Viral load assays
- Quantitative plasma HIV RNA assay.
- Most important indicator of response to ART. Goal of ART is to achieve and maintain viral suppression.
- Can be useful in predicting clinical progression. Viremia copy-years predicts mortality in ART-naive.
- Significant predictor of risk of transmission. Viral suppression prevents HIV transmission.
Commercial Viral Load Assays
- Real-time PCR
- TaqMan HIV-1 v2.0, Roche
- RealTime HIV-1, Abbott
- HIV RT-PCR, reverse transcriptase polymerase chain reaction (Amplicor HIV-1 Monitor v1.5/Ultrasensitive, Roche)
- Branched chain DNA (bDNA) (Versant HIV-1 RNA v3.0, Bayer)
- Nucleic acid sequence-based amplification (NucliSens HIV-1 EasyQ v2.0, bioMerieux)
Acute HIV Infection
- VL may be used to diagnose acute HIV infection prior to seroconversion. VL in acute infection is generally very high (>100,000 c/ml); low-level viremia (VL < 10,000 c/ml) may be a false-positive test.
- Fourth-generation Ag/Ab tests (ARCHITECT HIV Ag/Ab Combo and GS HIV Combo Ag/Ab) combine HIV-1/HIV-2 Ab and HIV-1 p24 Ag assays, decreasing the ’window period’ to 2 wks. Results may not distinguish positive antigen versus positive antibody. 4th generation Ag/Ab tests are typically positive during acute retroviral syndrome (ARS), but VL can be positive ~5 days before Ag/Ab tests.
- CDC compares FDA-approved assays for screening. Accessed 9/19/17 at https://www.cdc.gov…
- Confirm results by serologic methods (e.g., ELISA or Western blot) over the next 3-6 mos to document seroconversion.
- If serologic tests are negative or indeterminate and ARS is suspected, order VL. Serologic tests usually become positive within 1 month if negative at time of ARS.
Prognosis and Risk of Opportunistic Infection
- Decreased viremia associated with improved clinical outcome.
- Cumulative viremia copy-years associated with all-cause mortality.
- Viral set point is prognostic indicator of disease progression.
- VL predicts risk of OI, independent of CD4 when CD4 < 200.
Probability of Transmission
- VL directly correlated with probability of transmission in all types of exposure studied. Higher VL seen in early infection is associated with increased infectiousness. Suppressive ART prevents HIV transmission.
- When serially monitoring VL, use same laboratory and assay.
- Threefold change (0.5 log10 c/ml) considered significant.
- Sample collection in anticoagulant EDTA avoids heparin-associated inhibition of PCR.
VL Monitoring in Untreated Pts
- Perform at entry into care. ART recommended for all HIV+ patients.
- Baseline VL appears to be lower in women and African Americans compared to white men, although rates of disease progression are similar.
VL Monitoring in Treated Pts
- At ART initiation or modification, then 2 to 8 wks after initiation or modification. If detectable at 2-8 wks, repeat every 4-8 wks until suppressed < 200 c/ml, and then every 3-4 months.
- Optimal viral suppression: VL persistently below assay limit of detection.
- In adherent patients consistently suppressed with stable CD4 for more than 2 yrs, monitoring can be extended to every 6 months.
- After modification of ART for treatment failure, adverse effects, or regimen simplification, measure VL within 2-8 wks to confirm potency of new regimen.
- ACTG and DHHS guidelines-defined virologic failure: confirmed VL >200. Repeat assay when VL is unexpectedly detectable.
- Sustained viremia (>500) increases risk of drug resistance and viral failure.
Factors that Increase VL
Factors Not Measured by VL
- Immune function
- CD4 regenerative reserve
- Susceptibility to ARVs
- Syncytial vs. nonsyncytial forms; R5 vs X4 tropism
- VL in compartments other than blood (e.g., semen, lymph nodes, CNS, GI tract, and genital secretions)
Basis for recommendation
- Brooks JT, Kawwass JF, Smith DK, et al. Effects of Antiretroviral Therapy to Prevent HIV Transmission to Women in Couples Attempting Conception When the Man Has HIV Infection - United States, 2017. MMWR Morb Mortal Wkly Rep. 2017;66(32):859-860. [PMID:28817552]
Comment: Summary of 3 multinational studies HPTN 052, PARTNER, and Opposites Attract that followed 3,000 serodiscordant couples over >1,500 couple-years observed no HIV transmission while HIV+ partner was virally suppressed with ART.
