HIV is mostly diagnosed either by direct method such as detection of virus or viral genome by molecular test or by detection of p24 antigen or by indirect host response of rising antibodies by various serological tests.
Serology: Serologic tests are the most commonly employed tests for diagnosis of HIV infection. The detection of antibodies is available in various formats. Rapid test format, enzyme linked immunosorbent assay (ELISA) in conventional micro-ELISA format and western blot are the commonly used test formats whereas it is also available in chemiluminescence platform, line immune assay and enzyme-linked fluorescence assay for detection of antibody as well as antigen.
Nucleic acid amplification test (NAAT): This is highly sensitive test and is used for detection of proviral DNA or viral RNA. PCR is the most commonly employed test. Various structural genes such as gag, pol or env are the common target genes used in PCR assay. These tests are particularly useful to diagnose during the window period (before appearance of antibody) or in newborn or infants of <18 months in whom the presence of antibody cannot be differentiated from maternal IgG.
Sample: All HIV testings are done in blood sample. For serological assays, either serum, plasma or whole blood is used. Saliva and urine also can be used for antibody detection. For NAAT, either whole blood in EDTA vial or dried blood spot (DBS) can be used. For viral load assay, plasma is separated from the whole blood collected in EDTA. Pretest counselling and written informed consent must be obtained in all cases.
Serological tests for HIV diagnosis: Serological tests for detection of HIV antibody is the main stay of HIV diagnosis.
Indication of HIV testing:
• Screening before blood transfusion/trans plantation.
• Diagnosis in suspected asymptomatic/ symptomatic individuals.
• Prevention of transmission from mother to child
• Post-exposure prophylaxis
• Epidemiological surveillance
Several generations of serological tests have been evolved over the time for detection of HIV specific antibody which presently includes the detection of antibody to both HIV-1 and HIV-2 as well as detection of antigen. The purpose of development is to increase sensitivity, specificity and decrease the window period of detection. These different generations of tests are applicable mostly to ELISA and chemiluminescence assay platforms (Table 1).
Table1. Summary of 1st to 5th generation HIV serological tests
First generation HIV antibody test: The 1st generation ELISA was developed in 1985 using the crude virus lysate from virus infected tissue culture as antigen. The assay used to detect the IgG antibody to HIV-1. The test was sensitive but had a large antibody negative window period of up to 12 weeks post infection. The high sensitivity of the test was useful in screening the blood sample for transfusion purpose but was associated with false positives which led to the development of second generation assay. False positives were reported in autoimmune diseases, non HIV infections, pregnancy and several non specified conditions.
Second generation HIV antibody test: These assays added the recombinant proteins or synthetic peptides to increase the sensitivity and specificity of the assay. Some manufacturers added the proteins of HIV-2 and HIV-1 group O protein to make the assay enable to detect antibodies to these viruses as well. This brought down the window period of antibody detection from 12 to 6 weeks.
Third generation HIV antibody test: These assays use recombinant or synthetic peptides in an antigen sandwich configuration. It added the detection of IgM along with the existing IgG detection which decreases the window period further from 6 to 3 weeks.
Fourth generation HIV test: This assay combinedly detects the HIV-1 and 2 antibody and HIV-1 p24 antigen decreasing the window period from 3 to 2 weeks. However, the assay result does not differentiate whether the positivity is due to presence of antibody to HIV-1 or 2 or antigen. The first 4th generation assay was developed in late 1990. First FDA approved 4th generation test came in 2010 and currently several FDA approved tests are available from different manufacturer. The sensitivity and specificity of all FDA approved tests are >99–100%.
Fifth generation HIV test: This test like the 4th generation, detects the HIV-1 and 2 antibody and HIV-1 p24 antigen. The improvement here is it gives separate result for each analyte. Hence, the exact status of sample positivity regarding HIV-1 or 2 antibody or HIV p24 antigen is clear.
Rapid tests: Several rapid tests are available for HIV diagnosis. These tests can be done from serum, plasma as well as from the finger prick sample. Assay types of rapid tests are (Figs 1. to 3):
• Immunochromatographic test (lateral flow assay)
• Immunoconcentration/dot blot (vertical flow assay)
• Immunocomb test
• Particle agglutination
First three types are more commonly used. Advantages of these tests are:
• No equipment is required. Test results are visibly recorded.
• Can be performed with very minimum training. So, can be done in remote field area by field worker and even self-testing can be done.
