Download diagnosis of hiv infection the laboratory

DIAGNOSIS OF HIV INFECTION
THE LABORATORY
BY
DR. K.BUJJIBABU.MD.
METHODOLOGIES AVAILABLE
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SEROLOGICAL ASSAYS
SUPPLEMENTAL ASSAYS
ANTIGEN DETECTION
VIRAL CULTURE
MOLECULAR METHODS
SURROGATE MARKERS
SEROLOGICAL ASSAYS
Depends on rise of antibody levels to
detectable range.
2- 8 weeks of acquiring the infection( not
useful for early infections)
IgM- Gag proteins.
IgG- p 24 antigen and the gp120…. gp 41
Persistently undetectable antibodies more than
3 months – rare.
SEROLOGICAL ASSAYS
ELISA Format:
First generation: Whole viral lysate.
Second generation: Recombinant antigens
Third generation:- synthetic peptides.
Sens: >99% Specificity: >98.5%
Principles: Sandwich assay
Competitive assay
Capture assay principle
False negative serology results
Very early during the course of infection.
• Immunocompromised patients.
• Errors in collection, labeling and handling of
the specimens.
• Unusual HIV strains ( O group HIV strain)
• Recent Exchange transfusion.
Postulated to be due to a lack of B cell “ help”
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False positive Serology Results
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Human Error.
Lipaemic or hemolysed samples.
VDRL Positive.
Autoimmune disorders.
Hypergammaglobulinemia.
Multiple myeloma.
SEROLOGICAL ALTERNATIVES
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Latex agglutination
Immunochromatography.
Other spot tests.
SUPPLEMENTAL ASSAYS
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WESTERN BLOT ASSAY
INDIRECT IMMUNOFLUORESCENCE
RIPA ( Radio immunoprecipitation
assay)
Specific Blots for the differentiation of
the subtypes of HIV-1
WESTERN BLOT
Relevant antigens are separated from
electrophoresis and transferred to a
membrane.
Antibody reactivity is visible as bands.
Subjectivity is a problem and causes
diagnostic confusion.
Interpretation of Western Blot assay
CDC Criteria.
WHO Criteria.
American Red Cross Criteria.
ASPTHLD Criteria.
Reactive
Borderline reactive.
Non Reactive.
Problems in Interpretation
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False positive results- Hyperbilirubinemia,
Connective tissue disorders, Polyclonal
gammopathies.
Assays with no Group O proteins tend to miss the
Non Type B infections of HIV-1
20% of the HIV- 2 assays may give a false
negative result.
No clear patterns to predict seroconversion.
Problems in Interpretation
A single band in the blot.
Strong positive ELISA with a borderline
reactive blot.
Weak positive ELISA with a borderline
reactive blot.
Technical errors causing diagnostic
confusion.
ANTIGEN DETECTION
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Almost obscure today- replaced by the RNA
based molecular diagnostic tests.
Both false negative results and false positive
results seen.
Average sensitivity of the assays- 1030pg/ml.
Heat or glycine mediated dissociation of the
immune complexes- more sensitive.
VIRUS CULTURE
Cumbersome but highly specific for the
diagnosis of HIV-1 Infection.
Co-cultivation of the PBM’C of a donor with
those of the patient.
RT activity or the p 24 antigen are measured.
Drug resistance studies can be done with the
viral culture and strain typing with
differentiation can be performed.
Time consuming to be of clinical use.
MOLECULAR DIAGNOSIS
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Detection of HIV- 1 RNA in plasma using
RNA RT PCR( Lower limit of detection 2050 copies of the genome).
Detection of the HIV- 1 Subtypes is still
far from reality.
Window period diagnosis can be settled
with these assays, although not validated
by the US FDA for clinical use.
MOLECULAR
DIAGNOSIS
Detection of the proviral DNA in
PBMC’S 9 Helpful in the neonatal HIV
infection)
More sensitive and rapid than culture
Technically more cumbersome and
tedious than RNA RT PCR.
Quantification of the HIV- Viral oad
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Measurement of Virological set point
and prognosis.
Monitoring response to therapy.
Predicting resistance to antiretroviral drugs.
Predict progression of disease.
Predict maternal vertical
transmission.
Specialised Testing Procedures
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Screening for mutations in CCR5 gene(
homozygotes versus heterozygotes)
Detection of HIV strains with genomic
defects ( Deletions in the nef gene)
NSI/ SI phenotypes- inability to infect
lymphoblastoid cell lines- low
multiplication rate.
SURROGATE MARKERS
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CD4/ CD8 ratios ( useful but
superseded by the viral load tests)
Beta –2 microglobulin.
Serum neopterin levels.
Serum IgA levels.
High CD8 counts reflective of slower
disease progression.