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As dolutegravir (DTG)-containing HIV regimens are scaled up globally, monitoring for HIV drug resistance (HIVDR) will become increasingly important. We designed a partially multiplexed HIVDR assay using Sanger sequencing technology to monitor HIVDR mutations in the protease, reverse-transcriptase (PRRT), and integrase (INT). A total of 213 clinical and analytical plasma and dried blood spot (DBS) samples were used in the evaluation. The assay detected a wide range of known HIV-1 subtypes and circulating recombinant forms (CRFs) of group M from 139 samples. INT accuracy showed that the average nucleotide (nt) sequence concordance was 99.8% for 75 plasma samples and 99.5% for 11 DBS samples compared with the reference sequences. The PRRT accuracy also demonstrated the average nucleotide sequence concordance was 99.5% for 57 plasma samples and 99.2% for 33 DBS samples. The major PRRT and INT DR mutations of all samples tested were concordant with those of the reference sequences using the Stanford HIV database (db). Amplification sensitivity of samples with viral load (VL) >5000 copies/mL showed plasma exceeded 95% of positivity, and DBS exceeded 90% for PRRT and INT. Samples with VL (1000 to 5000 copies/mL) showed plasma exceeded 90%, and DBS reached 88% positivity for PRRT and INT. Assay precision and reproducibility showed >99% nucleotide sequence concordance in each set of replicates for PRRT and INT. In conclusion, this HIVDR assay met WHO HIVDR assay performance criteria for surveillance, worked for plasma and DBS, used minimal sample volume, was sensitive, and was a potentially cost-effective tool to monitor HIVDR mutations in PRRT and INT. IMPORTANCE This HIVDR genotyping assay works for both plasma and DBS samples, requires low sample input, and is sensitive. This assay has the potential to be a user-friendly and cost-effective HIVDR assay because of its partially multiplexed design. Application of this genotyping assay will help HIVDR monitoring in HIV high-burdened countries using a DGT-based HIV drug regimen recommended by the U.S. President's Emergency Plan for AIDS Relief and the WHO.

Background: Children living with human immunodeficiency virus (HIV) (CLHIV) receiving antiretroviral therapy (ART) in resource-limited settings are susceptible to high rates of acquired HIV drug resistance (HIVDR), but few studies include children initiating age-appropriate World Health Organization (WHO)-recommended first-line regimens. We report data from a cohort of ART-naive South African children who initiated first-line ART., Methods: ART-eligible CLHIV aged 0-12 years were enrolled from 2012 to 2014 at 5 public South African facilities and were followed for up to 24 months. Enrolled CLHIV received standard-of-care WHO-recommended first-line ART. At the final study visit, a dried blood spot sample was obtained for viral load and genotypic resistance testing., Results: Among 72 successfully genotyped CLHIV, 49 (68.1%) received ABC/3TC/LPV/r, and 23 (31.9%) received ABC/3TC/EFV. All but 2 children on ABC/3TC/LPV/r were <3 years, and all CLHIV on ABC/3TC/EFV were ≥3 years. Overall, 80.6% (58/72) had at least one drug resistance mutation (DRM). DRMs to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) were found among 65% and 51% of all CLHIV, respectively, with no statistical difference by ART regimen. More CLHIV on ABC/3TC/EFV, 47.8% (11/23), were found to have 0 or only 1 effective antiretroviral drug remaining in their current regimen compared to 8.2% (4/49) on ABC/3TC/LPV/r., Conclusions: High levels of NNRTI and NRTI DRMs among CLHIV receiving ABC/3TC/LPV/r suggests a lasting impact of failed mother-to-child transmission interventions on DRMs. However, drug susceptibility analysis reveals that CLHIV with detectable viremia on ABC/3TC/LPV/r are more likely to have maintained at least 2 effective agents on their current HIV regimen than those on ABC/3TC/EFV., (Published by Oxford University Press for the Infectious Diseases Society of America 2020.)

