High prevalence of pre-treatment HIV drug resistance in Papua New Guinea: findings from the first nationally representative pre-treatment HIV drug resistance study.

BACKGROUND: Determining the prevalence of pre-treatment HIV drug resistance (PDR) is important to assess the effectiveness of first-line therapies. To determine PDR prevalence in Papua New Guinea (PNG), we conducted a nationally representative survey. METHODS: We used a two-stage cluster sampling method to recruit HIV treatment initiators with and without prior exposure to antiretroviral therapies (ART) in selected clinics. Dried blood spots were collected and tested for PDR. RESULTS: A total of 315 sequences were available for analysis. The overall PDR prevalence rate was 18.4% (95% CI 13.8-24.3%). The prevalence of PDR to non-nucleoside analog reverse-transcriptase inhibitors (NNRTIs) was 17.8% (95% CI 13.6-23.0%) and of PDR to nucleoside reverse transcriptase inhibitors (NRTIs) was 6.3% (95% CI 1.6-17.1%). The PDR prevalence rate among people reinitiating ART was 42.4% (95% CI 29.1-56.4%). CONCLUSIONS: PNG has a high PDR prevalence rate, especially to NNRTI-based first-line therapies. Our findings suggest that removing NNRTIs as part of first-line treatment is warranted and will lead to improving viral suppression rates in PNG.

Drug Resistance Mutations Among South African Children Living With HIV on WHO-recommended ART Regimens.

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.

A single early introduction of HIV-1 subtype B into Central America accounts for most current cases.

Human immunodeficiency virus type 1 (HIV-1) variants show considerable geographical separation across the world, but there is limited information from Central America. We provide the first detailed investigation of the genetic diversity and molecular epidemiology of HIV-1 in six Central American countries. Phylogenetic analysis was performed on 625 HIV-1 pol gene sequences collected between 2002 and 2010 in Honduras, El Salvador, Nicaragua, Costa Rica, Panama, and Belize. Published sequences from neighboring countries (n = 57) and the rest of the world (n = 740) were included as controls. Maximum likelihood methods were used to explore phylogenetic relationships. Bayesian coalescence-based methods were used to time HIV-1 introductions. Nearly all (98.9%) Central American sequences were of subtype B. Phylogenetic analysis revealed that 437 (70%) sequences clustered within five significantly supported monophyletic clades formed essentially by Central American sequences. One clade contained 386 (62%) sequences from all six countries; the other four clades were smaller and more country specific, suggesting discrete subepidemics. The existence of one large well-supported Central American clade provides evidence that a single introduction of HIV-1 subtype B in Central America accounts for most current cases. An introduction during the early phase of the HIV-1 pandemic may explain its epidemiological success. Moreover, the smaller clades suggest a subsequent regional spread related to specific transmission networks within each country.

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.

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.

Prevalence of HIV drug resistance in Nigeria: results from a cross-sectional, population-based survey of Nigerian adults with unsuppressed viral load.

BACKGROUND: HIV drug resistance (HIVDR) surveillance is an important tool to monitor threats to progress towards epidemic control. The characterization of HIVDR in Nigeria at the national level is needed to inform both clinical decisions and population-level HIV policy strategies. This study uses data obtained from the Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS) to describe the prevalence and distribution of HIVDR in Nigeria. METHODS: NAIIS was a cross-sectional, population-based survey of households throughout Nigeria in 2018. NAIIS was designed to provide estimates of HIV prevalence and related health indicators from a nationally representative sample. The study population included participants aged 15-64 years who tested positive for HIV, had a viral load at least 1000 copies/ml, and had available HIV drug resistance genotypes. HIV isolates were genotyped to detect drug resistance mutations. Individual characteristics of study participants associated with HIVDR were identified using a weighted multivariable logistic regression model. RESULTS: Of 1355 respondents with available HIV genotypes, 293 (19%) had evidence of drug-resistant mutations (DRMs) that conferred resistance to at least one antiretroviral drug. The majority of DRMs observed conferred resistance to NNRTIs (17.6%) and NRTIs (11.2%). HIVDR was associated with being ART-experienced, longer duration on ART, and lower CD4+ count but not sociodemographic characteristics. CONCLUSION: The population level DRM prevalence in Nigeria was consistent with what would be expected in a mature HIV treatment landscape. The continued roll out of dolutegravir-anchored regimens should mitigate the impact of NNRTI resistance on population viral load suppression and progress towards epidemic control.

