. Grows very slowly,
which requires that drugs be present for long periods (months). Produces catalase, which is
required to activate isoniazid to the active drug. Habitat is the human lungs. Transmission is via
respiratory droplets produced by coughing.
Sometimes the results of the tuberculin skin test are equivocal, particularly in persons
who have been vaccinated with BCG or who live in areas where NTM mycobacteria are highly
prevalent in the environment. In an effort to improve diagnostic accuracy, whole-blood gamma
interferon release assays (IGRAs) have been commercially developed. These assays are based on
the host's immune responses to specific M tuberculosis antigens ESAT-6 (early secretory
antigenic target-6) and CFP-10 (culture filtrate protein-10), which are absent from most NTM
mycobacteria and BCG. The tests detectinterferon-gamma that is released by sensitized CD4 T
cells in response to these antigens. Currently, two commercial assays are available in the United
States. The Quantiferon-Gold In-Tube test (QFT-GIT) is an enzyme-linked immunoassay
(ELISA) that detects interferon gamma in whole blood. The T-SPOT-TB (Oxford Immunotec,
Oxford, UK) is an ELISA ImmunoSpot assay that uses purified peripheral blood mononuclear
cells. Results for both tests are reported as positive, negative, or indeterminate. These assays are
still undergoing extensive evaluation.

Neisseria gonorrhoeae
Neisseria gonorrhoeae is a gram-negative, nonmotile, non-spore-forming organism that
grows singly and in pairs (i.e., as monococci and diplococci, respectively). Exclusively a human
pathogen, the gonococcus contains, on average, three genome copies per coccal unit; this
polyploidy permits a high level of antigenic variation and the survival of the organism in its host.
Gonococci, like all other Neisseria species, are oxidase positive. They are distinguished from
other neisseriae by their ability to grow on selective media and to utilize glucose but not maltose,
sucrose, or lactose.
Organism invades mucous membranes and causes inflammation. Endotoxin present but
weaker than that of meningococcus, so less severe disease when bacteremia occurs. No
exotoxins identified. IgA protease and pili are virulence factors. It causes gonorrhea, neonatal
conjunctivitis and pelvic inflammatory disease.
Gram-stained smear and culture. Organism visible intracellularly within neutrophils in
urethral exudate. Oxidase-positive colonies on Thayer-Martin medium. Gonococci do not
ferment maltose, whereas meningococci do. Attempts to develop a serologic test for gonorrhea
have 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. Serologic tests not
useful. All patients should have a serologic test for syphilis and should be offered HIV/AIDS
testing.
Rickettsia rickettsii
Rickettsiae are small, intracellular coccobacilli that often have a transverse septum between
two bacilli, reflecting division by binary fission. They commonly measure no more than 0.3 to
0.5 m. Rickettsiae are obligate intracellular parasites, because they are unable to produce
sufficient energy to replicate extracellularly. Rickettsiae divide by binary fission within the host
cell, in contrast to chlamydiae, which are also obligate intracellular parasites but replicate by a
distinctive intracellular cycle. R. rickettsii, possess antigens that cross-react with antigens of the
OX strains of Proteus vulgaris. The Weil-Felix test, which detects antirickettsial antibodies in a
patient's serum by agglutination of the Proteus organisms, is based on this cross-reaction.
The most common serologic test for confirmation of the diagnosis is the indirect
immunofluorescence assay. Not until 710 days after onset is a diagnostic titer of 1:64 usually
detectable. The sensitivity and specificity of the indirect immunofluorescence assay are 94
100% and 100%, respectively. It is important to understand that serologic tests for RMSF are
usually negative at the time of presentation for medical care and that treatment should not be
delayed while a positive serologic result is awaited.
The only diagnostic test that is useful during the acute illness is immunohistologic
examination of a cutaneousbiopsy sample from a rash lesion for R. rickettsii. Examination of a 3-
mm punch biopsy from such a lesion is 70% sensitive and 100% specific. The recent dramatic
increase in the reported incidence of RMSF correlates with the use of single-titer spotted fever
group cross-reactive enzyme immunoassay serology, with which few cases are specifically
determined to be caused by R. rickettsii.

Salmonella typhi
Salmonellae are gram-negative rods that do not ferment lactose but do produce H
2
S
features that are used in their laboratory identification. Their antigenscell wall O, flagellar H,
and capsular Vi (virulence)are important for taxonomic and epidemiologic purposes. The O
antigens, which are the outer polysaccharides of the cell wall, are used to subdivide the
salmonellae into groups AI. There are two forms of the H antigens, phases 1 and 2. Only one of
the two H proteins is synthesized at any one time, depending on which gene sequence is in the
correct alignment for transcription into mRNA. The Vi antigens (capsular polysaccharides) are
antiphagocytic and are an important virulence factor for Sal. typhi, the agent of typhoid fever.
The Vi antigens are also used for the serotyping of Sal. typhi in the clinical laboratory.
The Widal test is a presumptive serological test for enteric fever or undulant
fever whereby bacteria causing typhoid fever are mixed with serum containing specific
antibodies obtained from an infected individual. In case of Salmonella infections, it is a
demonstration of the presence of O-soma false-positive result. Test results need to be interpreted
carefully in the light of past history of enteric fever, typhoid vaccination, and the general level of
antibodies in the populations in endemic areas of the world. Typhidot is the other test used to
ascertain the diagnosis of typhoid fever. As with all serological tests, the rise in antibody levels
needed to perform the diagnosis takes 714 days, which limits it applicability in early diagnosis.
Other means of diagnosing Salmonella typhi (and paratyphi) include cultures of blood, urine
and faeces.

Shigella dysenteriae
Shigella dysenteriae is a nonspore-forming, gram-negative bacterium that, unlike E. coli,
is nonmotile and does not produce gas from sugars, decarboxylate lysine, or hydrolyze arginine.

