Академический Документы
Профессиональный Документы
Культура Документы
DIAGNOSIS OF INFECTIOUS
DISEASES AND TUMOR MARKERS
• SEROLOGY
• The scientific study of blood sera and their effects
• Subdivision of immunology concerned with in-vitro
Ag-Ab reaction
• Concerned with the laboratory study of the
activities of the components of serum that
contribute to immunity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• IMMUNOLOGY
• The study of the molecules, cells, organs and
systems responsible for the recognition and
disposal of foreign (non-self) material
• The study of how the body components respond
and interact
• The desirable and undesirable consequences of
immune interactions
• The ways in which the immune system can be
manipulated to protect or treat disease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• IMMUNITY
• The ability of an organism to resist infection by
means of the presence of circulating antibodies
and white blood cells
• Distinctive characteristics of the immune system
Specificity
Memory
Mobility
Replicability
cooperativity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
• Nucleic acid amplification
• DNA sequencing and typing
• Direct molecular probe (in situ hybridization)
• Nucleic acid quantitation
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
– Uses:
• Cases requiring increased sensitivity and specificity of
identification
• Cases requiring faster report turnaround time
• Confirmation of culture
• Identification of organisms that are non-viable or cannot
be cultured
• Identification of fastidious, slow growing organisms
• Identification of organisms that are dangerous to culture
• Identification of organisms in small numbers or in small
volume specimens
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Molecular Biology
– Uses:
• Density of amplifiable DNA correlates with microbial
density
• Monitoring of disease progression or initiation or
modification of therapy
• Drug susceptibility testing
• Differentiation of antigenically similar organisms
• Molecular epidemiology and infection control
• Disease diagnosis by characterization of genetic materials
without direct identification of infectious agent
• Determination of virulence of antimicrobial resistance
genes
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• The most commonly acquired spirochete disease in the
U.S.
• A complex sexually transmitted disease that has a highly
variable clinical course
• Over 50,000 cases reported in 1990 in the U.S.
• Causative agent is Treponema pallidum
• No natural reservoir in the environment, requires living
host
• Spiral shaped and motile due to peri-plasmic flagella
• Variable length
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Three other pathogens in the group Treponema
which are morphologically and anti-genetically
similar to T. pallidum, differences are in
characteristics of lesions, amount of systemic
involvement and course of the disease
T. pertenue (Yaws)
T. endemicum (non-venereal syphilis)
T. carateum (pinta)
T. cuniculi (rabbit syphilis)
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Mode of Transmission
– Organism is very fragile, destroyed rapidly by heat, cold
and drying
– Sexual transmission most common, occurs when
abraded skin or mucous membranes come in contact
with open lesion
– Can be transmitted to fetus
– Rare transmission from needle stick and blood
transfusion
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS
• Congenital Syphilis
Transmitted from mother to fetus
Fetus affected during the second or third trimester
40% result in syphilitic stillbirth
Live-born infants show no signs during first few
weeks
= 60-90% develop clear or hemorrhagic rhinitis
= skin eruptions (rash) especially around mouth,
palms of hands and soles of feet
= general lymphadenopathy, hepatosplenomegaly,
jaundice, anemia, painful limbs & bone abnormality
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
• Evaluation based on 3 factors
Clinical findings
Demonstration of spirochetes in clinical specimen
Present of antibodies in blood or CSF
= more than one test should be performed
= no serological test can distinguish between
other treponemal infections
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
• Laboratory Testing
A. Direct examination of clinical specimen by dark-field
microscopy or fluorescent antibody testing of sample
B. Non-specific or non-treponemal serological test to
detect reagin, utilized as screening test only, not
diagnostic
= Reagin is an antibody formed against cardiolipin
