Indian J Sex Transm Dis Indian J Sex Transm Dis
Official Publication of the Indian Association for the Study of Sexually Transmitted Diseases
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Year : 2008  |  Volume : 29  |  Issue : 1  |  Page : 42-45

Laboratory diagnosis of HIV

Department of Skin V.D., Medical College and S.S.G. Hospital, Vadodara, Gujarat, India

Correspondence Address:
Y S Marfatia
OPD-1, Department of Skin and V.D. Medical College, Vadodara
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Source of Support: None, Conflict of Interest: None

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How to cite this article:
Sharma A, Marfatia Y S. Laboratory diagnosis of HIV. Indian J Sex Transm Dis 2008;29:42-5

How to cite this URL:
Sharma A, Marfatia Y S. Laboratory diagnosis of HIV. Indian J Sex Transm Dis [serial online] 2008 [cited 2023 Jun 6];29:42-5. Available from:

   When to Offer Human Immunodeficiency Virus (HIV) Test ? Top

  • Clinical features suggestive of HIV infection.
  • Patients with tuberculosis, especially in young patients
  • Patients having sexually transmitted diseases (STDs)
  • Antenatal Care (ANC) patients
  • Hepatitis B and C coinfection
  • History of high-risk behavior/transfusion

Laboratory tests employed for the diagnosis of HIV infection may be classified into the following groups

Antibody based tests [1],[2],[3]

Screening tests

ELISA (Enzyme-linked immunosorbent assay)
Rapid tests

Supplemental or confirmatory

Immunofluorescent assay (IFA)
Western blot
Lineimmuno assay (LIA)
Radioimmuno precipitation assay (RIPA)

Other tests

p24 antigen
Polymerase chain reaction (PCR)
Plasma/ serum viral load

Alternative to classical tests

Oral fluid (saliva) HIV tests
Urine tests

Screening tests


This test is most widely used for screening people with HIV. Because of its high sensitivity, it is suitable for testing large number of samples.

Generations [1]

  1. First generation - whole viral lysates
  2. Second generation - recombinant antigen
  3. Third generation - synthetic peptide
  4. Fourth generation - antigen + antibody (Simultaneous detection of HIV antigen and antibody) - HIV duo

Antibody can be detected in a majority of individuals within 6-12 weeks after infection using the earlier generation of assays. But it can be detected within 3-4 weeks when using the newer third generation ELISA. Due to their ability to detect p24 antigen, the fourth-generation ELISA will be of value in detecting early infection. The window period can be shortened to two weeks using p24-antigen assay. [4] The principles of ELISA are classified as indirect, competitive, sandwich, and capture assay.

As per National HIV testing policy, two ELISA using different principles are required to diagnose HIV infection(in clinical setting). [1]

False positive results [1],[2]

  • Autoimmune disorders
  • Hematological malignancies (Plasmacytoma)
  • Alcoholic hepatitis
  • Connective tissue disorders
  • Acute rheumatic fever
  • Multiple pregnancies
  • Multiple transfusions
  • Post-vaccination including HIV vaccine
  • Chronic renal failure
  • Positive rapid plasma reagin test
  • Technical errors, etc.

False negative results

  • Window period (up to 3 months)
  • Immunosuppressive therapy
  • Replacement transfusion
  • B-cell dysfunction
  • Malignant disorder
  • Late stage disease (immune collapse)
  • Technical error

Rapid tests [4]

These are tests that can yield results in < 30 min. The results are read by naked eye. When performed correctly, these are accurate and have wide utility in a number of testing situations. Application includes emergency room, physician's office, autopsy room, and smaller blood banks.

Types [1]

  1. Dot blot assays/ tridot
  2. Particle agglutination
  3. HIV spot and comb tests
  4. Fluorimetric microparticle technologies

One class of rapid tests is the 'dot blot' or 'immunoblot'; they produce a well-circumscribed dot on the solid phase surface if the test is positive. There is a color change because of antigen-antibody reaction.


  1. Subjective interpretation
  2. Difficult to read if the laboratorian is color blind


  1. Rapid HIV assays have proven particularly useful for testing pregnant women in labor who have not received prenatal care.
  2. It is helpful in detecting HIV-2 infection which can not be detected by ELISA.

Why is it important to know about HIV-2? [2]

  1. NNRTI'S do not work against HIV-2.
  2. A patient doing well on ART, if super infected with HIV-2, will start failing immunologically (CD4 count decreases), but viral load will stay undetectable (as viral load detects HIV-1).

   Western Blot (WB) Top
[Figure 1]

It is a more specific assay. In this antibodies against numerous proteins are detected.

Env (gp160, gp120, and gp41)

gag (p55, p24, and p17)

pol (p66, p51, and p31)

If the sample has antibodies, colored bands will appear wherever human IgG binds to the viral protein on the strip. In the absence of colored bands, WB is interpreted as negative.


  • Negative - no bands
  • Positive - various criteria [Table 1]
  • Intermediate - bands present but doesn't satisfy the criteria

The commonest is WHO criteria [Table 1]. HIV-2 confirmations can be done by WB, but is not available in India (except NARI, Pune). Western blot is used only in cases of unequivocal /discordant result.