Shigella can cause shigellosis (bacillary dysentery). Shigellae are Gram-negative, non-spore-
forming, facultatively anaerobic, non-motile bacteria. S. dysenteriae, spread by contaminated
water and food, causes the most severe dysentery because of its potent and deadly Shiga toxin,
but other species may also be dysentery agents. Shigella infection occurs essentially through oral
contamination via direct fecal-oral transmission, the organism being poorly adapted to survive in
the environment. Resistance to low-pH conditions allows shigellae to survive passage through
the gastric barrier, an ability that may explain in part why a small inoculum (as few as 100 CFU)
is sufficient to cause infection.
Normal persons often have agglutinins against several Shigella species. However, serial
determinations of antibody titers may show a rise in specific antibody. Serology is not used to
diagnose Shigella infections.

VIRUS
Herpes Simplex Virus Type 1
Enveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. No
virion polymerase. One serotype; cross-reaction with HSV-2 occurs. No herpes groupspecific
antigen.Commonly transmitted by saliva or direct contact with virus from the vesicle. It causes
herpeslabialis (fever blisters or cold sores), keratitis, encephalitis.
Initial vesicular lesions occur in the mouth or on the face. The virus then travels up the
axon and becomes latent in sensory (trigeminal) ganglia. Recurrences occur in skin innervated
by affected sensory nerve and are induced by fever, sunlight, stress, etc. Dissemination to
internal organs occurs in patients with depressed cell-mediated immunity with life-threatening
consequences. HSV-1 encephalitis often affects the temporal lobe.
Virus causes cytopathic effect (CPE) in cell culture. It is identified by antibody
neutralization or fluorescent antibody test. Tzanck smear of cells from the base of the vesicle
reveals multinucleated giant cells with intranuclear inclusions. These giant cells are not specific
for HSV-1; they are seen in the vesicular lesions caused by HSV-2 and varicella-zoster virus as
well. A rise in antibody titer can be used to diagnose a primary infection but not recurrences.
HSV encephalitis can be diagnosed using a PCR assay to detect HSV-1 DNA in spinal fluid.

Herpes Simplex Virus Type 2
Enveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. No
virion polymerase. One serotype; cross-reaction with HSV-1 occurs. No herpes groupspecific
antigen.Transmission is by sexual contact in adults and during passage through the birth canal in
neonates. Diseases associated include Herpes genitalis, aseptic meningitis, and neonatal
infection.
Initial vesicular lesions occur on genitals. The virus then travels up the axon and becomes
latent in sensory (lumbar or sacral) ganglion cells. Recurrences are less severe than the primary
infection. HSV-2 infections in neonate can be life-threatening because neonates have reduced
cell-mediated immunity. Asymptomatic shedding of HSV-2 in the female genital tract is an
important contributing factor to neonatal infections.
Virus causes CPE in cell culture. Identify by antibody neutralization or fluorescent
antibody test. Tzanck smear reveals multinucleated giant cells but is not specific for HSV-2. A
rise in antibody titer can be used to diagnose a primary infection but not recurrences.

Varicella-Zoster Virus
Enveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. No
virion polymerase. One serotype.Varicella is transmitted primarily by respiratory droplets.
Zoster is not transmitted; it is caused by a reactivation of latent virus. It causes varicella
(chickenpox) in children and zoster (shingles) in adults.
Initial infection is in the oropharynx. It spreads via the blood to the internal organs such
as the liver and then to the skin. After the acute episode of varicella, the virus remains latent in
the sensory ganglia and can reactivate to cause zoster years later, especially in older and
immunocompromised individuals.
Virus causes CPE in cell culture and can be identified by fluorescent antibody test.
Multinucleated giant cells seen in smears from the base of the vesicle.Intranuclearinclusions seen
in infected cells. A four-fold or greater rise in antibody titer in convalescent-phase serum is
diagnostic.



Cytomegalovirus
Enveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. No
virion polymerase. One serotype.Virus is found in many human body fluids, including blood,
saliva, semen, cervical mucus, breast milk, and urine. It is transmitted via these fluids, across the
placenta, or by organ transplantation. It is the most common cause of congenital abnormalities
in the United States, cytomegalic inclusion body disease in infants, mononucleosis in transfusion
recipients, pneumonia and hepatitis in immunocompromised patients, retinitis and enteritis,
especially in AIDS patients.
Initial infection usually in the oropharynx. In fetal infections, the virus spreads to many
organs (e.g., central nervous system and kidneys). In adults, lymphocytes are frequently
involved. A latent state occurs in monocytes. Disseminated infection in immunocompromised
patients can result from either a primary infection or reactivation of a latent infection.
The virus causes CPE in cell culture and can be identified by fluorescent antibody test.
"Owl's eye" nuclear inclusions are seen. A four-fold or greater rise in antibody titer in
convalescent-phase serum is diagnostic.

Epstein-Barr Virus
Enveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. No
virion polymerase. One serotype.Virus found in human oropharynx and B lymphocytes. It is
transmitted primarily by saliva. It causes infectious mononucleosis and it is associated with
Burkitt's lymphoma in East African children.
Infection begins in the pharyngeal epithelium, spreads to the cervical lymph nodes, then
travels via the blood to the liver and spleen. EBV establishes latency in B lymphocytes.
The virus is rarely isolated. Lymphocytosis, including atypical lymphocytes, occurs.
Heterophil antibody is typically positive (Monospot test). Heterophil antibody agglutinates sheep
or horse red blood cells. A significant rise in EBV-specific antibody to viral capsid antigen is
diagnostic.
Human Papillomavirus
Nonenveloped virus with icosahedral nucleocapsid and circular double-stranded DNA.
No virion polymerase. There are at least 60 types, which are determined by DNA sequence not
by antigenicity. Many types infect the epithelium and cause papillomas at specific body sites.
Transmission is by direct contact of skin or genital lesions. Diseases include papillomas (warts);
condylomataacuminata (genital warts); associated with carcinoma of the cervix and penis.
Two early viral genes, E6 and E7, encode proteins that inhibit the activity of proteins
encoded by tumor suppressor genes (e.g., the p53 gene and the retinoblastoma gene,
respectively).
Diagnosis is made clinically by finding koilocytes in the lesions. DNA hybridization tests
are available. Virus isolation and serologic tests are not done.

