HIV AIDS laboratory findings

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]; Associate Editors-in-Chief: Ujjwal Rastogi, MBBS, Ammu Susheela, M.D. [2]; Jesus Rosario Hernandez, M.D. [3]

Overview

Important laboratory tests for the initial evaluation of patients with suspected HIV infection include screening tests with high sensitivity such as ELISA, dot blot, and latex agglutination assays, and confirmatory tests with high specificity such as western blot assays, P24 antigen assays, and nucleic acid testing. Two surrogate markers, the CD4 T-cell count (CD4 count) and the plasma HIV RNA viral load, are routinely used to asses immune function and levels of viremia. Resistance testing is becoming of greater importance in the management of HIV/AIDS patients given the increased resistance to certain antiretroviral agents.

Laboratory Findings

Sequence of Appearance of Laboratory Markers for HIV-1 Infection
Note. Units for vertical axis are not noted because their magnitude differs for RNA, p24 antigen, and antibody. Modified from MP Busch, GA Satten (1997)[1] with updated data from Fiebig (2003),[2] Owen (2008),[3] and Masciotra (2011, 2013). [4][5]
- Source: https://www.cdc.gov/

]

Types of HIV Tests

  • Diagnostic Testing
  1. Screening tests
  2. Supplemental tests
  3. Confirmatory tests
  • Follow-up Testing
  1. CD4 count
  2. HIV viral load
  • Resistance Testing
  1. Phenotypic tests
  2. Genotypic tests
  3. Tropism assays

Testing Schedule

Laboratory testing is important at several stages during an HIV infection:

  1. At diagnosis
  2. During follow-up if antiretroviral therapy (ART) has not been initiated
  3. Prior to and after initiation or modification of therapy to assess virologic and immunologic efficacy of ART
  4. To monitor for laboratory abnormalities that may be associated with ART


By WHO infographics
By WHO infographics

Diagnostic Testing

Screening Tests

Most HIV tests used to screen for HIV infection detect the presence of antibodies against HIV. Detectable antibodies usually develop within 2–8 weeks after infection, but may take longer. There are three different screening tests:

  1. ELISA test (based on antigen-antibody and enzyme substrate reactions).
  2. Rapid Tests (Dot blot and Latex Agglutination Tests).
  3. Simple Tests (Particle agglutination tests).

Both simple and rapid tests are readily available and cheaper as compared to ELISA although they may not be as sensitive.

Supplemental Tests

These are used to validate results obtained by the screening tests and are of two types:

  1. Western blot tests
  2. Immunofluorescence tests

Confirmatory Tests

These test aim at the following:

  1. Demonstration of Viral Antigen (P24).
  2. Isolation of HIV.
  3. Detection of viral nucleic acid.

The confirmatory tests can diagnose HIV infection even during the window period (initial two to three weeks of infection), in which both the screening and the supplemental tests fail to diagnose the infection. However these are done in the reference centers thus time consuming and costly.

Important Considerations

  • The window period is the time between the initial infection and the development of detectable antibodies against the virus. This period varies between 1 to 6 months prior to which antibody testing is generally negative and a possible HIV infection can be missed. Detection of the virus using polymerase chain reaction (PCR) during the window period is preferable, and evidence suggests that an infection may often be detected earlier than when using a fourth generation enzyme immunoassay screening test. Positive results obtained by PCR are later confirmed by antibody tests.[6]
  • Routinely used HIV tests cannot be used in neonates born to HIV-positive mothers because of the presence of maternal antibodies to HIV in the child's blood. Prior to 18 months, HIV infection can only be diagnosed by PCR, testing for HIV pro-viral DNA.[7]

Follow-up Testing

CD4 T-Cell Count

The CD4 count serves as the major laboratory indicator of immune function in patients who have HIV infection. It is one of the key factors in deciding whether to initiate ART and prophylaxis for opportunistic infections, and it is the strongest predictor of subsequent disease progression and survival according to clinical trials and cohort studies.[8][9]

A significant change (2 standard deviations) between two tests is approximately a 30% change in the absolute count or an increase or decrease in CD4 percentage by 3 percentage points. The CD4 count is one of the most important factors in the decision to initiate ART and/or prophylaxis for opportunistic infections. All patients should have a baseline CD4 count at entry into care. An adequate CD4 response for most patients on therapy is defined as an increase in CD4 count in the range of 50–150 cells/mm3 per year, generally with an accelerated response in the first 3 months. Subsequent increases in patients with good virologic control show an average increase of approximately 50–100 cells/mm3 per year for the subsequent years until a steady state level is reached.[10] Patients who initiate therapy with a low CD4 count or at an older age may have a blunted increase in their count despite virologic suppression. The CD4 cell count response to ART varies widely, but a poor CD4 response is rarely an indication for modifying a virologically suppressive ARV regimen. In patients with consistently suppressed viral loads who have already experienced ART-related immune reconstitution, the CD4 cell count provides limited information, and frequent testing may cause unnecessary anxiety in patients with clinically inconsequential fluctuations. Thus, for the patient on a suppressive regimen whose CD4 cell count has increased well above the threshold for opportunistic infection risk, the CD4 count can be measured less frequently than the viral load. In such patients, CD4 count may be monitored every 6 to 12 months, unless there are changes in the patient’s clinical status, such as new HIV-associated clinical symptoms or initiation of treatment with interferon, corticosteroids, or anti-neoplastic agents.