Other antibody band tests such as immunofluoresent assay are seldom used.

   Viral Detection Top

  • HIV Ag detection -p24
  • HIV culture


  1. Acute HIV infection
  2. Indeterminate serology
  3. Neonatal infection


  1. Repeat tests needed for confirmation
  2. Expensive

None of these are superior to routine serology.

   P24 Antigen Top

The antigen test detects HIV free antigen (p24) in the serum.

This test is useful:

  • During window period.
  • To detect HIV infection in newborn because diagnosis is difficult due to presence of maternal antibodies.
  • During late disease when patient is symptomatic.

Unfortunately, this test has a low sensitivity and not routinely recommended. The reason for the lack of sensitivity of this test is that the free antigen (p24) in serum may be complexed with p24 antibody. Antigens although transient can appear as early as two weeks after infection and lasts 3-5 months, so this method can shorten the window period by one week.

   PCR Top

In this the target HIV RNA or proviral DNA is amplified enzymatically in vitro by chemical reaction. It is an extremely sensitive assay because a single copy of proviral DNA can be amplified. Qualitative PCR is useful for diagnostic purposes.

Three different techniques namely RT-PCR, nucleic acid sequence based amplification (NASBA) and branched-DNA (b-DNA) assay have been employed to develop commercial kits.

These kits shorten the window period between infection and detectability to about 12 days.

Role in post exposure prophylaxis (PEP)

Both these tests (p24 and PCR) are not routinely recommended in case of occupational exposure because of its low positive predictive value. Hence, negative test must not be a ground to discontinue PEP.

   HIV Diagnosis in Newborns Top

Serological diagnosis of HIV infection in babies born to HIV infected mothers is difficult because of presence of maternal antiHIV-antibody up to 18 months.

If DNA PCR is positive within the first 48 hours of life, it indicates in-utero infection. In intrapartum infection, PCR is negative at 48 hours and positive within one month.

In case of p24 Ag, false positive results are common at less than one month of age. Sensitivity in infants more than six months of age is 50-75%. It is clearly an inferior test compared to HIV culture/PCR.

Thus, in case of neonatal diagnosis, PCR should be done at 48 hours, 1 week, 3 months, and 6 months. It should always be confirmed by serology at 18 months.

   Orasure - (Saliva) HIV Tests Top

Noninvasively collected specimens of oral fluids are used, although generally referred to as saliva; the fluid used for testing is actually oral mucosal transudate. This system obtains antibodies that are comparable to or exceed those from serum samples.

This test first employs ELISA, and then WB.

   Oraquick Advance Rapid HIV Test Top

This test was approved in 2004. It provides results in 20 min. The blood, plasma, or oral fluid is mixed in a vial with developing solution and the results are read from a stick-like testing device.

   Urine Tests Top

Intact IgG antibodies are found in urine, but their exact origin is unknown. The collection of urine is simple, noninvasive, and inexpensive. The use of urine is appropriate for physicians, officers, health clinics, and developing countries where healthcare personnel may not be trained professionally or where clean needles to withdraw blood may not be available.

Major disadvantage

There is no approved urine-based confirmatory assay, necessitating collection of blood when results are reactive. The USA FDA has approved an ELISA and WB for use to test urine for antibodies to HIV-1.

   Nucleic Acid Amplification Testing (NAAT) Top

Although standard tests that measure antibody response to the HIV virus have become increasingly sensitive, cases of HIV are occasionally missed because individuals can have negative antibody tests during the early stages of infection. Also, a few people with long-term HIV infection may have false negative antibody tests or may be chronic carriers who are clinically asymptomatic. The NAAT test helps avoid these problems as it amplifies the HIV viral RNA and detects viral genes instead of viral antibodies or antigens. Adding this new HIV screening method, to standard HIV testing, researchers were able to uncover six percent more cases of HIV infection in urban STD and drug treatment clinics and HIV testing sites in Atlanta than with standard HIV antibody tests alone.

   References Top

1.HIV Antibody testing with special reference to HIV-1.NACO, HIV Testing Manual. p. 45-67.  Back to cited text no. 1    
2.Pujari S, editor. Cipla e-workshop Module 1: HIV diagnosis, monitoring and initial evaluation.   Back to cited text no. 2    
3.Seth P. Laboratory diagnosis of HIV infection. In: Sharma VK, editor. Sexually transmitted diseases and AIDS, 1st ed. New Delhi: Viva Books Private Limited; 2003. p. 87-94.  Back to cited text no. 3    
4.Neil Constantine, HIV antibody assays. Available from: [cited on 2008 Jun 17].  Back to cited text no. 4    
5.Neil Constantine, HIV antigen assays. Available from [cited on 2008 Jun 17].  Back to cited text no. 5    
6.Prahraj AK. Problems in diagnosis of HIV infection in babies. Med J Armed Forces India 2006;62:363-6.  Back to cited text no. 6    
7.Meldung N. Genetic amplification (NAAT) test detects HIV more effectively than standard tests in urban study. Available from: [cited on 2008 Jun 17].   Back to cited text no. 7    


  [Figure 1]

  [Table 1]


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