Influenza Virus
Enveloped virus with a helical nucleocapsid and segmented, single-stranded RNA of
negative polarity. RNA polymerase in virion. The two major antigens are the hemagglutinin
(HA) and the neuraminidase (NA) on separate surface spikes. Antigenic shift in these proteins as
a result of reassortment of RNA segments accounts for the epidemics of influenza caused by
influenza A virus. Influenza A viruses of animals are the source of the new RNA segments.
Antigenic drift due to mutations also contributes. The virus has many serotypes because of these
antigenic shifts and drifts. The antigenicity of the internal nucleocapsid protein determines
whether the virus is an A, B, or C influenza virus.Transmitted by respiratory droplets from
human to human. H5N1 strains transmitted from birds to humans.
It causes influenza. Influenza A virus is the main cause of worldwide epidemics
(pandemics). A pandemic caused by a swine-origin strain of H1N1 influenza A virus began in
2009. Infection is limited primarily to the epithelium of the respiratory tract.
A rapid ELISA test to detect influenza viral antigen in respiratory secretions is often
used. Virus grows in cell culture and embryonated eggs and can be detected by hemadsorption or
hemagglutination. It is identified by hemagglutination inhibition or complement fixation. A four-
fold or greater antibody titer rise in convalescent-phase serum is diagnostic.

Measles Virus
Enveloped virus with a helical nucleocapsid and one piece of single-stranded, negative-
polarity RNA. RNA polymerase in virion. It has a single serotype. Transmission is by serologic
droplets. It causes measles. Subacutesclerosingpanencephalitis is a rare late complication.
Initial site of infection is the upper respiratory tract. Virus spreads to local lymph nodes
and then via the blood to other organs, including the skin. Giant cell pneumonia and encephalitis
can occur. The maculopapular rash is due to cell-mediated immune attack by cytotoxic T cells on
virus-infected vascular endothelial cells in the skin.
The virus is rarely isolated. Serologic tests are used if necessary. PCR assay is available.
Mumps Virus
Enveloped virus with a helical nucleocapsid and one piece of single-stranded, negative-
polarity RNA. RNA polymerase in virion. It has a single serotype. Transmission is by respiratory
doplets. Sterility due to bilateral orchitis is a rare complication.
The initial site of infection is the upper respiratory tract. The virus spreads to local lymph
nodes and then via the bloodstream to other organs, especially the parotid glands, testes, ovaries,
meninges, and pancreas.
The virus can be isolated in cell culture and detected by hemadsorption. Diagnosis can
also be made serologically. PCR assay is available.

Rubella Virus
Enveloped virus with an icosahedral nucleocapsid and one piece of single-stranded
positive-polarity RNA. No polymerase in virion. It has a single serotype. Transmission is by
respiratory droplets and across the placenta from mother to fetus. It causes Rubella. Congenital
rubella syndrome is characterized by congenital malformations, especially affecting the
cardiovascular and central nervous systems, and by prolonged virus excretion. The incidence of
congenital rubella has been greatly reduced by the widespread use of the vaccine.
The initial site of infection is the nasopharynx, from which it spreads to local lymph
nodes. It then disseminates to the skin via the bloodstream. The rash is attributed to both viral
replication and immune injury. During maternal infection, the virus replicates in the placenta and
then spreads to fetal tissue. If infection occurs during the first trimester, a high frequency of
congenital malformations occurs. Maternal antibody protects against fetal infection.
Virus is detected by PCR assay. To determine whether an adult woman is immune, a
single serum specimen to detect IgG antibody in the hemagglutination inhibition test is used. To
detect whether recent infection has occurred, either a single serum specimen for IgM antibody or
a set of acute-and convalescent-phase sera for IgG antibody can be used.

Poliovirus
Naked nucleocapsid with single-stranded, positive-polarity RNA. Genome RNA acts as
mRNA and is translated into one large polypeptide, which is cleaved by virusencoded protease
to form functional viral proteins. No virion polymerase. There are three serotypes. Transmission
is by Fecaloral route. Humans are the natural reservoir. It causes paralytic poliomyelitis and
aseptic meningitis. Poliomyelitis has been eradicated in the western hemisphere and in many
other countries.
The virus replicates in the pharynx and the GI tract. It can spread to the local lymph
nodes and then through the bloodstream to the central nervous system. Most infections are
asymptomatic or very mild. Aseptic meningitis is more frequent than paralytic polio. Paralysis is
the result of death of motor neurons, especially anterior horn cells in the spinal cord.
Pathogenesis of postpolio syndrome is unknown.
Recovery of the virus from spinal fluid indicates infection of the central nervous system.
Isolation of the virus from stools indicates infection but not necessarily disease. It can be found
in the GI tract of asymptomatic carriers. The virus can be detected in cell culture by CPE and
identified by neutralization with type-specific antiserum. A significant rise in antibody titer in
convalescent-phase serum is also diagnostic.
Parainfluenza Virus
Enveloped virus with helical nucleocapsid and one piece of single-stranded, negative-
polarity RNA. RNA polymerase in virion. Unlike influenza viruses, the antigenicity of its
hemagglutinin and neuraminidase is stable. There are four serotypes. Transmission is by
respiratory droplets. It causes bronchiolitis in infants, croup in young children, and the common
cold in adults. Infection and death of respiratory epithelium without systemic spread of the virus.
Isolation of the virus in cell culture is detected by hemadsorption. Immunofluorescence is
used for identification. A four-fold or greater rise in antibody titer can also be used for diagnosis.
PCR assay is available.







