A qualitative PCR minipool strategy to screen for virologic failure and antiretroviral drug resistance in South African patients on first-line antiretroviral therapy

https://doi.org/10.1016/j.jcv.2014.05.011Get rights and content

Highlights

Abstract

Background

The high cost of commercial HIV-1 viral load tests for monitoring of patients on antiretroviral treatment limits their use in resource-constrained settings. Commercial genotypic antiretroviral resistance testing is even more costly, yet it provides important benefits.

Objectives

We sought to determine the sensitivity and negative predictive value of a qualitative PCR targeting partial reverse transcriptase for detection of virologic failure when 5 patient specimens are pooled.

Study design

A total of 300 South African routine patient samples were included and tested in 60 pools of 5 samples each. A qualitative nested PCR was optimised for testing pools and individual samples from positive pools. All positive samples were sequenced to detect drug resistance-associated mutations. Results were compared to those of conventional viral load monitoring.

Results

Twenty-two of 60 pools tested positive. Individual testing yielded 29 positive individual samples. Twenty-six patients had viral loads of above 1000 copies/ml. The pooling algorithm detected 24 of those 26 patients, resulting in a negative predictive value of 99.3%, and a positive predictive value of 89.7%. The sensitivity for detecting patients failing therapy was 92%, with a specificity of 98.9%. Of the patients failing first-line ART, 83.3% had NRTI and 91.7% NNRTI resistance mutations.

Conclusions

The pooled testing algorithm presented here required 43% fewer assays than conventional viral load testing. In addition to offering a potential cost saving over individual viral load testing, it also provided drug resistance information which is not available routinely in resourced-limited settings.

Section snippets

Background

The enormous increase in the number of patients receiving antiretroviral therapy (ART) worldwide is a remarkable success story but also poses a considerable burden on resource-limited countries due to the cost of drugs, medical care and laboratory monitoring [1].

As part of its public health approach, the World Health Organisation (WHO) recommends non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line ART regimens for adult patients and for those failing first-line ART a boosted

Objectives

This study aimed to validate and adapt this approach for a Southern African setting, where HIV-1 subtype C is most prevalent, with the specific aim of determining the sensitivity and negative predictive value the qualitative PCR targeting partial reverse transcriptase for detection of virologic failure when 5 patient specimens are pooled, and to determine the prevalence of mutations detected by sequencing the PCR product of individual specimens from positive pools.

Study population

Samples received at the Diagnostic Virology Laboratory of the National Health Laboratory Service (NHLS) Tygerberg in Cape Town, South Africa, between May 2013 and June 2013 for routine HIV viral load testing were sequentially selected if they met the following inclusion criteria: adult patient; on first-line ART; no HIV viral load testing in the previous 4 months; sufficient specimen volume left after routine testing.

Specimens

Routine viral load testing, using the Abbott RealTime HIV-1 assay with a limit

Results

Table 2 shows the ART data for the study population. The majority of patients were on the current first-line ART regimen in South Africa, consisting of tenofovir, lamivudine and efavirenz.

Twenty-two of the 60 pools were positive. Individual testing revealed 29 (9.7%) samples with a detectable viral load. Of these, 26 samples (8.7%) had viral loads of above 1000 viral RNA copies/ml and were therefore defined as failing ART. Pooled testing detected 24 of those 26 patients. The two samples that

Discussion

The pooled testing algorithm presented here was able to detect 24 of 26 adult patients failing first-line ART according to WHO and South African national criteria and provided drug resistance information on the failing patients.

Pooled viral load testing has been shown to decrease the cost of virological monitoring in adults on first-line ART who have a low prevalence of failure [14], [23]. Qualitative rather than quantitative detection of failure has previously been shown to reduce the cost of

Funding

We were assisted with financial support from the National Health Laboratory Service Research Trust, the Polio Research Foundation and the International Research Training Group (IRTG) 1522 “HIV/AIDS and associated infectious diseases in Southern Africa”.

Competing interests

We hereby declare that there is no conflict of interest.

Ethical approval

The study protocol was approved by the health research ethics committee of Stellenbosch University (reference S12/03/064).

Acknowledgements

We would like to express our gratitude to the NHLS staff that assisted in the study. GvZ initiated the study, HN and LB performed the experiments and analysed the results, under guidance and supervision by GvZ, WP and GS. HN, WP, GvZ, GS, LB wrote and revised the manuscript.

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