Background: Virologic failure (VF) is highly prevalent in sub-Saharan African children on antiretroviral therapy (ART) and is often associated with human immunodeficiency virus drug resistance (DR). Most children still lack access to routine viral load (VL) monitoring for early identification of treatment failure, with implications for the efficacy of second-line ART., Methods: Children aged 1 to 14 years on ART for ≥12 months at 6 public facilities in Maputo, Mozambique were consecutively enrolled after informed consent. Chart review and caregiver interviews were conducted. VL testing was performed, and specimens with ≥1000 copies/mL were genotyped., Results: Of the 715 children included, the mean age was 103 months, 85.8% had no immunosuppression, 73.1% were taking stavudine/lamivudine/nevirapine, and 20.1% had a history prevention of mother-to-child transmission exposure. The mean time on ART was 60.0 months. VF was present in 259 patients (36.3%); 248 (95.8%) specimens were genotyped, and DR mutations were found in 238 (96.0%). Severe immunosuppression and nutritional decline were associated with DR. M184V and Y181C were the most common mutations. In the 238 patients with DR, standard second-line ART would have 0, 1, 2, and 3 effective antiretrovirals in 1 (0.4%), 74 (31.1%), 150 (63.0%), and 13 (5.5%) patients, respectively., Conclusion: This cohort had high rates of VF and DR with frequent compromise of second-line ART. There is urgent need to scale-up VL monitoring and heat-stable protease inhibitor formulations or integrase inhibitorsfor a more a durable first-line regimen that can feasibly be implemented in developing settings., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

Background: The main objective of this study was to determine the frequency and patterns of HIV drug resistance-associated mutations among children under 18 months of age born to HIV-1-positive mothers enrolled in the prevention of mother-to-child transmission services in Haiti., Methods: Between January 1, 2013 and December 31, 2014, HIV-positive remnant dried blood spots collected from children under 18 months of age for Early Infant Diagnosis at the National Public Health Laboratory were used for HIV-1 genotyping. HIV drug resistance mutations were analyzed using the Stanford Drug Resistance HIVdb program., Results: Of the 3555 dried blood spots collected for Early Infant Diagnosis, 360 (10.1%) were HIV-positive and 355 were available for genotyping. Of these, 304 (85.6%) were successfully genotyped and 217 (71.4%) had ≥1 drug resistance mutation. Mutations conferring resistance to nucleoside reverse transcriptase inhibitor (NRTIs) and non-NRTIs were present in 40.5% (123) and 69.1% (210), respectively. The most frequent mutations were K103N/S (48.0%), M184V (37.5%), G190A/S (15.1%), and Y181C/G/V (14.1%). Predicted drug resistance analysis revealed that 68.8% of the children had high-level resistance to non-NRTIs and 11.5% had intermediate to high-level resistance to abacavir., Conclusions: This study showed high rates of resistance to NRTIs and non-NRTIs among newly HIV-diagnosed children in Haiti, suggesting that in the era of "Option B+" (initiation of lifelong combination antiretroviral therapy to pregnant women with HIV), the majority of children who acquire HIV infection through mother-to-child transmission of HIV have resistant HIV. These results have led the National HIV Program to revise the pediatric guidelines to include protease inhibitors in first-line regimens for all HIV-positive newborns.

Background: Continued use of standardized, first-line ART containing NNRTIs and NRTIs may contribute to ongoing emergence of HIV drug resistance (HIVDR) in Namibia., Methods: A nationally representative cross-sectional survey was conducted during 2015-16 to estimate the prevalence of significant pretreatment HIV drug resistance (PDR) and viral load (VL) suppression rates 6-12 months after initiating standardized first-line ART. Consenting adult patients (≥18 years) initiating ART were interviewed about prior antiretroviral drug (ARV) exposure and underwent resistance testing using dried blood spot samples. PDR was defined as mutations causing low-, intermediate- and high-level resistance to ARVs according to the 2014 WHO Surveillance of HIV Drug Resistance in Adults Initiating ART. The prevalence of PDR was described by patient characteristics, ARV exposure and VL results. Results were weighted to be nationally representative., Results: Successful genotyping was performed for 381 specimens; 144 (36.6%) specimens demonstrated HIVDR, of which 54 (12.7%) demonstrated PDR. Resistance to NNRTIs was most prevalent (11.9%). PDR was higher in patients with previous ARV exposure compared with no exposure (30.5% versus 9.6%) (prevalence ratio = 3.17; P < 0.01)., Conclusions: This survey demonstrated overall PDR at >10% among adults initiating ART in Namibia. Patients with prior ARV exposure had higher rates of PDR. Introducing a non-NNRTI-based regimen for first-line ART should be considered to maximize benefit of ART and minimize the emergence of HIVDR.

