Performance characteristics of a modified HIV-1 drug resistance genotyping method for use in resource-limited settings

Samples

A total of 26 blood samples were collected in EDTA tubes from patients attending Kenyatta National Hospital HIV clinic laboratory for routine viral load monitoring. Plasma was separated by centrifugation at 2000g for 10 minutes. The plasma samples were stored at -80°C freezer (Nuve, Ankara, Turkey). Remnant plasma samples were used for this study.

Ethical approval

Ethical clearance for the study was obtained from University of Nairobi-Kenyatta National Hospital Ethics Review Committee (UoN-KNH-ERC). The need for consent was waived since remnant plasma from HIV viral load testing were used in this study.

Viral RNA extraction

HIV-1 RNA was extracted from 500 µl plasma using the PureLink^TM extraction kit according to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Briefly, 25 μl proteinase K and 500 μl plasma were transferred into 2-ml microcentrifuge tube. Lysis buffer (500 μl) was added to the mixture, vortexed for 15 seconds, and incubated at 56°C for 15 minutes. Next, 500 μl of 96% ethanol was added to the reaction tube, and vortexed for 15 seconds and incubated at room temperature for 5 minutes. The lysate was transferred to sterile viral spin column, washed twice with 500 μl of wash buffer and finally eluted in 40 μl of elution buffer. RNA was stored at -80°C. The extraction procedure was similar for both the original and modified method.

HIV DRT

Original genotyping method. The Thermo Fisher Scientific^TM HIV Genotyping workflow that amplifies a 1.1-kb fragment covering codons 6–99 of the protease gene and codons 1–251 of the reverse transcriptase (RT) gene was implemented according to manufacturer’s instructions¹⁰. Briefly, reverse transcriptase PCR and nested PCR were performed using the HIV-1 Genotyping Kit: Amplification Module. Primers used are shown in Table 1. Cycle sequencing was performed using the HIV-1 Genotyping Kit: Cycle Sequencing Module. The resulting cycle sequencing products were analyzed using an ABI 3730 genetic analyzer. The consensus sequences were generated using ReCall (requires free registration) and drug resistance mutations were interpreted using the Stanford HIVdb genotyping resistance interpretation algorithm.

Modified genotyping system Specific reaction steps of the original protocol were modified by reducing the reagent volumes by half.

The HIV Genotyping kit: Amplification module was used for RT-PCR according to the manufacturer’s instructions. Briefly, 10 µl RNA was denatured by incubating at 65°C for 10 minutes and added to 40 µl RT-PCR Master Mix containing SuperScript^TM III One-Step RT-PCR with Platinum^TMTaq High Fidelity Enzyme. The reaction conditions for RT-PCR were 50°C for 45 minutes where first-strand cDNA synthesis was performed. Enzyme inactivation and denaturation of cDNA-RNA hybrid was accomplished by incubating the reaction at 94°C for 2 minutes. Second-strand synthesis and PCR amplification was carried out in 40 cycles of 94°C for 15 seconds, 50°C for 20 seconds, 72°C for 2 minutes and a final extension for 10 minutes at 72°C using Veriti thermocycler.

Nested PCR reaction mix was prepared by adding 2 μl RT-PCR product to 23.7 μl of nested mastermix containing 0.12 mM of each of inner primers, 1X GeneAmp Gold Buffer II, 2 mM MgCl₂, 400 mM each dNTP and 0.25 μl AmpliTaq Gold™ LD DNA Polymerase enzyme (Applied Biosystem, CA, USA). The target pol region was amplified using Veriti Thermocycler in a final reaction volume of 25.7 µl (Applied Biosystems, CA, USA). The reaction conditions were 94⁰C for 4 minutes, 40 cycles of 94°C for 15 seconds, 55°C for 20 seconds, 72°C for 2 minutes and a final extension at 72°C for 10 minutes. Nested PCR products of 1.1 kb were confirmed by gel electrophoresis. A 5 μl aliquot of nested PCR product was added to 4 μl ExoSAP-IT^TM PCR Purification Reagent) and incubated at 37°C for 15 min and 80°C for 15 minutes in a Veriti thermocycler.

Cycle sequencing was performed using the HIV Genotyping kit: Cycle Sequencing module. Six overlapping primers, labelled F1, F2, F3, R1, R2, and R3 were used. One microliter nested PCR product was added to 9 µl of each cycle sequencing mix. The cycle sequencing reaction conditions were 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes.

The Big Dye XTerminator purification kit was used to purify the sequencing reaction by adding 10 µl of the Big Dye XTerminator and 45 µl SAM solution to cycle sequencing products. The reaction plate was vortexed at 1,800 rpm for 30 minutes. The plate was then centrifuged at 1000g for 2 minutes at room temperature. Next, 30 μl of purified cycle sequencing products were transferred to a reaction plate and analyzed using an ABI 3730 genetic analyzer (Applied Biosystem, CA, USA). Sequences were generated using ReCall and drug resistance mutations interpreted using the Stanford HIVdb genotyping resistance interpretation algorithm (output files available as Extended data¹²) and the International AIDS Society (IAS) 2011 mutation list¹³.

