HIV‐1 viral load determination based on reverse transcriptase activity recovered from human plasma

Abstract

We describe a procedure (ExaVir Load) to carry out human immunodeficiency virus-1 (HIV-1) viral load testing using reverse transcriptase (RT) recovered from HIV-1 virions in plasma. Samples from individuals infected with HIV-1 were treated with a sulphydryl-reactive agent to inactivate endogenous polymerases. Virions were then immobilised on a gel and washed in individual mini columns to remove RT-inhibiting antibodies, antiviral drugs, and other RT inhibitors. Immobilised virions were lysed finally, and the viral RT eluted. The amount of RT recovered was quantified by a sensitive RT activity assay using either colorimetry or fluorimetry to detect DNA produced by RT. The "RT load" values of 390 samples from 302 HIV-1 patients living in Sweden were compared to results obtained with an HIV-1 RNA viral load assay. The correlation between the two tests was r = 0.90, P < 0.0001. Four of 202 samples from healthy blood donors gave low positive values in the RT test. All samples in a panel with 10 HIV-1 subtypes were positive by the RT load. The RT load test provides a technically less demanding and cost-effective alternative to methods based on nucleic acid amplification. Being insensitive to genetic drift occurring in HIV, the assay should be of particular use in resource-limited settings, where different subtypes and recombinant HIV strains occur.

Establishment of In-House Quantitative Real-Time RT-PCR Assay for HIV-1 Viral Load Measurement: Application to Evaluate Efficacy of ART in Ghanaian Patients in an Urban Setting

“…CRF02_AG is the major driver of HIV infection in Ghana and others in minor proportion of circulation such as CRF06_cpx, sub-subtype A3, CRF09_cpx and subtypes A, G and D had been identified. Studies have shown that RT activity is independent of the HIV-subtype [40][41][42][43][44][45]. This is in agreement with other studies that evaluated an in-house real-time RT-PCR assay that targeted the same Long Terminal Repeat (LTR) region probing for viral RNA.…”

“…The mean age of the HIV-1 seronegative subjects was 27.5 years (S.D ± 5.9), 11 (55.0%) of them were women. The mean age of the HIV-1-infected patients was 36.9 (S.D ± 9.2) years, 286 (70.6%) were women and 51.3% of all infected patients were in clinical stage 3 or 4 [27]. Median CD4 cell counts, available for 143 of the HIV-infected patients, was 243 cells/mm 3 (IQR 138-405 cells/ mm 3 ).…”

Evaluation of Two Commercially Available, Inexpensive Alternative Assays Used for Assessing Viral Load in a Cohort of Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients from South Africa

“…Viral RT activity was expressed as femtograms per milliliter. These dUTP values were then converted to an equivalent number of copies per milliliter by the software provided (15).…”

Transfer and Evaluation of an Automated, Low-Cost Real-Time Reverse Transcription-PCR Test for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection in a West African Resource-Limited Setting

“…Unfortunately, in resource-constrained settings, these commercial assays are available only in reference laboratories because they are sophisticated and very expensive (for instance, between $50 and $100 per test in Côte d'Ivoire), largely exceeding the monthly cost of a standard HAART regimen (19). Two cheaper alternatives have been recently evaluated: the heat-dissociated HIV-1 p24 antigen enzymelinked immunosorbent assay (2,32) and the reverse transcriptase activity assay kit (Cavidi ExaVir) (3,24). These commercial assays may be useful in provincial laboratories but have not been widely implemented because they are still labor intensive, relatively expensive ($21 and $15, respectively), and need more clinical validations.…”