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1.  Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay 
Journal of Clinical Microbiology  2013;51(11):3666-3674.
High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.
doi:10.1128/JCM.01669-13
PMCID: PMC3889749  PMID: 23985909
2.  Prevalence of HIV Drug Resistance Before and 1 Year After Treatment Initiation in 4 Sites in the Malawi Antiretroviral Treatment Program 
Since 2004, the Malawi antiretroviral treatment (ART) program has provided a public health–focused system based on World Health Organization clinical staging, standardized first-line ART regimens, limited laboratory monitoring, and no patient-level monitoring of human immunodeficiency virus drug resistance (HIVDR). The Malawi Ministry of Health conducts periodic evaluations of HIVDR development in prospective cohorts at sentinel clinics. We evaluated viral load suppression, HIVDR, and factors associated with HIVDR in 4 ART sites at 12–15 months after ART initiation. More than 70% of patients initiating ART had viral suppression at 12 months. HIVDR prevalence (6.1%) after 12 months of ART was low and largely associated with baseline HIVDR. Better follow-up, removal of barriers to on-time drug pickups, and adherence education for patients 16–24 years of age may further prevent HIVDR.
doi:10.1093/cid/cir987
PMCID: PMC3338306  PMID: 22544204
3.  Prevalence of Transmitted HIV Drug Resistance Among Newly Diagnosed Antiretroviral Therapy–Naive Pregnant Women in Lilongwe and Blantyre, Malawi 
In 2006, a survey of transmitted human immunodeficiency virus (HIV) drug resistance (TDR) was conducted in Lilongwe, Malawi. The survey followed the World Health Organization method to classify TDR to nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) among primigravid women aged <25 years. Results of the 2006 survey showed <5% TDR in all drug classes. In 2009, TDR surveys using the same method were repeated in Lilongwe and expanded to Blantyre. Findings show that in Lilongwe TDR to NRTIs and PIs was <5%, whereas TDR to NNRTIs was 5%–15%. In Blantyre, TDR was <5% to all drug classes. Observed moderate TDR in Lilongwe is cause for concern and signals the need for closer monitoring of Malawi’s antiretroviral therapy program.
doi:10.1093/cid/cir993
PMCID: PMC3338309  PMID: 22544197
4.  A Retrospective Survey of HIV Drug Resistance Among Patients 1 Year After Initiation of Antiretroviral Therapy at 4 Clinics in Malawi 
In 2004, Malawi began scaling up its national antiretroviral therapy (ART) program. Because of limited treatment options, population-level surveillance of acquired human immunodeficiency virus drug resistance (HIVDR) is critical to ensuring long-term treatment success. The World Health Organization target for clinic-level HIVDR prevention at 12 months after ART initiation is ≥70%. In 2007, viral load and HIVDR genotyping was performed in a retrospective cohort of 596 patients at 4 ART clinics. Overall, HIVDR prevention (using viral load ≤400 copies/mL) was 72% (95% confidence interval [CI], 67%–77%; range by site, 60%–83%) and detected HIVDR was 3.4% (95% CI, 1.8%–5.8%; range by site, 2.5%–4.7%). Results demonstrate virological suppression and HIVDR consistent with previous reports from sub-Saharan Africa. High rates of attrition because of loss to follow-up were noted and merit attention.
doi:10.1093/cid/cis004
PMCID: PMC3338311  PMID: 22544203
5.  Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings 
PLoS ONE  2011;6(11):e28184.
Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.
Conclusions
The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
doi:10.1371/journal.pone.0028184
PMCID: PMC3223235  PMID: 22132237
6.  Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance ▿  
Journal of Clinical Microbiology  2010;48(9):3158-3164.
As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.
doi:10.1128/JCM.00564-10
PMCID: PMC2937690  PMID: 20660209

Results 1-6 (6)