PARASITES

Entamoebahistolytica
It utilizes a number of strategies to circumvent the immune defenses of the host. It resists
complement-mediated lysis during hematogenous spread by proteolytic degradation of C3 and
C5. In addition, the Gal/GalNaclectin binds to C8 and C9, preventing assembly of the C5b-9
membrane attack complex. Cytolysis by E. histolytica may occur via necrosis and apoptosis; the
use of the host's apoptotic machinery abrogates the local inflammatory response. Trophozoites
also inhibit the macrophage respiratory burst and the production of IL-1 and TNF-. A protective
antibody response is subverted by the degradation of IgA and IgG by amebic cysteine proteases,
and by capping, ingesting or shedding ameba-specific antibodies. E. histolyticais the causative
agent of Amebiasis.
Serologic testing uses enzyme immunoassay, DFA (Direct fluorescent antibody) wherein
monoclonal antibody labelled with fluorescein isothiocyanate is used against the parasites cell
wall to visualize the initial antibody-parasite complex.Complement fixation and
immunodifussion test are also used.Normal Range is titer-1:8

Strongyloidesstercoralis
It isan intestinal nematode of humans. It is estimated that tens of millions of persons are
infected worldwide, although no precise estimate is available. Although most infected
individuals are asymptomatic, S. stercoralis is capable of transforming into a fulminant fatal
illness under certain conditions associated with a compromise of host immunity. Such conditions
have commonly been summarized as defects in cell-mediated immunity, although the specific
circumstances under which S. stercoralis hyperinfection develops are not always predictable. It
causes Strongyloidiasis.
Immunodiagnostic tests for strongyloidiasis are indicated when the infection is suspected
and the organism cannot be demonstrated by duodenal aspiration, string tests, or by repeated
examinations of stool. Antibody detection tests should use antigens derived
from Strongyloidesstercoralis filariform larvae for the highest sensitivity and specificity.
Although indirect fluorescent antibody (IFA) and indirect hemagglutination (IHA) tests have
been used, enzyme immunoassay (EIA) is currently recommended because of its greater
sensitivity (90%). Immunocompromised persons with disseminated strongyloidiasis usually
have detectable IgG antibodies despite their immunodepression. Cross-reactions in patients with
filariasis and some other nematode infections may occur. Antibody test results cannot be used to
differentiate between past and current infection. A positive test warrants continuing efforts to
establish a parasitological diagnosis followed by antihelminthic treatment. Serologic monitoring
may be useful in the follow-up of immunocompetent treated patients: antibody levels decrease
markedly within 6 months after successful chemotherapy.

Toxoplasma gondii
It is a ubiquitous protozoan parasite that infects humans by ingestion of infective cysts
and transferred by hand-to-mouth contact or through blood transfusion. Cat is the definitive host
of the parasite. It is the causative agent of Toxoplasmosis. This disease is may be present with a
mild lymphadenopathy. Toxoplasma species rarely reaches the CNS but it can cross the placenta.
It is capable of replicating inside the macrophages and prevent the fusion of lysosome and
phagosomes. T. gondii is a potent activator of CD4 and CD8 T cells. Infection with T.
gondii induces IL-12-dependent activation of NK cells which when activated produces IFN-,
that activates macrophages to limit parasite replication.
Isolation of the parasites requires tissue biopsy. In seroconversion to an antibody-positive
state, a fourfold increase in the titer of IgG, or high levels of IgM and IgG antibodies are
considered diagnostic. Concurrent elevations of IgM and IgA antibodies indicate early infection
and used to screen pregnant women.
Enzyme immunoassays (EIA) for IgM, IgG or IgA and indirect fluorescent
antibody(IFA) are performed when there is congenital toxoplasmosis. Elevated titer of IgG, IgM,
or IgA antibody classes indicates that infection has occurred within the previous 3 to 9 months.
Elevated IgGtiters without IgM antibody suggests older infection.

Trichinella spiralis
It possess a capacity to retune the immune cell repertoire, acting as a moderator of the
host response not only to itself but also to third party antigens. T. spiralis-stimulated dendritic
cells has the ability to polarize the immune response toward Th2 and regulatory mode in vitro
and in vivo and also on the capacity of this parasite to modulate autoimmune disease. The
causative agent of Trichinellosis (Trichinosis)
Trichinella antibody testing by ELISA or IFA is detectable 2-4 weeks after infection
(may be >1,000/mL), Cross reacts with other parasitic infections and uncommonly, positive in
early disease

Trypanosomacruzi
A bloodstream trypomastigotes resist complement-mediated lysis because the parasite
hasa complement regulatory protein (GP-160), which is functionally similar to mammalian
decay-accelerating factor in that it inhibits C3 convertase formation and activation of the
alternate complement pathway. It causes Chagas disease.
Serologic testing includes IFA (Indirect fluorescent antibody). IFA was assayed using
fixed epimastigotes and anti-human immunoglobulin G-fluorescein conjugate. Specimens were
considered reactive when fluorescence was observed at a 1:20 or higher dilution.IHA (indirect
hemagglutination assay) was conducted using two commercially available kits. For the former
test kit, IHA was performed with specimens treated with 2-mercaptoethanol (2-ME) at a dilution
of 1:40 according to the manufacturer's instructions. The latter test was performed according to
the manufacturer's instructions, including the absence of 2-ME treatment. Both assays were read
and interpreted manually. In ELISA (enzyme-linked immunosorbent assay), four commercially
available enzyme-linked immunosorbent assay (ELISA) kits for detection of antibodies to T.
cruzi were used. In RIPA testing all specimens were assayed in parallel with three negative- and
three positive-control specimens, the latter obtained from parasitologically confirmed cases of
Chagas' disease. Diagnostic confirmation of reactivity by RIPA was defined as the presence of
bands in autoradiographs indicative of antibodies specific for the 72- and 90-kDa glycoproteins
of T. cruzi.



