Frequency of CD4 Count Monitoring

In general, CD4 counts should be monitored every 3–4 months to:

  1. Determine when to start ART in untreated patients.
  2. Assess immunologic response to ART.
  3. Assess the need for initiation or discontinuation of prophylaxis for opportunistic infections .
Factors that affect absolute CD4 count

The absolute CD4 count is a calculated value based on the total white blood cell (WBC) count and the percentages of total and CD4+ T lymphocytes. This absolute number may fluctuate among individuals or may be influenced by factors that may affect the total WBC and lymphocyte percentages, such as use of bone marrow–suppressive medications or the presence of acute infections. Splenectomy [11][12] or coinfection with human T-lymphotropic virus type I (HTLV-1) [13] may cause misleadingly elevated absolute CD4 counts. Alpha-interferon, on the other hand, may reduce the absolute CD4 number without changing the CD4 percentage.[14] In all these cases, CD4 percentage remains stable and may be a more appropriate parameter to assess the patient’s immune function.

Plasma HIV RNA Testing (HIV Viral Load)

  • Plasma HIV RNA (viral load) should be measured in all patients at baseline and on a regular basis thereafter, especially in patients who are on treatment, because viral load is the most important indicator of response to antiretroviral therapy (ART) (AI).
  • Analysis of 18 trials that included more than 5,000 participants with viral load monitoring showed a significant association between a decrease in plasma viremia and improved clinical outcome.[15]
  • The viral load testing serves as a surrogate marker [16] for treatment response and can be useful in predicting clinical progression.[17][18]
  • The minimal change in viral load considered to be statistically significant (2 standard deviations) is a threefold, or a 0.5 log 10 copies/mL change.
  • Optimal viral suppression is generally defined as a viral load persistently below the level of detection (<20–75 copies/mL, depending on the assay used).
  • However, isolated “blips” (viral loads transiently detectable at low levels, typically <400 copies/mL) are not uncommon in successfully treated patients and are not thought to represent viral replication or to predict virologic failure.[19]
  • In addition, low-level positive viral load results (typically <200 copies/mL) appear to be more common with some viral load assays than others, and there is no definitive evidence that patients with viral loads quantified as <200 copies/mL using these assays are at increased risk for virologic failure.[20][21][22]
  • For the purposes of clinical trials the AIDS Clinical Trials Group (ACTG) currently defines virologic failure as a confirmed viral load >200 copies/mL, which eliminates most cases of apparent viremia caused by blips or assay variability. This definition may also be useful in clinical practice.

Resistance Testing

Phenotypic Testing

Phenotypic assays measure the ability of a virus to grow in different concentrations of ARV drugs. RT and PR gene sequences and, more recently, integrase and envelope sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient-derived HIV genes of interest. Replication of these viruses at different drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference HIV strain. The drug concentration that inhibits viral replication by 50% (i.e., the median inhibitory concentration [IC50]) is calculated, and the ratio of the IC50 of test and reference viruses is reported as the fold increase in IC50 (i.e., fold resistance).

Genotypic Testing

Genotypic assays detect drug-resistance mutations present in relevant viral genes. Most genotypic assays involve sequencing of the RT and PR genes to detect mutations that are known to confer drug resistance. Genotypic assays that assess mutations in the integrase and gp41 (envelope) genes are also commercially available. Genotypic assays can be performed rapidly and results are available within 1 to 2 weeks of sample collection. Interpretation of test results requires knowledge of the mutations selected by different antiretroviral (ARV) drugs and of the potential for cross resistance to other drugs conferred by certain mutations.