- Panel on Antiretroviral Guidelines for Adults and Adolescents; Guidelines for the use of antiretroviral agents in HIV-1-infected adults & adolescents; last updated 5/1/2014. Accessed 9/7/2017. http://aidsinfo.nih.gov…
Comment: DHHS guidelines state plasma HIV-1 RNA should be measured in all HIV-1-infected patients at entry into care, at ART initiation or modification, and 2 to 8 weeks after ART initiation or modification. If detectable at 2-8 wks, repeat every 4-8 wks until supressed < 200 c/ml, and then every 3-4 months. In patients consistently suppressed with stable CD4 for more than 2 years, monitoring can be extended to every 6 months. Optimal viral suppression defined as below the level of detection (VL< 20-75 copies/ml, depending on assay). Virologic failure defined as confirmed viral load > 200 copies/ml.
- Kulkarni S, Jadhav S, Khopkar P, et al. GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India. BMC Infect Dis. 2017;17(1):506. [PMID:28732472]
Comment: Rapid assay uses reverse transcriptase polymerase chain reaction (RT-PCR) technology to achieve high sensitivity with dynamic range of 40-10,000,000 copies/ml for all HIV-1 Group M, N, and O subtypes. Results in 90 mins, fully automated integrated system. Study compared GeneXpert HIV-1 Quant assay with gold standard, Abbott m2000rt RealTime HIV-1 assay in 151 HIV+ ART-naive, 129 on ART, 34 with suspected virologic failure, and 20 HIV- individuals. Bland-Altman plots showed differences were within the limits of agreement, mean bias was positive with overestimation by HIV-1 Quant assay. With cut-off defined as 200 copies/ml, sensitivity to classify virologic failure was 97%, specificity was 100%, PPV was 100%, and NPV was 88%.
- Templer SP, Seiverth B, Baum P, et al. Improved Sensitivity of a Dual-Target HIV-1 Qualitative Test for Plasma and Dried Blood Spots. J Clin Microbiol. 2016;54(7):1877-82. [PMID:27194686]
Comment: Early confirmation of pediatric HIV infection requires viral testing as passively transferred maternal HIV Ab may be detectable in HIV-exposed infant for up to 18 months of life or longer. TaqMan HIV-1 Qual Test, v2.0 is total nucleic acid amplification test for the qualitative detection of HIV-1 DNA and RNA. Investigators compared Abbott RealTime HIV-1 Qual assay (Abbott) and TaqMan HIV-1 Qual Test v2.0 (Roche). In EDTA plasma and dried blood spots, sensitivity was greater as measured by copies/ml for TaqMan HIV-1 Test v2.0 adn attributed to dual target region: gag gene and LTR region.
- Álvarez Estévez M, Chueca Porcuna N, Guillot Suay V, et al. Quantification of viral loads lower than 50 copies per milliliter by use of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, can predict the likelihood of subsequent virological rebound to >50 copies per milliliter. J Clin Microbiol. 2013;51(5):1555-7. [PMID:23390288]
Comment: Retrospective cohort study of 290 pts evaluated confirmed rebound VL > 50 c/ml as measured by Cobas Ampliprep/Cobas TaqMan HIV-1, v2.0. Time to viral rebound was significantly shorter for those with two consecutive viral loads of 20-39 and 40-49 c/ml as compared to those with VL < 20 c/ml. In this study, patients with VL < 20 c/ml had been suppressed on ART for a longer duration.
- Wojewoda CM, Spahlinger T, Harmon ML, et al. Comparison of Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0) with other real-time PCR assays in HIV-1 monitoring and follow-up of low-level viral loads. J Virol Methods. 2013;187(1):1-5. [PMID:23098667]
Comment: Newer VL assays use automatic nucleic acid extraction and real-time PCR detection of nucleic acid during each cycle of amplification. Compared to TaqMan v1.0, v2.0 is more sensitive with a lower level of quantification (20 c/ml vs 48 c/ml). TaqMan v2.0 targets two separate regions in the HIV-1 RNA (gag gene and LTR region) and optimizes quantification by better accommodating viral genetic variability and primer and probe binding polymorphisms. Due to increased test specificity of TaqMan v2.0, this study reported increased frequency of negative or "undetected" results in samples that were reported < 48 c/ml by TaqMan v1.0 (N=10).