FUNGI

Aspergillus
It is a filamentous, cosmopolitan and ubiquitous fungus found in nature that causes
Aspergillosis. It is commonly isolated from soil, plant debris, and indoor air environment. While
a teleomorphic state has been described only for some of the Aspergillus spp., others are
accepted to be mitosporic, without any known sexual spore production.
For serologic testing of Aspergillus,Immunodiffusion (ID) and Counterimmuno-
electrophoresis Tests (CIE) are used. The presence of one or more lines of serum precipitins is
suggestive but not conclusive evidence of an active infection. Three or more lines are associated
with aspergilloma or with invasive aspergillosis if the patient is not anergic. For test result to be
valid, the Aspergillus reference antiserum must demonstrate three or more bands with
Aspergillus reference antigen. Nonspecific banding can be caused by C-reactive protein. The
immunodiffusion tests are positive only in immunocompetent patients. Another test is Enzyme
immunoassay which is developed to detect serum galactomannan antigen of Aspergillus. Results
are available in about 3 hours rather than the usual 4 weeks required for culture. Invasive
aspergillosis has a mortality rate of 50-100 percent. Earlier identification of the organism
expedites the administration of antifungal drugs and reduces mortality.

Candida albicans
It is yeast found primarily in mucosal tissues such as the oral cavity and female genital
tract, colonizing these regions by binding to glycoproteins present in the secretions of mucus
membranes, or on the epithelial cell surface. It may invade the underlying basement membrane
by forming budding protrusions termed hyphae.Conditions leading to the loss of epithelial cells
allowsCandida albicans to bind to the glycoproteins in the basement membrane and invade the
tissues below the basement membrane by forming hyphae. It causes Candidiasis.
Immunodiffusion (ID) and Counterimmunoelectrophoresis Tests (CIE) detect antibodies
in systemic candidiasis in immunocompetent hosts. A heat-stable cytoplasmic antigen is used
with positive-control sera containing at least three of the seven known precipitins. Formation of
one or more bands between the ragnet antigen and patients serum is considered a positive
reaction. In Latex agglutination (LA), latex particles sensitized with a homogenate antigen of C.
albicans are reacted with patient sera and control sera. When this screening test is positive, a
serial dilution is performed and reported as the highest dilution giving a 2+ reaction. A titer of
1:4 suggests an early infection, colonization, or a nonspecific reaction. A titer of 1:8 or greater,
conversion from a negative to a positive test, or a fourfold increase in titer is presumptive
evidence of invasive infection. A fourfold decrease in titer may indicate successful therapy.


Coccidioidesimmitis
Its cell wall is rich in chitin and chitin metabolism is a reasonable target for the design of
antifungal agents. The antigenic composition of an alkali-soluble, water-soluble cell wall extract
of Coccidioidesimmitis is heterogeneous in composition,containing two distinct antigenic
components. One is present as a polymer that is antigenically identical to a polymeric antigen in
coccidioidin, designated antigen 2. The other component presented an unusual precipitin pattern
in that a cathodal leg was demonstrable in the absence of an anodal leg. The fungal cell wall also
contains mannoproteins and glucans. Any disruption in its integrity should affect growth. The
cell wall provides a unique therapeutic opportunity for antifungal agents by targeting a structure
not found in mammalian cells. The echinocandins are cyclic hexapeptides, members of a new
class of antifungal agents. They appear to inhibit the synthesis of 1, 3--d-glucan, a major cell
wall component which provides structural integrity and osmotic stability in most pathogenic
fungi. It causes Coccidioidomycosis.
Complement-fixing antibodies of the IgG class develop in 3-6 months after onset of
symptoms. Titers 1:2 to 1:4 are presumptive evidence of an early infection. Titer 1:16 is
indicative of an active infection. Titers greater than 1:16 occur with disseminated
coccidioidomycosis.Tube Precipitation (TP) Testpositive result can be an early indication of a
primary infection because precipitating IgM antibodies appear in 1 to 3 weeks after infection.
InImmunodiffusion, when diffusion bands form lines of identity to reference antisera, the bands
represent the reaction of coccidioidin antigen and patient antibody. Single bands indicate chronic
infection. Two or more bands indicate active disease.Another test isLatex Agglutinationwherein
latex particles sensitized with coccidioidin are reacted with inactivated patient serum to detect
antibodies to the organism. The LA test is positive early in the course of the disease, but as many
as 10 percent of the cases of coccidioidomycosis confirmed by culture or serology give false-
negative results with an LA test. Because false-positive results are common, a CF test or an ID
test should be performed for confirmation when LA screening tests are positive.19,22,29Enzyme
ImmunoassayIgG and IgM antibodies are available for use with serum or CSF. Positive EIA tests
should be confirmed with CF or TP tests, because the EIA test is not absolutely specific.

Cryptococcus neoformans
Has the presence of a polysaccharide capsule surrounding the organism protects it from
phagocytosis. Opsoninmediataed phagocytosis occurs when IgG antibodies are secreted
following B-cell differentiation to antibody secreting plasma cells. B-cell activation leading to
isotype switching requires cytokines. Low endogenous levels of cytokines probably serve to
promote sufficient IgG production for opsonisation of the organism. Following sequestration in
macrophages, other CD4 derived cytokines are required for optimal activation of intracellular
killing mechanisms. It causes Cryptococcosis
In Latex Particle Agglutination (LPA) Antigen Test, inactivated serum or spinal fluid and
positive and negativehuman reference sera are each mixed with latex particlessensitized with
rabbit anticryptococcus globulin in rings ona test slide. The test is read macroscopically for
agglutination.To be valid, the negative control must be negative, andthe positive control must
show 2_ agglutination. The presenceof any agglutination when assaying a patient specimenis
considered a positive test if controls are acceptable. Resultsmay be quantitated by performing a
serial dilution. The titeris reported as the highest dilution showing 2_ agglutination.A titer of 1:2
suggests infection, but a titer of 1:4 orgreater is evidence of an active infection. Higher titers
correlatewith the severity of infection. Positive titers are found in CSF. For Enzyme-Linked
Immunoassay, it is available to detect antigens of C. neoformansin both serum and CSF. The
EIA method for antigen detectionis more sensitive than the latex agglutination procedurebut
takes more time to perform. In Tube Agglutination (TA) Test, serum or CSF from patients with
suspectedC. neoformansinfection is heat inactivated to destroy complement. The serum or spinal
fluid, an anticryptococcuspositive control, and a negative control of rabbit serum areeach mixed
in test tubes with a Cryptococcus antigen suspensionof weakly encapsulated yeast cells. The
tubes areincubated and then refrigerated overnight. Reactions areread for agglutination. The
positive control must read 3_ to4_ in order for the test to be valid. Tests with reactionsgreater
than the negative control are considered positive.Indirect Fluorescent Antibody Testarealso
available for thedetection of antibodies to C. neoformans. A positive test suggests a recent or
current infectionwith C. neoformansor a cross-reaction with another fungus.IFA tests have a
specificity of 77 percent and a sensitivity of50 percent. Both false-negative and false-positive
resultsmay occur.

