To the Editors:
HIV viral load (VL) in conjunction with CD4+ T-cell counts are routine laboratory tests for monitoring HIV-infected patients.1,2 Conventional nucleic acid HIV VL assays are expensive, require skilled technicians, and are subject to laboratory contamination, making their use less feasible within many resource-constrained countries.2-4 An alternative low-cost HIV VL assay, developed mainly for use in resource-constrained countries, is the Cavidi ExaVir Load (Cavidi) reverse transcriptase (RT) assay,5 which has been shown to compare well to nucleic acid-based assays.6,7 As the Cavidi assay measures RT activity, the question has been raised as to whether drugs that bind tightly to HIV RT (such as efavirenz) may be incorporated into the virus and interfere with this assay. Nucleoside analogue reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are known to decrease the activity of RT. Many of these mutations directly affect the ability of RT to incorporate nucleoside analogs,8-10 and therefore, the possibility exists that these mutations may also negatively impact on the enzyme's ability to incorporate the Bromo-deoxyuridine-triphosphate (BrdUTP) substrate in the Cavidi assay.5 We have compared VL results obtained using the Cavidi assay and conventional nucleic acid-based VL testing from patients being treated with RT inhibitors or with resistance mutations within RT to investigate the influence of antiretroviral drugs and mutations on the performance of the Cavidi assay.
Plasma from EDTA anticoagulated blood was collected from HIV-seropositive patients with a previous VL between 50 and 100 000 copies per milliliter as determined by the Roche COBAS Amplicor Monitor v1.5, ultrasensitive assay, reverse transcriptase-polymerase chain reaction (RT-PCR; Roche Diagnostics, Branchburg, NJ). Samples were tested by RT-PCR and the Cavidi Exavir Load RT assay version 2 (Cavidi AB, Uppsala, Sweden), according to manufacturer's instructions.5 The Cavidi version 2 assay has a lower limit of quantitation of 400 copies per milliliter equivalents (the RT assay result is converted to RNA copies/mL equivalents using supplied software). Genotype analysis was performed using the ViroSeq HIV-1 genotyping system V 2.0 (Celera Diagnostics, LLC, Alameda, CA) and submitted to the Stanford HIV Resistance Database for drug resistance interpretation. Patients were classified as NRTI or NNRTI users if they had been prescribed at least 1 medication of that class at the time of resistance testing, whereas patients defined as receiving “no therapy” included both therapy naive and those undergoing treatment interruptions. Pearson correlation was used to measure association. All other analyses were completed using clustered linear regression.
Plasma samples (n = 396) from 261 HIV-seropositive patients were analyzed by both RT-PCR and Cavidi assays with a strong and significant correlation between the 2 assays (r = 0.87; P < 0.0001). The difference between the Cavidi and RT-PCR VL result was not significant between patients receiving NRTIs (n = 187) versus those receiving no therapy (n = 99; P = 0.24; 95% CI: −0.04 to 0.18). Furthermore, Cavidi versus RT-PCR results for patients receiving both NRTIs and NNRTIs (n = 108) were also found not to differ significantly from patients receiving no therapy (P = 0.71; 95% CI: −0.10 to 0.15). The median log10 transformed difference of the assays showed minimal variation between patients taking no therapy, NRTI alone, or NRTI + NNRTI (0.01; 0.11; and 0.09 log10 copies/mL, respectively; Fig. 1). As there were only 2 patients receiving only NNRTI therapy, a statistical analysis was not performed for this group. An additional statistical analysis comparing samples from patients receiving either NRTI or NRTI + NNRTI therapy to all other results confirmed that there was no significant difference between any therapy regimen. Further separation of the “no therapy” group into treatment-naive patients and treatment-experienced patients did not reveal any significant differences between the treatment groups. These results suggest that the performance of the Cavidi assay is not significantly affected by either NRTI or NNRTI therapy.