= Found in sera of patients with syphilis as well as
other diseases
= Non-treponemal tests become positive 1-4 weeks
after appearance of primary chancre, in secondary
stage may have false positive due to prozone, in
tertiary 25% are negative, after successful
treatment will become non-reactive after 1 to 2
years
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• SYPHILIS - - DIAGNOSIS
• Laboratory Testing
C. Specific Treponemal antibody tests are used as a
confirmatory test for a positive reagin test
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
1. Biologic False Positives (BFP)
A. Collagen diseases such as arthritis, LE,
etc., sometimes result in increased
amount of reagin
B. Certain infections : IM, malaria, leprosy
C. Other treponemal infections
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
2. False negatives
A. Very early in disease or latent, inactive
stage
B. Immunosuppressed patients
C. Consumption of alcohol prior to testing
(temporary)
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
3. Congenital syphilis
A. Non-treponemal tests on cord blood or
baby serum detect IgG antibody,
maybe of maternal origin
B. Detection of IgM lacks sensitivity
C. Western blot has demonstrated high
sensitivity and specificity
D. Recommended that all mothers be tested
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• PROBLEM AREAS
4. Cerebrospinal Fluid tests
A. Used to determine if Treponemes have
invaded the CNS
B. VDRL utilized to confirm neurosyphilis
C. Lacks sensitivity
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = SYPHILIS
• LYME’S DISEASE
= Disease first recognized in 1977 in Lyme, Connecticut
= Causative organism is Borrelia burgdorferi
= Can be cultured but it is very difficult
= Organism has been isolated from blood, CSF, skin lesions
and joint fluid
= Can be transmitted perinatally, causing intrauterine death
= Vector of transmission is the Ixodes tick
= Must remain attached a minimum of 24-48 hours for
transmission to occur
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = LYME’S DISEASE
• Diagnostic criteria
• Isolation of organism from clinical specimen or
• Diagnostic titers of IgG and IgM in serum or CSF
or
• Significant change in serum titers of IgG or IgM
in paired acute and convalescent sera
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = LYME’S DISEASE
• LABORATORY DIAGNOSIS
• Diagnosed clinically, confirmed serologically
• Antibodies to antigens of B. burgdorferi can be detected by latex
agglutination, IFA, ELISA, and Western Blot
• Serological tests are often falsely negative during early weeks.
Specific IgM Abs usually appear 2- 4 weeks after erythema
migrans, peak after 3-6 weeks of illness, decline to normal after
4-6 months
IgG titers appears more slowly (4-8 weeks after the rash), peak
after 4-6 months, may remain high for months or years
• Western Blot is most sensitive
• IFA and ELISA are more commonly performed due to ease of
procedure, but are subject to false positives due to either
spirochete diseases and some autoimmune diseases
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• STREPTOCOCCAL SEROLOGY
• Streptococci are gram (+), beta-hemolytic, spherical,
ovoid, or lancet-shaped organisms which are catalase
negative and seen in pairs or chains
• Divided into groups or serotypes based on cell wall
components Streptococcus pyogenes belongs to
Lancefield group A and it is believed the M protein is the
chief virulent factor of this group
• Numerous exo-antigens are produced and excreted as the
cell metabolizes (Streptolysin O, DNase, Hyaluronidase,
Nicotinamide, Adenine dinucleotidase (NADase),
Streptokinase)
• Culture and rapid screening tests detect early infection
• Sequelae include Rheumatic Fever and Acute GN
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
• Most reliable test is culture and identification of the
organism from infected site
• Rapid streptococcal screening tests from the throat
exudates have high specificity but low sensitivity, 60-85%
• Detection of Streptococcal antibodies most useful in
Streptococcal sequelae
• The most useful antibodies are : ASO, anti-DNase B, anti-
NADase, anti-Hyaluronidase
• Serological evidence of disease is based on elevated or
rising titer of Streptococcal antibodies
• Four-fold (2 tube dilution) rise in titer is considered
clinically significant
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
1. Anti-Streptolysin O Titer (ASO Titer)
= two of the toxins produced are Streptolysin S, which is oxygen
