Reverse transcriptase activity for quantitation of HIV-1 subtype C in plasma: relation to RNA copy number and CD4 T-cell count.

The present study monitored the changes in human immunodeficiency virus (HIV) viral load using a reverse transcriptase (RT) assay and an HIV-1 RNA based assay, and relates these data to the dynamics of CD4 cell counts. The samples examined originate from a prospective study of HIV-1 subtype C infected, untreated Ethiopians followed twice yearly over a period of up to 5 years. The ExaVir Load test, version 1, was used for isolation and quantitation of HIV-1 RT in plasma. The RT activities recovered were compared to the HIV-1 RNA copy numbers, which had been determined previously by the NucliSens HIV-1 QT Test. There was a significant correlation between the data obtained in the two tests (r = 0.65, P < 0.0001). During follow-up, the median RT and RNA levels increased more or less in parallel up to approximately four times the values at admittance. CD4 cell counts, which had also been determined previously, decreased slowly but continuously from approximately 310 to 190 CD4 cells/ml. In the majority of individual patients, there was an inverse correlation between CD4 T-cell counts and RT activity, and with the RNA copy number, and the data obtained by either test could be used to predict CD4 T-cell counts. The ExaVir Load test thus provides data equivalent to the estimation of the number of HIV-1 RNA copies for the prediction of CD4 T-cell counts. It is based on a simple technique, can be run in any routine diagnostic laboratory, and is a competitive alternative for use in resource limited settings.

Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay.

Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000 copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r = 0.83 to 0.96, r = 0.84 to 0.99, r = 0.58 to 0.67, and r = 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.

Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma.

A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.

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

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.

Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing.

OBJECTIVE: To demonstrate the use of HIV-1 reverse transcriptase (RT) recovered directly from plasma for phenotypic drug susceptibility testing. METHODS: Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RT eluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits. RESULTS: The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC(50)) of 1.5 +/- 0.93 microM for nevirapine, 0.21 +/- 0.099 microM for efavirenz, 7.1 +/- 3.2 microM for delavirdine, 0.42 +/- 0.15 microM for azidothymidine triphosphate and 0.059 +/- 0.018 microM for didehydrothymidine triphosphate. The increase in IC(50) value for RT with drug resistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs. CONCLUSION: RT derived from virions recovered from the plasma of HIV infected individuals can be used for analysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysing phenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs.

Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3'-azido-2',3'-deoxythymidine-resistant HIV isolates.

Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC(50)), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC(50) values and the sensitivity of the corresponding virus to AZT in cell culture (r=0.60, P<0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT(50)), revealed a more than 600-fold difference between the different isolate RTs. For the majority of enzymes there was a strict correlation between the results from the two assays; however, four isolates exhibited significantly higher CT(50)/IC(50) ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215-->Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39-->Ala (isolates 80 and 157). The Thr-39-->Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.