There was no significant difference between patients receiving no therapy (n = 99) and those treated with either efavirenz (n = 45; P = 0.56; 95% CI: −0.13 to 0.24) or nevirapine (n = 65; P = 0.92; 95%CI: −0.15 to 0.13, Fig. 1). Minimal variation was observed in the median log10 transformed difference of the assays between patients taking no therapy, efavirenz, or nevirapine (0.01; −0.01; and 0.10 log10 copies/mL respectively; Fig. 1). There was also no significant difference observed between the nevirapine and efavirenz groups (P = 0.54) suggesting that neither efavirenz nor nevirapine significantly affected the Cavidi assay results despite efavirenz binding particularly tightly to the HIV RT enzyme.
There was no significant difference between the Cavidi and RT-PCR assay results for patients with (n = 32) or without (n = 6) NRTI resistance-associated mutations (P = 0.24; 95% CI: −0.89 to 0.23; data not shown). When data were analyzed from patients with 1-3 (n = 10), 4-6 (n = 6), and 7 (n = 16) individual NRTI mutations, there was no significant difference between 1-3 mutations (P = 0.35; 95% CI: −0.94 to 0.34), 4-6 mutations (P = 0.38; 95% CI: −0.88 to 0.35), and 7 mutations (P = 0.20; 95% CI: −0.95 to 0.20) compared with patients with no mutations (n = 6). Similarly, the difference between the Cavidi and RT-PCR VL assay results for samples with NNRTI resistance-associated mutations (n = 28) did not vary significantly from those samples without NNRTI mutations (n = 10; P = 0.43; 95% CI: −0.42 to 0.18). The most common mutations observed were at positions 41, 103, 184, and 215 and were observed in 63%, 61%, 84%, and 66% of patients, respectively. Although the sample size tested here was small, the data suggest that the presence of resistance-associated mutations within RT does not affect the performance of the Cavidi assay, even within samples containing up to 7 mutations.
The Cavidi assay is currently being implemented in countries with limited resources. Our data confirm previous findings from our group and others that the Cavidi assay shows an excellent association with the RT-PCR assay.5-7,11,12 This study shows that although the Cavidi assay measures RT activity, current antiretroviral therapies that inhibit RT (NRTIs and NNRTIs) do not significantly interfere with the assay. Our data show no difference between the Cavidi and RT-PCR assay results for samples containing either NRTI or NNRTI mutations as compared with those containing no mutations. In our earlier study,6 we reported a decrease in VL of 0.20 log10 when NNRTI mutations were present, which we considered is unlikely to be of clinical significance. The results presented here are likely to be more accurate as only samples where a genotype was performed on the same sample assessed for VL were used, whereas our earlier study used samples where the “matching” genotype was performed on a sample within 100 days of the VL sample date.
Our data suggest that the Cavidi assay is an acceptable assay for monitoring VL in patients on RT inhibitor medication and carrying HIV quasispecies with RT mutations.
We would like to acknowledge Cavidi for kindly donating the kits for this study and thank Prof Ben Berkhout and Erna Albers for support of Lisette B. van Rooijen.
Lisette B. van Rooijen, MSc*†
Vicki Greengrass, BSc*
Lisa M. Morris, BSc, MBA*
Megan M. Plate, BSc*
Maelenn Gouillou, MSc‡
Gilda Tachedjian, PhD§‖¶
Nicolas Sluis-Cremer, PhD#
Anna C. Hearps, PhD*
Suzanne M. Crowe, MD*¶
*Clinical Research Laboratory, Burnet Institute, Melbourne, Victoria, Australia
†VU University Medical Centre, Amsterdam, The Netherlands
‡Centre for Population Health, Burnet Institute, Melbourne, Victoria, Australia
§Molecular Interactions Group, Burnet Institute, Melbourne, Victoria, Australia
‖Department of Microbiology, Monash University, Clayton, Victoria, Australia
¶Department of Medicine, Monash University, Melbourne, Victoria, Australia
#Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA.
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