stable, non-antigenic and Streptolysin O (SLO), which is
oxygen labile and antigenic
= SLO is a hemolysin which is toxic to many tissues, including heart
and kidneys
= evokes an antibody response (anti-SLO) which neutrolizes the
hemolytic action of SLO
= the test is specific for ASO, it does not test for antibodies to any
other Streptococcal exotoxins
= normal values will vary, <125 Todd units for adults, 5-125 Todd
units for children, recent Strep infections 250 Todd units for
adults, 333 Todd units for children
= a single titer is of little significance unless extremely elevated,
titers performed over a period of time will give the most information
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
2. Anti-DNase B Testing
= may appear earlier than ASO
= increased sensitivity for detection of
glomerulonephritis preceded by streptococcal
skin infection
= macro- and micro-titer, ELISA, and neutralization
techniques are available
= Neutralization technique has advantage of
stability of reagents
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
3. Anti-Hyaluronidase Testing
= test patient serum for antibodies which inhibit
action of Hyaluronidase
= after performance of the test, a clot will form into
the tubes where enzyme activity of
Hyaluronidase has been neutralized by
patient antibody
= Hyaluronidase produced by patients with throat
or skin infections, ASO produced in response
to throat infections only
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = STREPTOCOCCAL INFECTION
• LABORATORY TESTING
4. Streptozyme Testing
= hemagglutination procedure to detect antibodies
to numerous Streptococcal antigens
= sheep RBC’s are coated with Streptolysin,
Streptokinase, Hyaluronidase, DNase, and
NADase
= patient serum diluted 1 : 100, mixed with sheep
RBC’s and observed for agglutination
= rapid and simple to perform, more false positive
and negative results occur
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES
• Hepatitis G virus
• Independently discovered 1995-1996 by 2
separate research groups
• RNA virus
• Transmissible by blood-borne route
• Found in patients with acute or chronic liver dse.
• Exact clinical significance needs to be further
defined
• ELISA and Western Blot methods have been
developed
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS
• Cytomegalovirus
• Transmission occurs from person to person
• Symptoms resemble IM but has negative test for
EBV
• In babies may cause life-threatening illness
resulting in CNS involvement, hearing loss, and
mental retardation
• Seen in patients with deficient immune system,
AIDS, transplantation
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Immunologic response
• For best diagnostic results, lab tests for CMV antibody should
be performed by using paired serum samples
• One blood sample should be taken upon suspicion of CMV,
and another one taken within 2 weeks. A virus culture can be
performed at any time the pt. is symptomatic
• IgM antibodies produced against early and intermediate-early
(IE) CMV antigens, last for 3 to 4 months
• IgG appear shortly after and peak at 2 to 3 months
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Laboratory Diagnosis
• Range from culture and cytologic techniques to
DNA probes, PCR and serologic techniques
• Detection of antibodies indicator of recent infection
• Viral culture lack sensitivity and are time
consuming and expensive
• Microscopic examination of biopsy specimens,
urine sediment or peripheral blood may reveal the
typical cytomegalic cell with “owl’s eye” inclusion
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Cytomegalovirus
– Laboratory Diagnosis
• Detection of CMV Ag in cells more appropriately detected by
immunofluorescent techniques using monoclonal antibodies
• ELISA is the most commonly available serologic test for
measuring antibody to CMV
• The result can be used to determine if acute infection, prior
infection, or passively acquired maternal antibody in an
infant is present
• Other tests include various fluorescence assays, indirect
hemagglutination, and latex agglutination
• Screening tests using coated latex particles compare
favorably to more complex tests for antibody detection
• False positives can occur = RA and Ebstein-Barr antibodies
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HERPES VIRUS GROUP
• Varicella-Zoster Virus
– Laboratory testing important to distinguish
VZV from other infections, selection of
antiviral drugs, or determining immune status
of individuals