Histoplasmacapsulatum strains
They were classified into two types based on the polysaccharide composition of the cell
walls. G217B belongs to type I, and it lacks -(1,3)-glucan in its cell wall, while G186A belongs
to type II because it has large amounts of -(1,3)-glucan in its cell wall. This was interesting
because for G186A variants that lack -(1,3)-glucan are avirulent whereas G217B contains no -
(1,3)-glucan but is fully virulent. Therefore, the relationship between -(1,3)-glucan and the H.
capsulatum pathogenesis is relatable.It causes Histoplasmosis or Darlings disease
Immunodiffusion (ID) test is a qualitatively measures precipitating antibodies (H and M
precipitin lines or bands). ID is highly specific for the detection of anti-M and anti-H antibodies
but shows low sensitivity principally in acute manifestation of the disease. In general, ID is
useful for detecting antibodies 46 weeks after infection. While the ID test is more specific for
histoplasmosis than the complement-fixation test, it is less sensitive. The H band appears after
the M band and can be found in sera from patients during acute and/or progressive disease.
However, this band is present in only 7% of serum from patient with acute infection. Antibodies
to the H antigen may be detected 12 years after the resolution of the disease, but antibodies to
the H antigen usually disappear more quickly than the anti-M antibodies. Complement-fixation
(CF) test measures antibodies to either the intact yeast form or mycelial (HMIN) antigen.
Complement-fixing antibodies may appear 3 to 6 weeks (sometimes as early as 2 weeks)
following infection by H. capsulatum in 95% of the patients with histoplasmosis, and repeated
tests will give positive results for months. Titers between 1:8 and 1:16 are considered weakly
positive and are observed in almost one quarter of patients. However, these titers may represent
past infection and can also be detected in the serum of healthy persons from regions where
histoplasmosis is endemic. A high titer (1:32 or greater) or a fourfold increase in titer over time
is indicative of active histoplasmosis. Antibody titers will gradually decline and eventually
disappear months to years following recovery in the absence of re-exposure, but persist for years
in individuals with recurrent exposures.Enzyme-linked immunosorbent assays (ELISA)
methodology has been evaluated and validated as a specific and sensitive immunoassay for the
detection of the antibodies in sera from patients with all the clinical manifestations of
histoplasmosis.


PRIONS

Prions are protein-containing particles with no detectable nucleic acid that are highly
resistant to inactivation by heat, formaldehyde, and ultraviolet light at doses that will inactivate
viruses. Note that prions are resistant to the temperatures usually employed in cooking, a fact
that may be important in their suspected ability to be transmitted by food. Prions are, however,
inactivated by protein- and lipid-disrupting agents such as phenol, ether, NaOH, and
hypochlorite .
The prion protein is encoded by a normal cellular gene and is thought to function in a
signal transduction pathway in neurons. The normal prion protein (known as PRPC, or prion
protein cellular) has a significant amount of alpha-helical conformation. When the alpha-helical
conformation changes to a beta-pleated sheet (known as PrPSC, or prion protein scrapie), these
abnormal forms aggregate into filaments, which disrupt neuron function and cause cell
death. Prions, therefore, reproduce" by the abnormal beta-pleated sheet form recruiting normal
alpha-helical forms to change their conformation. Note that the normal alpha-helical form and
the abnormal beta-pleated sheet form have the same amino acid sequence. It is only their
conformation that differs. A specific cellular RNA enhances this conformational change.
Prion diseases are also referred to as transmissible spongiform encephalopathies (TSE).
They occur in humans and animals, primarily affecting the central nervous system. They can be
sporadic (spontaneous), familial (genetic/inherited) or acquired (transmitted by infection). The
hallmark of these diseases is the presence of microscopic vacuolization of the brain tissue, called
spongiform degeneration (meaning that the tissue deteriorates, developing a spongy texture), and
an abnormal protein, called scrapie prion protein (PrP
Sc
), prion or abnormal prion protein. The
PrP
Sc
, unlike other known infectious diseases, is believed to result from a change in the
conformation or shape of a normal protein called cellular prion protein (PrP
C
), which is present
in large amounts in the brain as well as in other tissues. Since the abnormal prion protein cannot
be broken down through the bodys normal process, it aggregates mostly in the brain causing
degeneration and disease. The abnormal prion protein is often infectious and, under certain
conditions, can transmit the disease. Currently, there are no cures for prion diseases. The average
world-wide occurrence of prion diseases is approximately one case per million people per year.