- Naeth G, Ehret R, Wiesmann F, et al. Comparison of HIV-1 viral load assay performance in immunological stable patients with low or undetectable viremia. Med Microbiol Immunol. 2013;202(1):67-75. [PMID:22699843]
Comment: Study compares Abbott m2000 RealTime assay versus Roche Cobas TaqMan v2.0 for concordance in 150 samples. Results from 50 samples in Group 1 (undetectable by m2000 at all 5 time points), 50 samples in Group 2 (undetectable or detected < 40 c/ml), and 50 samples in Group 3 (VL detected < 40 c/ml at all 5 time points) were compared. Group 1 had high concordance, 90%. For groups 2 and 3, concordance was 56%. Longitudinal follow-up of VL quantification, especially in pts with low or undetectable viremia, may vary if derived from different assays.
- Manlutac AL, Giesick JS, McVay PA. Identification of early HIV infections using the fourth generation Abbott ARCHITECT HIV Ag/Ab Combo chemiluminescent microparticle immunoassay (CIA) in San Diego County. J Clin Virol. 2013;58 Suppl 1:e44-7. [PMID:24342477]
Comment: Assessment of 4th generation Abbott ARCHITECT HIV Ag/Ab Combo assay in 14,517 specimens collected by San Diego County Public Health Laboratory found 279 (1.9%) repeatedly positive. Of these, 240 were confirmed by HIV-1 immunofluorescence Ab assay (IFA). Of those deemed IFA negative or inconclusive (39), 30 samples were further tested. Thirteen were considered false positive and 17 specimens had detectable HIV-1 RNA and were classified as acute infections that would have been missed by by the prior screening assay.
- Mitchell EO, Stewart G, Bajzik O, et al. Performance comparison of the 4th generation Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA on the EVOLIS™ automated system versus Abbott ARCHITECT HIV Ag/Ab Combo, Ortho Anti-HIV 1+2 EIA on Vitros ECi and Siemens HIV-1/O/2 enhanced on Advia Centaur. J Clin Virol. 2013;58 Suppl 1:e79-84. [PMID:24342482]
Comment: Industry-sponsored comparison of two 4th generation Ag/Ab assays that detect HIV p24 Ag and HIV Ab and reduce the "window period’ between infection and detection to within 2 weeks. Authors emphasize potential of assay with higher specificity to have greater utility in low prevalence settings.
- Taylor N, Grabmeier-Pfistershammer K, Egle A, et al. Cobas Ampliprep/Cobas TaqMan HIV-1 v2.0 Assay: Consequences at the Cohort Level. PLoS One. 2013;8(8):e74024. [PMID:24023696]
Comment: Retrospective study of 373 HIV-infected patients on stable ART regimens with undetectable VL (< 50 c/ml) by Amplicor Monitor v1.5 assay for ≥ 1 year followed by TaqMan v2.0 assay. Data were based on serial not repeat measurements. The majority (59%) were < 20 c/ml (N=221), 18% were 20-49 c/ml, 17% were 50-499 c/ml, and 6% were >500 c/ml. Transition to TaqMan v2.0 resulted in greater frequency of measurable VL. Clinically important information from genotype testing included: a new M184V mutation in 1 pt and 2 new minor PI mutations in another pt. Six pts had subsequent ART regimen changes.
- Mugavero MJ, Napravnik S, Cole SR, et al. Viremia copy-years predicts mortality among treatment-naive HIV-infected patients initiating antiretroviral therapy. Clin Infect Dis. 2011;53(9):927-35. [PMID:21890751]
Comment: US-based observational cohort study (CFAR Network of Integrated Clinical Systems) compared cumulative viremia copy-years in 2027 patients followed from 2000-2008 and contributing median 8 (IQR, 4-15) VL measures with associated all-cause mortality (HR, 1.8, 95%CI, 1.5-2.2). The authors cite the relationship between plasma HIV VL and systemic inflammation or immune system activation as an important predictor of mortality after adjustment for most recent CD4 count.
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