Coreceptor Tropism Assays

  • HIV enters cells by a complex process that involves sequential attachment to the CD4 receptor followed by binding to either the CCR5 or CXCR4 molecules and fusion of the viral and cellular membranes.[23]
  • CCR5 inhibitors (i.e., maraviroc [MVC]), prevent HIV entry into target cells by binding to the CCR5 receptor.[24]
  • Phenotypic and, to a lesser degree, genotypic assays have been developed that can determine the coreceptor tropism (i.e., CCR5, CXCR4, or both) of the patient’s dominant virus population.
  • The vast majority of patients harbor a CCR5-utilizing virus (R5 virus) during acute/recent infection, which suggests that the R5 variant is preferentially transmitted compared with the CXCR4 (X4) variant.
  • Viruses in many untreated patients eventually exhibit a shift in coreceptor tropism from CCR5 to either CXCR4 or both CCR5 and CXCR4 (i.e., dual- or mixed-tropic; D/M-tropic).
  • This shift is temporally associated with a more rapid decline in CD4 T-cell counts, although whether this shift is a cause or a consequence of progressive immunodeficiency remains undetermined.[25][26]

Phenotypic Tropism Assays

  • There are at least two high-throughput phenotypic assays that can quantify the coreceptor characteristics of plasma-derived virus.
  • Both involve the generation of laboratory viruses that express patient-derived envelope proteins (i.e., gp120 and gp41).
  • These pseudoviruses are either replication competent (Phenoscript assay, VIRalliance, Paris, France) or replication defective (Trofile assay, Monogram Biosciences, Inc.).
  • These pseudoviruses then are used to infect target cell lines that express either CCR5 or CXCR4. In the Trofile assay, the coreceptor tropism of the patient-derived virus is confirmed by testing the susceptibility of the virus to specific CCR5 or CXCR4 inhibitors in vitro.
  • The Trofile assay takes about 2 weeks to perform and requires a plasma HIV RNA level ≥1,000 copies/mL.

Genotypic Tropism Assays

  • Genotypic determination of HIV-1 coreceptor usage is based on sequencing the V3-coding region of HIV-1 env, the principal determinant of coreceptor usage.
  • A variety of algorithms and bioinformatics programs can be used to predict coreceptor usage from the V3 sequence. When compared to the phenotypic assay, genotypic methods show high specificity (~90%) but only modest sensitivity (~50%–70%) for the presence of a CXCR4-utilizing virus.
  • Given these performance characteristics, these assays may not be sufficiently robust to completely rule out the presence of an X4 or D/M variant.[27]

Recommendations for Coreceptor Tropism Assays

  • Coreceptor tropism assay should be performed whenever the use of a CCR5 inhibitor is being considered.
  • Coreceptor tropism testing might also be considered for patients who exhibit virologic failure on a CCR5 inhibitor.