• Result in these tests is readable in 20 30 minutes. These criteria make these tests suitable for point of care test.
Fig1. Interpretation of dot blot test for HIV diagnosis
Fig2. Interpretation of immunochromatography test for HIV diagnosis
Fig3. Interpretation of immunocomb test for HIV diagnosis
Presently both third and fourth generation tests are available in rapid test format for detection of HIV-1 and 2 antibody and detection of free HIV p24 antigen along with HIV-1 and 2 antibody, respectively. For detection of HIV-1 specific antibody, the device is coated with HIV-1gp120 and gp41 synthetic peptides and for detection of HIV-2 specific antibody it is coated with gp136 synthetic peptide.
In general, the sensitivity and specificity of rapid tests are comparable with that of ELISA. HIV tests in rapid test formats are available up to fourth generation assay. However, in a comparative assay between various 4th generation rapid tests, product of only one manufacturer was found to give additional 28% positivity than 3rd generation test result but products of all other manufacturer were not found to be suitable. Reasons of false positive HIV antibody test:
• Pre-analytical error
• Hypergammaglobulinemia
• Influenza vaccination
• Recipient of HIV vaccine as a trial participant
Reasons of false negative HIV antibody test:
• Prior to seroconversion
• Early acute infection
• Infants
• Immunosuppressive conditions
• Hypogammaglobulinemia
• Advance AIDS
Western blot test (WB): In the Western blot assay, the various HIV proteins are blotted to nitrocellulose paper by electrophoresis. Separate Western blot strips for HIV-1 and 2 are available. The HIV-1 viral antigens are separated according to their molecular weight (from above downwards): gp160, gp120, p66, p55, p51, gp41, p31, p24, p17, and p15. On incubation with patient’s serum, the HIV specific antibody present in the serum will bind to the corresponding proteins. On further addition of enzyme labelled secondary antibody and substrate, a colorimetric band will be produced at the site of antigen antibody reaction (Fig. 4).
Fig4. Western blot assay for HIV diagnosis
Interpretation: Reactivity of HIV antibodies present in the patient sample with the different antigenic components on the WB strip should be interpreted as per manufacturer instructions.
According to the CDC guidelines: Positive when reactive to at least 2 of the following antigens: p24, gp41, gp120/160.
WHO recommendations: Positive when reactive to at least two envelop proteins (gp41, gp120/ 160). Presence of very weak p17 band is regarded as negative.
Reactivity to antigens not fulfilling WHO/ CDC criteria is classified as indeterminate result and requires repetition of the test.
The HIV-1 Western blot was previously recommended by CDC and NACO (National AIDS Control Organization, Govt of India) to make a confirmatory laboratory diagnosis of HIV-1 infection. However, due to several reports on the false detection of HIV-2 as HIV-1 by western blot test, the current CDC guidelines have removed HIV-1 Western blot as a confirmatory test from the recommended HIV testing algorithm. Considering the rapid, simple to perform reliable RDTs and enzyme immune assays WHO in its “2019 consolidate guideline on HIV testing services for changing epidemic” recommends countries to move away from Western blotting.
In general, antibody detection tests are not useful for diagnosis during the early part of infection because of window period of 3 4 weeks. The test is also not helpful to diagnose the infection in infants born to HIV positive mothers (till <18 months) as they may be positive due to transplacental transfer of maternal antibody.
Ultrasensitive p24 antigen detection test: p24 antigen can be detected in the whole blood, plasma or serum either in the free form or in the bound form with the bound antibody as antigen–antibody complex. After the appearance of antibody, p24 antigen is mostly present in the form of antigen–antibody complex and hence may not be detectable. Ultrasensitive antigen detection tests have been developed which includes dissociation of antigen from the antigen–antibody complex along with signal amplification to detect smaller amount of antigen. The performance of these tests is almost comparable with that of HIV PCR both in terms of sensitivity and time of detection. The advantages of antigen detection tests are, these tests do not require sophisticated lab facilities like molecular technique and can be done in a set up where enzyme immune assay can be performed.
Virus isolation: HIV is cultivated by co cultivating peripheral blood mononuclear cells (PBMC) from an infected individual and a mitogen stimulated PBMC from a non infected individual in presence of interleukin 2 (IL-2) at 37p C with 5% CO2 . Replication of HIV is detected by PCR or p24 antigen detection. This method is not used for diagnosis.
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