Introduction: Surveillance of HIV drug resistance (HIVDR) is crucial to ensuring the continued success of antiretroviral therapy (ART) programs. With the concern of reduced genotyping sensitivity of HIV on dried blood spots (DBS), DBS for HIVDR surveillance have been limited to ART-naïve populations. To investigate if DBS under certain conditions may also be a feasible sample type for HIVDR testing in ART patients, we piloted nationwide surveys for HIVDR among ART patients using DBS in two African countries with rapid scale-up of ART., Methods: EDTA-venous blood was collected to prepare DBS from adult and pediatric ART patients receiving treatment during the previous 12-36 months. DBS were stored at ambient temperature for two weeks and then at -80°C until shipment at ambient temperature to the WHO-designated Specialized HIVDR Laboratory at CDC in Atlanta. Viral load (VL) was determined using NucliSENS EasyQ® HIV-1 v2.0 kits; HIVDR genotyping was performed using the ATCC HIV-1 Drug Resistance Genotyping kits., Results: DBS were collected from 1,368 and 1,202 ART patients; 244 and 255 these specimens had VL ≥1,000 copies/mL in Kenya and Tanzania, respectively. The overall genotyping rate of those DBS with VL ≥1,000 copies/mL was 93.0% (95% CI: 89.1%-95.6%) in Kenya and 91.8% (87.7%-94.6%) in Tanzania. The turnaround times for the HIVDR surveys from the time of collecting DBS to completing laboratory testing were 6.5 months and 9.3 months for the Kenya and Tanzania surveys, respectively., Conclusions: The study demonstrates a favorable outcome of using DBS for nationwide surveillance of HIVDR in ART patients. Our results confirm that DBS collected and stored at ambient temperature for two weeks, and shipped with routine courier services are a reliable sample type for large-scale surveillance of acquired HIVDR., Competing Interests: Chunfu Yang, Nicolas Wagar and Joshua DeVos are the inventors in U.S. patent application PCT/US2012/045523. This does not alter the authors’ adherence to all the journal’s policies on sharing data and materials.

A survey of 461 HIV-infected Kenyan children receiving antiretroviral therapy found 143 (31%) failing virologically. Drug resistance mutations were found in 121; 37 had L74V/I mutations, with 95% receiving abacavir (ABC)-containing regimens. L74V/I was associated with current ABC usage (P = 0.0001). L74V/I may be more prevalent than previously realized in children failing ABC-containing regimens, even when time on treatment has been short. Ongoing rigorous pediatric drug resistance surveillance is needed.

Background: Zimbabwe set up 12 sentinel sites to monitor HIV drug resistance (HIVDR) following the international standards for prevention of HIVDR from 2008 to 2010., Methods: Participants were consecutively enrolled. Blood was collected and used for CD4 count, viral load (VL) and pre-treatment DR (PDR) tests besides routine baseline tests. We analyzed the characteristics of participants enrolled into the survey and estimated the point prevalence of PDR and its associated factors among ART initiators in a cross-sectional analysis using the baseline data collected from a prospective cohort in 12 purposefully selected sentinel sites., Results: A total of 1728 participants (96 % response rate) were enrolled and 1610 had complete data. Of the 1610 there were more females (68.7 %) than males (31.3 %). The median CD4 count was 168 cells/mm(3) with males having lower values (P = 0.003). Ninety-six percent of participants had a VL ≥ 1000 copies/ml and the median VL was 128,000. Previous exposure to antiretroviral drugs (ARVs) was mainly through PMTCT (5 % of the participants). Overall, PDR mutations were detected in 6.3 % (95 % CI 5.2-7.7) of the 1480 successfully genotyped participants. However, the prevalence of PDR mutations was double for those with previous exposure (12.1 %) to ARVs compared with those without previous exposure (5.7 %, P = 0.002)., Conclusions: The results show a moderate level of PDR prevalence among ART initiators. To maintain the efficacy of the current first-line regimens, there is need to strengthen all HIVDR prevention efforts and to conduct further studies to investigate optimal strategies that can prolong the efficacy of first-line ARV regimens in the country.

Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at -80 °C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.