Gallery

References

  1. ↑ Busch MP, Satten GA (1997). "Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure". Am J Med. 102 (5B): 117–24, discussion 125-6. PMID 9845513.
  2. ↑ Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L; et al. (2003). "Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection". AIDS. 17 (13): 1871–9. doi:10.1097/01.aids.0000076308.76477.b8. PMID 12960819.
  3. ↑ Owen SM, Yang C, Spira T, Ou CY, Pau CP, Parekh BS; et al. (2008). "Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States". J Clin Microbiol. 46 (5): 1588–95. doi:10.1128/JCM.02196-07. PMC 2395119. PMID 18322061.
  4. ↑ Masciotra S, McDougal JS, Feldman J, Sprinkle P, Wesolowski L, Owen SM (2011). "Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections". J Clin Virol. 52 Suppl 1: S17–22. doi:10.1016/j.jcv.2011.09.011. PMID 21981983.
  5. ↑ Masciotra S, Luo W, Youngpairoj AS, Kennedy MS, Wells S, Ambrose K; et al. (2013). "Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast". J Clin Virol. 58 Suppl 1: e54–8. doi:10.1016/j.jcv.2013.07.002. PMID 23911678.
  6. ↑ Weber B (2006). "Screening of HIV infection: role of molecular and immunological assays". Expert Rev. Mol. Diagn. 6 (3): 399–411. doi:10.1586/14737159.6.3.399. PMID 16706742.
  7. ↑ Tóth FD, Bácsi A, Beck Z, Szabó J (2001). "Vertical transmission of human immunodeficiency virus". Acta Microbiol Immunol Hung. 48 (3–4): 413–27. PMID 11791341.
  8. ↑ Mellors JW, Muñoz A, Giorgi JV, Margolick JB, Tassoni CJ, Gupta P et al. (1997) Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 126 (12):946-54. PMID: 9182471
  9. ↑ Egger M, May M, Chêne G, Phillips AN, Ledergerber B, Dabis F et al. (2002) Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies. Lancet 360 (9327):119-29. PMID: 12126821
  10. ↑ Kaufmann GR, Perrin L, Pantaleo G, Opravil M, Furrer H, Telenti A et al. (2003) CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the Swiss HIV Cohort Study. Arch Intern Med 163 (18):2187-95. DOI:10.1001/archinte.163.18.2187 PMID: 14557216
  11. ↑ Zurlo JJ, Wood L, Gaglione MM, Polis MA (1995) Effect of splenectomy on T lymphocyte subsets in patients infected with the human immunodeficiency virus. Clin Infect Dis 20 (4):768-71. PMID: 7795071
  12. ↑ Bernard NF, Chernoff DN, Tsoukas CM (1998) Effect of splenectomy on T-cell subsets and plasma HIV viral titers in HIV-infected patients. J Hum Virol 1 (5):338-45. PMID: 10195261
  13. ↑ Casseb J, Posada-Vergara MP, Montanheiro P, Fukumori LM, Olah I, Smid J et al. (2007) T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Rev Inst Med Trop Sao Paulo 49 (4):231-3. PMID: 17823752
  14. ↑ Berglund O, Engman K, Ehrnst A, Andersson J, Lidman K, Akerlund B et al. (1991) Combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon-alpha and zidovudine. J Infect Dis 163 (4):710-5. PMID: 1672701
  15. ↑ Murray JS, Elashoff MR, Iacono-Connors LC, Cvetkovich TA, Struble KA (1999) The use of plasma HIV RNA as a study endpoint in efficacy trials of antiretroviral drugs. AIDS 13 (7):797-804. PMID: 10357378
  16. ↑ Hughes MD, Johnson VA, Hirsch MS, Bremer JW, Elbeik T, Erice A et al. (1997) Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team. Ann Intern Med 126 (12):929-38. PMID: 9182469
  17. ↑ Marschner IC, Collier AC, Coombs RW, D'Aquila RT, DeGruttola V, Fischl MA et al. (1998) Use of changes in plasma levels of human immunodeficiency virus type 1 RNA to assess the clinical benefit of antiretroviral therapy. J Infect Dis 177 (1):40-7. PMID: 9419168
  18. ↑ Thiébaut R, Morlat P, Jacqmin-Gadda H, Neau D, Mercié P, Dabis F et al. (2000) Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment. Groupe d'Epidémiologie du SIDA en Aquitaine (GECSA). AIDS 14 (8):971-8. PMID: 10853978
  19. ↑ Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, Hirsch MS, Ignacio C, Condra J, Günthard HF, Richman DD, Wong JK (2001). "Prevalence and predictive value of intermittent viremia with combination hiv therapy". JAMA. 286 (2): 171–9. PMID 11448280. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  20. ↑ Gatanaga H, Tsukada K, Honda H, Tanuma J, Yazaki H, Watanabe T, Honda M, Teruya K, Kikuchi Y, Oka S (2009). "Detection of HIV type 1 load by the Roche Cobas TaqMan assay in patients with viral loads previously undetectable by the Roche Cobas Amplicor Monitor". Clin. Infect. Dis. 48 (2): 260–2. doi:10.1086/595707. PMID 19113986. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  21. ↑ Damond F, Roquebert B, Bénard A, Collin G, Miceli M, Yéni P, Brun-Vezinet F, Descamps D (2007). "Human immunodeficiency virus type 1 (HIV-1) plasma load discrepancies between the Roche COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 assays". J. Clin. Microbiol. 45 (10): 3436–8. doi:10.1128/JCM.00973-07. PMC 2045351. PMID 17715371. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  22. ↑ Willig JH, Nevin CR, Raper JL, Saag MS, Mugavero MJ, Willig AL, Burkhardt JH, Schumacher JE, Johnson VA (2010). "Cost ramifications of increased reporting of detectable plasma HIV-1 RNA levels by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 version 1.0 viral load test". J. Acquir. Immune Defic. Syndr. 54 (4): 442–4. doi:10.1097/QAI.0b013e3181d01d1d. PMC 2901889. PMID 20611035. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  23. ↑ Moore JP, Kitchen SG, Pugach P, Zack JA (2004). "The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection". AIDS Res Hum Retroviruses. 20 (1): 111–26. doi:10.1089/088922204322749567. PMID 15000703.
  24. ↑ Fätkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, Goebel FD, Rockstroh JK, Dezube BJ, Jenkins TM, Medhurst C, Sullivan JF, Ridgway C, Abel S, James IT, Youle M, van der Ryst E (2005). "Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1". Nat. Med. 11 (11): 1170–2. doi:10.1038/nm1319. PMID 16205738. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  25. ↑ Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR (1997). "Change in coreceptor use correlates with disease progression in HIV-1--infected individuals". J. Exp. Med. 185 (4): 621–8. PMC 2196142. PMID 9034141. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  26. ↑ Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M (1993). "Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS". Ann. Intern. Med. 118 (9): 681–8. PMID 8096374. Unknown parameter |month= ignored (help); |access-date= requires |url= (help)
  27. ↑ Lin NH, Kuritzkes DR (2009). "Tropism testing in the clinical management of HIV-1 infection". Curr Opin HIV AIDS. 4 (6): 481–7. doi:10.1097/COH.0b013e328331b929. PMC 2874683. PMID 20048714. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  28. ↑ 28.0 28.1 28.2 28.3 28.4 28.5 "Public Health Image Library (PHIL)".
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