• PCR is now the routine testing method for VZV
• Direct fluorescent antibody staining and viral
culture techniques may be used for the detection
of VZV in most specimen types
• IgG and IgM antibody tests by ELISA may be used
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = GERMAN MEASLES
• Rubella Virus
– Laboratory testing
• Performed primarily for diagnosis of acquired infections and
to determine immune status of pregnant patients
• Some tests detect IgG antibodies, other IgM
• Methods include : hemagglutination inhibition, passive
hemagglutination, neutralization, hemolysis in gel,
complement fixation, fluorescent immunoassay, RIA, ELISA
and latex agglutination
• Method depends on volume of testing, turn around time,
complexity, expense and whether a qualitative or quantitative
test is needed
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = MEASLES
• Rubeola
• Serology testing provides best means of confirming a
measles diagnosis
• Methods to detect Rubeola antibodies include :
hemagglutination inhibition, endpoint neutralization,
complement fixation, IFA and ELISA
• In addition to signs and symptoms, diagnosis confirmed by
presence of Rubeola specific IgM antibodies or four-fold rise
in IgG antibody titer in paired samples taken after rash to 10
to 30 days later
• IgM test highly depended on time of sample collection with
3-11 days after rash being optimal
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = MUMPS
• Mumps
• Methods to detect mump antibodies include : complement
fixation, hemagglutination inhibition, hemolysis-in-gel,
neutralization assays, IFA and ELISA
• Current or recent infections indicated by presence of specific
IgM antibody in single sample which can be detected within
5 days of illness
• Fourfold rise in specific IgG antibody in 2 samples collected
during acute and convalescent phases
• Fluorescent antibody staining for mumps antigens developed
but not widely used
• Cross-reactivity between antibodies to mumps and
parainfluenza viruses has been reported in test for IgG
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Human Immunodeficiency Virus (HIV)
• Etiologic agent of AIDS
• Discovered independently by Luc Montagnier of France and
Robert Gallo of the US in 1983-1984
• Former names of the virus include :
Human T cell Lymphotrophic virus (HTLV-III)
Lymphadenopathy associated virus (LAV)
AIDS associated retrovirus (ARV)
• HIV-2 discovered in 1986, antigenically distinct virus endemic
in West Africa
• One million people infected in US, 30 Million worldwide are
infected
• Leading cause of death of men aged 25-44 and 4th leading
cause of death of women in this age group in the US
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Structural genes
• Gag is p55 from which three core proteins (p15,
p17 and p24) are formed
• Env gene codes for envelope proteins gp160,
gp120 and gp41
• Pol codes for p66 and p51 subunits of reverse
transcriptase and p31 an endonuclease
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Immunologic Manifestations
• Early stage slight depression of CD4 count, few symptoms,
temporary
• Window of up to 6 weeks before antibody is detected, by 6
months 95% positive
• During window p24 antigen present, acute viremia and
antigenemia
• Antibodies produced to all major antigens
First antibodies detected produced against gag proteins
p24 and p55
Followed by antibody to p51, p120 and gp41
As disease progresses, antibody levels decreases
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Immunologic Manifestations
• Immune abnormalities associated with increased
viral replication
Decrease in CD4 cells
B cells have decreased response to antigen
CD8 cells initially increase and may remain elevated
As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections
Death comes due to immuno-incompetence
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV
• Dengue fever
• Transmitted by mosquitoes
• There are 4 known distinct serotypes ( dengue
virus 1, 2, 3 and 4)
• In children , infection is often sub-clinical or causes
a self-limited febrile disease
• Secondarily infected with a different serotype,
dengue hemorrhagic fever or dengue shock
syndrome
Algorithm for Serologic Testing for AIDS
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = DENGUE
• Dengue fever
• Dengue IgG/IgM Rapid Test is a solid phase
immunochromatographic assay for the rapid
qualitative and differential detection of IgG and
IgM antibodies to dengue virus in human serum,
plasma or whole blood. This test can also detect all