Classification of Human Prion Diseases
FORM PHENOTYPE (Clinical and Pathological Features)
Sporadic Sporadic Creutzfeldt-Jakob Disease (sCJD)
Sporadic Familial Insomnia (sFI)
Familial Familial Creutzfeldt-Jakob Disease (fCJD)
Fatal Familial Insomnia (FFI)
Gerstmann-Strussler-Scheinker Syndrome (GSS)
Acquired Iatrogenic Creutzfeldt-Jakob Disease (iCJD)
variant Creutzfeldt-Jakob Disease (vCJD)
Kuru
Four new concepts have emerged from studies of prions:
(1) Prions are the only known infectious pathogens that are devoid of
nucleic acid; all other infectious agents possess genomes composed of either RNA
or DNA that direct the synthesis of their progeny.
(2) Prion diseases may be manifest as infectious, genetic, and sporadic
disorders; no other group of illnesses with a single etiology presents with such a
wide spectrum of clinical manifestations.
(3) Prion diseases result from the accumulation of PrP
Sc
, the conformation
of which differs substantially from that of its precursor, PrP
C
.
(4) PrP
Sc
can exist in a variety of different conformations, each of which
seems to specify a particular disease phenotype. How a specific conformation of a
PrP
Sc
molecule is imparted to PrP
C
during prion replication to produce nascent
PrP
Sc
with the same conformation is unknown.
Additionally, it is unclear what factors determine where in the CNS a particular
PrP
Sc
molecule will be deposited.
Sporadic Creutzfeldt-Jakob Disease (sCJD) is the most common of the human prion
diseases, accounting for approximately 85% of all cases. The sCJD includes five distinct types,
which differ according to clinical characteristics (observable physical and subjective symptoms)
and neuropathological characteristics (tissue changes that occur in the brain). The molecular
features of the different types of sCJD also vary, such as the genotype at codon 129 of the prion
protein gene and the length of the scrapie prion protein.
Sporadic Fatal Insomnia (sFI) has clinical and histopathological features
indistinguishable from those of Fatal Familial Insomnia (FFI) but does not have the mutation on
the prion gene that characterizes FFI.
Familial CJD (fCJD) is the second most common type of CJD, accounting for
approximately 10-15% of cases worldwide. This hereditary form of CJD is caused by a genetic
mutation in the prion protein gene, which causes a change in the amino acid sequence of the
normal prion protein. This change is believed to cause the mutated prion protein to take on the
scrapie prion protein conformation. DNA extracted from blood or brain tissue obtained at biopsy
or autopsy may be used to test for mutations in persons with suspected fCJD. Currently, there are
over 55 mutations of the prion gene that are known to cause fCJD and other familial prion
diseases in humans, including Fatal Familial Insomnia (FFI) and Gerstmann -Strussler-
Scheinker disease (GSS).
Iatrogenic CJD (iCJD) is a form of acquired CJD and one of the least common types,
accounting for less than 1% of cases. Both laboratory and clinical research has determined that
human-to-human transmission of CJD can occur as the result of tissue implant, use of
contaminated neurosurgical instruments, or administration of human hormones extracted from
the organs of human cadavers. Although blood transmission of CJD has been reported only in
variant CJD, the American Red Cross currently does not accept donations from persons with a
history of permanence in certain foreign countries or family history of CJD.
Variant CJD (vCJD) is also a form of acquired CJD, and is thought to be transmitted
through the ingestion of contaminated meat. In 1996, the first ten cases of vCJD were reported in
the UK. As of November 2004, 151 cases have been reported in the UK. In addition, six have
been reported in France, and one each in Italy, Ireland, Canada, and the USA. The cases reported
in Canada and the USA very likely acquired the disease in the UK. There is strong evidence that
vCJD has been acquired from cattle affected by bovine spongiform encephalopathy, or mad
cow disease, which occurred with epidemic proportions in the UK in the 1980s. vCJD has well
defined and consistent clinical and pathological features that make it relatively easy to identify
and distinguish from sporadic CJD. Furthermore, vCJD is the only type of prion disease in which
a definitive diagnosis can be made with a biopsy of the tonsils.
Kuru is an acquired prion disease that is virtually extinct. It was originally described in
members of a native tribe in New Guinea known to practice cannibalism. The epidemics
probably originated from the consumption of contaminated meat by a member of the tribe
affected by sporadic CJD. Clinically and pathologically, Kuru is fairly different from vCJD.
Bovine Spongiform Encephalopathy (BSE) is a prion disease of cattle. Since 1986,
when BSE was recognized, over 180,000 cattle in the UK have developed the disease, and
approximately one to three million are likely to have been infected with the BSE agent, most of
which were slaughtered for human consumption before developing signs of the disease. The
origin of the first case of BSE is unknown, but the epidemic was caused by the recycling of
processed waste parts of cattle, some of which were infected with the BSE agent and given to
other cattle in feed. Control measures have resulted in the consistent decline of the epidemic in
the UK since 1992. Infected cattle and feed exported from the UK have resulted in smaller
epidemics in other European countries, where control measures were applied later.
Chronic Wasting Disease (CWD) is a prion disease of elk and deer, both free range and
in captivity. CWD is endemic in areas of Colorado, Wyoming, and Nebraska, but new foci of
this disease have been detected in Nebraska, South Dakota, New Mexico, Wisconsin, Mississippi
Kansas, Oklahoma, Minnesota, Montana, and Canada. Since there are an estimated 22 million
elk and deer in the USA and a large number of hunters who consume elk and deer meat, there is
the possibility that CWD can be transmitted from elk and deer to humans.
Presently, the most widely used diagnostic tests exploit the relative protease resistance of
PrP
Sc
in brain samples to discriminate between PrP
C
and PrP
Sc
, in combination with
immunological (anti-PrP-antibody-mediated) detection of the proteinase K-resistant part of
PrP
Sc
(PrP2730). Sample preparation is one crucial part of such diagnostic tests and influences
the diagnostic sensitivity and specificity as well as the through-put. Because of the membrane
attachment of PrP
C
and PrP
Sc
, tissue solubilisation has to include detergent extraction. In
addition, conditions for proteinase K digestion have to be set such that the N-terminus of
PrP
Sc
and the whole PrP
C
peptide can efficiently be digested while PrP2730 remains resistant.
Various other proteases can be used for discriminating between the protease-sensitive
PrP
C
molecules and the protease-resistant PrP
Sc
structure. However, while proteinase K digests
the whole N-terminus of PrP
Sc
, other proteases (e.g. trypsin or pronase) do not result in such a
well-defined removal of the N-terminus but rather leave the whole PrP
Sc
molecule intact or digest
away a few amino acids only. Currently, 5 test kits use the detection of protease-resistant PrP as
an assay principle and have been positively evaluated by the European Union in 1999 and 2003.
Prionics-Check Western (Western blot, Prionics AG). Simple automated one-step
sample preparation followed by protease treatment. Detection occurs after the separation of the
treated sample by denaturing polyacrylamide gel electrophoresis and transfer to a membrane
using a PrP-specific antibody and an alkaline phosphatase-coupled secondary antibody detection
system generating chemiluminescence. The presence of a PrP-immunoreactive signal with the
additional two criteria of a reduced molecular weight (due to digestion of the N-terminus of
PrP
Sc
) and a typical 3-band pattern (due to different glycosylation forms of PrP) results in a TSE-
positive diagnosis.
Platelia test (ELI SA, BioRad). Sample preparation involving protease treatment
followed by precipitation and a centrifugation step for enrichment of the analyte. Detection by
sandwich ELISA using a colour-converting enzyme-coupled detection antibody. Cut-off setting
with a grey zone that requires repetition of the assay.
Enfer test (ELI SA, Enfer) One-step sample preparation involving sample transfer into
and out of a plastic bag followed by protease treatment. The analyte is coated individually onto
ELISA plates followed by detection via polyclonal antibodies and an enzyme-coupled secondary
antibody. Detection is by chemiluminescence.
Prionics-Check LI A (ELI SA, Prionics AG). Simple one-step sample preparation
procedure followed by protease treatment. Detection occurs via sandwich ELISA using
monoclonal antibodies, one of which is enzyme-labelled. Detection is with chemiluminescence
using a plate-specific cut-off setting.
CDI (I npro). In addition to a mild protease-treatment, the CDI uses the differential
binding of antibodies to native or denatured PrP
Sc
. The detection antibody recognises a
conformation-dependent epitope that, while always exposed in the non-infectious form (PrP
C
),
only becomes exposed in the infectious form of PrP (PrP
Sc
) upon denaturation. Quantification of
the binding events leads to a difference between the signals given by denatured PrP
Sc
and native
PrP
Sc
, which is used as a diagnostic criterion.
Currently, the most widely used BSE tests in Europe are the Prionics-Check Western test
and the Platelia test. In total, about 8 million BSE tests are performed per year.




