Modified assay validation

Performance characteristics of the modified assay were assessed using the WHO/HIV ResNet guidelines, including accuracy, precision, reproducibility and amplification sensitivity¹⁴.

Accuracy. Accuracy refers to the agreement between a result and an expected reference value. In genotyping assays, nucleotide sequence identity is used. Ten samples were analyzed using both methods and the degree of concordance in mutations identified was compared based on the 2017 IAS mutations list¹³. Nucleotide sequence identity between the paired sequences was assessed using the EMBOSS pairwise alignment tool. The WHO recommends 90% nucleotide sequence similarity as the cutoff point for assay performance characteristics¹⁴.

Precision. This is the ability of an assay to generate the same result on multiple replicates of the same sample within a test run. Three samples were analyzed using the modified method in n=4 replicates. The degree of concordance of detected mutations within replicates was determined. Nucleotide sequence identity was also determined using the EMBOSS program for pairwise alignment¹⁴.

Reproducibility. This is the ability of a test to produce the same result on multiple aliquots of the same sample in different test runs. Ten samples were analyzed in duplicates using the modified method on different days. Nucleotide sequence identity of sequences obtained was assessed using EMBOSS program for pairwise alignment¹⁴.

Amplification sensitivity. Amplification sensitivity is defined as the percentage of successful genotyping tests amongst specimens with a specific viral load range. In this study, sixteen samples with viral loads ranging between 207 and 86,040 copies/ml were analyzed using the modified assay to determine the viral load ranges at which ≥95% of the samples were successfully genotyped¹⁴.

Reagent cost comparison

An ingredient costing approach was utilized to estimate reagent costs for both original and modified assays at RNA extraction, DNA/RNA amplification, gel electrophoresis, and sequencing steps. All costs were converted to US dollar using 2019 conversion rate.

Statistical analyses

Quantitative variables were expressed as mean ± standard deviations (SD). The McNemar test was used to assess significance in the discordant mutations between the modified and the original assay. Precision and reproducibility were assessed using the Cohen kappa statistic. Wilcoxon signed-rank test was used to compare the original and modified assays in base calling for mixed bases between the two methods. Statistical Package for Social Sciences (SPSS) version 22 (IBM, NY, USA) was used for all data analysis.

Validation of the modified HIV-1 drug resistance genotyping assay

Accuracy of sequence identity and mutation detection. The mean nucleotide identity was 98.5% (confidence interval (CI), 97.92–99.1%). A total of 68 mutations were detected, including 9 (13%) mutations in the protease gene and 59 (87%) in the reverse transcriptase (RT) gene as shown in Table 2. The original assay detected 67 drug resistance mutations, including 2 mutations (V106I and K225H) which were missed by the modified assay. On the other hand, the modified assay detected 68 drug resistance mutations, including four mutations (D67DG, K101KPQT, K70KN and I50IL) which were not detected by the original method. A total of six mutations were discordant (V106I, P225H, D67DN, I50IL, K101KPQT, and K70KN). Of the six discordant drug resistance mutations, two were completely discordant (V106I and P225H) while four (D67DN, K101KPQT, K70KN and I50IL) were partially discordant. Three of the discordant mutations (D67DN, K101KPQT, and K70KN) were detected as mixtures in the modified assay as non-mixtures (D67N, K101P and K70N) in the original assay and as shown in Table 3. A high mutation concordance was obtained between the two assays at χ² = 2.36, p = 0.26. The significance of mixture base-calling between the two methods was determined using the Wilcoxon signed rank test, which showed no significant difference in mixtures detected by the two methods at p = 0.089.

Precision and reproducibility of the modified assay. Assessment to examine the precision (intra-assay precision) and reproducibility (inter-assay precision) of the modified assay were performed by analyzing n=3 samples in quadruplicate in a single test run for precision. The mean nucleotide sequence identity within the replicates was 98.67% (CI, 98.1–99.3). Reproducibility was assessed by testing 10 samples in duplicate on different days using the modified assay. The mean nucleotide sequence identity was 98.6% (CI, 98.2–99.0). The overall agreement of drug resistance mutations detected by precision and reproducibility was significant at kappa value of 0.792 and 0.778, respectively, as shown in Table 4.

Amplification sensitivity of the modified assay. A total of 16 samples with a median viral load of 832.5 copies/ml (IQR 403.5–2454.25) were used to assess amplification sensitivity. The modified assay showed an amplification sensitivity of 100% for samples with viral load ≥1000 copies/ml and 62.5% for samples with viral load <1000 copies/ml as shown in Table 5.

Comparison of costs of reagents between the original and modified assays

After a 50% reduction in reagent volumes in the amplification and sequencing reactions, we compared the cost between the original and the modified method. The cost of analyzing one sample from extraction to sequencing was decreased from $97 to $59. This represents about 39.2% cost reduction as shown in Table 6.

Underlying data

GenBank accession numbers for HIV-1 pol and gag sequences isolated from each participant using each technique are available in Table 7.

Extended data

Figshare: Performance Characteristics of a Modified HIV-1 Drug Resistance Genotyping Method for use in Resource Limited Settings: https://doi.org/10.6084/m9.figshare.9114833¹².

This study contains the following extended data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).