4 Dengue serotypes by using a mixture of
recombinant Dengue envelop proteins
Rapid Test for Dengue
Rapid Test for Dengue
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES =DENGUE
• Dengue fever
– Interpretation of the test
• IgG and IgM positive = indicative of a late primary or early
secondary dengue infection
• IgM positive = indicative of primary Dengue infection
• IgG positive = indicative of secondary or past dengue
infection
• Negative = retest in 3-5 days if Dengue infection is
suspected
• Invalid = insufficient specimen volume or incorrect
procedural technique. Repeat the test using a new test
device
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = Typhoid Fever
• Typhoid Fever
• Caused by Salmonella typhi
• Rapid detection is now available in the market
Typhidot = a qualitative detection test against a specific
antigen of Salmonella typhi. It can detect both IgG and
IgM separately and simultaneously. Thus, indicating the
status of acute infection, convalescence or previous
exposure
Salmonella typhi IgG/IgM Rapid test = an
immunochromatographic assay for rapid, qualitative and
differential detection of IgG and IgM antibodies to
Salmonella typhi in human serum, plasma or whole blood
Typhidot
Typhoid Fever Rapid test
Typhoid Fever Rapid Test
H. Pylori Rapid test
Malaria Ab Rapid test
Rapid test for TB
Rapid test for Chlamydia
Rotavirus/Adenovirus Rapid test
Rapid test for Rubella
Rapid test for RSV
Rapid test for Tetanus
Rapid test for Legionella
Rapid test
TUMOR MARKERS
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
• An oncofetal protein that was first discovered in
1963 in the serum of mice with hepatoma
• Normal fetal protein synthesized by the liver, yolk
sac, and GIT that shares sequence homology with
albumin
• A major component of fetal plasma, reaching a
peak concentration of 3mg/ml at 12 weeks of
gestation -- following birth, it clears rapidly from
the circulation, having a half-life of 3.5 days
• Concentration in adult serum <20ng/ml
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
– Benign conditions causing elevation of AFP
• 2nd and 3rd trimesters of pregnancy
• Cirrhosis
• Acute and chronic hepatitis
• Hepatic necrosis
TUMOR MARKERS
• Alpha-FETOPROTEIN (AFP)
– Malignant conditions causing elevation of AFP aside
from hepatoma
• Teratocarcinoma of the testis and embryonal Ca (70%)
• Carcinoma of the pancreas (23%)
• Carcinoma of the stomach (18%)
• Carcinoma of the lung (7%)
• Carcinoma of the colon (5%)
*** In patients with hepatoma, the incidence of elevation of
levels of AFP correlates with tumor burden
TUMOR MARKERS
• CALCITONIN
– Other neoplasms less frequently associated
with increased levels
• Small cell carcinoma of the lung
• Carcinoma of the breast
• Carcinoid
• Hepatoma
• Renal cell carcinoma
• Zollinger-Ellison syndrome
TUMOR MARKERS
• CALCITONIN
– Benign conditions associated with increased
level
• Pancreatitis
• Hyperparathyroidism (primary and secondary)
• Paget’s disease of bone
• Pulmonary disease
TUMOR MARKERS
• CATECHOLAMINE METABOLITES
• Most commonly assayed catecholamine metabolites are
vanillylmandelic acid (VMA) and homovanillic acid (HVA),
which are metabolites of norepinephrine and dopamine,
respectively
• Urinary levels of this metabolites can be accurately measured
from a single urine specimen using gas chromatographic
techniques – requires avoidance of tea, coffee, fruit and
vanilla from the diet 72 hours before urinary sampling
• Most useful in diagnosing and monitoring patients with
NEUROBLASTOMA
TUMOR MARKERS
• CATECHOLAMINE METABOLITES
• Neuroblastoma is a malignant lesion of the neural
crest tissue, which most commonly occurs in
children
• Elevated urinary levels of VMA and HVA are
observed in 75 to 95% of patients
• Improved survival time was reported in patients
with a ratio of urinary VMA to HVA of ≥1.5
TUMOR MARKERS
• CA 125
• An antigen present on 80% of nonmucinous
ovarian carcinomas
• Defined by a monoclonal antibody (OC125) that
was generated by immunizing laboratory mice with
a cell line established from human ovarian
carcinoma
• Elevated in other cancers – endometrial,
pancreatic, lung, breast, and colon
• Elevated in benign conditions – menstruation,
pregnancy, endometriosis
TUMOR MARKERS
• CA 19-9
• A monoclonal antibody generated against a colon
carcinoma cell line to detect a
monosialoganglioside found in patients with
gastrointestinal adenocarcinoma
• Elevated in gastric cancer (21-42%), colon cancer
(20-40%), pancreatic cancer (71-93%)
TUMOR MARKERS