References:
Brooks, G. F. (2013). Jawetz, Melnick & Adelberg's medical microbiology (26th ed.). New York:
McGraw-Hill.

Dyck, E. v., & Meheus, A. (1999). Laboratory diagnosis of sexually transmitted diseases.
Geneva: World Health Organization.

Fauci, A.S., Braunwald, E., Kasper, D.L., Hauser, S.L., Longo, D.L., Jameson, J.L., Loscalzo, J.
(Eds.). (2008). Harrisons principles of internal medicine (17 th ed.). New York:
McGraw Hill.

CDC - Prion Diseases. (n.d.). Centers for Disease Control and Prevention. Retrieved October 6,
2013, from http://www.cdc.gov/ncidod/dvrd/prions/
Gorczynski, R. and Stanley, J. (1999).Clinical Immunology. Austin, Texas: Landes Bioscience.
Greenberger, N. J., Blumberg, R. S., & Burakoff, R. (2009). Current diagnosis & treatment (3rd
ed.). New York, NY: McGraw-Hill.
Grey, M. R., & Spaeth, K. R. (2006). The bioterrorism sourcebook. New York: McGraw-Hill
Medical Pub. Division.
Harkness, J., Marriott, D. and Stark, R.(2007).Laboratory Diagnostic Techniques for Entamoeba
Species. Retrieved from http://cmr.asm.org/content/20/3/511.full
Ilic, N. ,Gruden-Movsesijan, A. and Sofronic-Milosavljevic L. (2012). Trichinellaspiralis:
shaping the immune response. Retrieved from
http://link.springer.com/article/10.1007%2Fs12026-012-8287-5.
Immunoloy test (2013). Antigens and Antibodies. Retrieved from
http://www.medindia.net/bloodtest/immunology/immunology.htm Beebe, N., Ellis, J.,
Fotedar, R.,
Kubler, E ; Oesch, B ; Alex, JR. (2003).Diagnosis of prion diseases. British Medical Bulletin;
66: 267279
MicrobeWiki (2010). Study Microbes. Retrieved from
http://microbewiki.kenyon.edu/index.php/Study_Microbes
Murray, P. R., & Rosenthal, K. S. (2005). Review of medical microbiology. Philadelphia: Mosby.
Nicoll, D. (2013). Pocket guide to diagnostic tests (6th ed.). New York: McGraw-Hill Medical.
Ryan, K. J., Ray, C. G., & Sherris, J. C. (2010). Sherris medical microbiology (5th ed.). New
York: McGraw Hill Medical.
Stevens, C. (2010). Clinical & Immunology Serology: A Laboratory Perspective Third Edition.
Philadelphia: F.A. Davis Company.
DEAR WAIT,, HAHAHA APKIEDIT MY REFERNCE THANK YOU AND I LOVE
YOU MMMMMMMMMMMMMMMMMWAH.. JEJEJE
References:

AccessMedicine | Haemophilus influenzae. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=57032843&searchStr=haemophilus
+influenzae#57032843
AccessMedicine | Pseudomonas aeruginosa. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=57032606&searchStr=pseudomonas
+aeruginosa#57032606
AccessMedicine | Streptococcus pyogenes. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=57032151&searchStr=streptococcus
+pyogenes#57032151
AccessMedicine | Treponema Pallidum and Syphilis. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=57033576&searchStr=treponema+p
allidum#57033576
AccessMedicine | enterotoxigenic escherichia coli. (n.d.). Retrieved from
http://accessmedicine.com/search/searchAMResult.aspx?rootterm=enterotoxigeni
c+escherichia+coli&searchType=1&rootID=42034&searchStr=Enterotoxigenic+
Escherichia+coli&modifier=yes&subID=167
AccessMedicine | Staphylococcus. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=56757125&searchStr=staphylococc
us+aureus#56757125
AccessMedicine | Vibrio. (n.d.). Retrieved from
http://accessmedicine.com/content.aspx?aID=56757714&searchStr=vibrio+choler
ae