Dried blood spot specimens are a suitable alternative sample type for HIV-1 viral load measurement and drug resistance genotyping in patients receiving first-line antiretroviral therapy.

BACKGROUND: Antiretroviral therapy (ART) is being administered in developing nations at unprecedented numbers following the World Health Organization's (WHO) development of standardized first-line drug regimens. To ensure continued efficacy of these drug regimens, WHO recommends monitoring virological responses and development of human immunodeficiency virus (HIV) drug resistance (HIVDR) in HIV-infected patients in a prospective cohort. The current study compared dried fluid spot specimens with the reference standard plasma specimens as a practical tool for viral load (VL) and HIVDR genotyping in resource-limited settings. METHODS: Dried blood spot (DBS), dried plasma spot (DPS), and plasma specimens were collected from 173 -patients receiving ART at 2 hospital sites in Abuja, Nigeria. HIV-1 VL analysis was performed using NucliSENS EasyQ HIV-1 v1.1 RUO test kits. Genotyping of the HIV-1 pol gene was performed using a broadly sensitive in-house assay. RESULTS: Direct comparison of VL levels showed that DBS specimens, and not DPS specimens, gave results comparable to those of plasma specimens (P = .0619 and .0007, respectively); however, both DBS and DPS specimens had excellent correlation with plasma specimens in predicting virological failure (VL, ≥1000 copies/mL) in patients (κ = 0.78 and 0.83, respectively). Of the 18 specimens with a plasma VL ≥1000 copies/mL, HIVDR genotyping rates were 100% in DBS and 38.9% in DPS specimens, and DBS specimens identified 61 of 65 HIVDR mutations (93.8%) identified in plasma specimens. CONCLUSIONS: Our results indicate that DBS specimens could be used for surveys to monitor HIVDR prevention failure in resource-limited settings.

Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus.

BACKGROUND: A new pandemic influenza A (H1N1) virus has emerged, causing illness globally, primarily in younger age groups. To assess the level of preexisting immunity in humans and to evaluate seasonal vaccine strategies, we measured the antibody response to the pandemic virus resulting from previous influenza infection or vaccination in different age groups. METHODS: Using a microneutralization assay, we measured cross-reactive antibodies to pandemic H1N1 virus (2009 H1N1) in stored serum samples from persons who either donated blood or were vaccinated with recent seasonal or 1976 swine influenza vaccines. RESULTS: A total of 4 of 107 persons (4%) who were born after 1980 had preexisting cross-reactive antibody titers of 40 or more against 2009 H1N1, whereas 39 of 115 persons (34%) born before 1950 had titers of 80 or more. Vaccination with seasonal trivalent inactivated influenza vaccines resulted in an increase in the level of cross-reactive antibody to 2009 H1N1 by a factor of four or more in none of 55 children between the ages of 6 months and 9 years, in 12 to 22% of 231 adults between the ages of 18 and 64 years, and in 5% or less of 113 adults 60 years of age or older. Seasonal vaccines that were formulated with adjuvant did not further enhance cross-reactive antibody responses. Vaccination with the A/New Jersey/1976 swine influenza vaccine substantially boosted cross-reactive antibodies to 2009 H1N1 in adults. CONCLUSIONS: Vaccination with recent seasonal nonadjuvanted or adjuvanted influenza vaccines induced little or no cross-reactive antibody response to 2009 H1N1 in any age group. Persons under the age of 30 years had little evidence of cross-reactive antibodies to the pandemic virus. However, a proportion of older adults had preexisting cross-reactive antibodies.

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine.

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-microg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999-2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

A Partially Multiplexed HIV Drug Resistance (HIVDR) Assay for Monitoring HIVDR Mutations of the Protease, Reverse-Transcriptase (PRRT), and Integrase (INT).

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.

High Level of HIV Drug Resistance and Virologic Nonsuppression Among Female Sex Workers in Ethiopia: A Nationwide Cross-Sectional Study.

OBJECTIVE: To determine viral load (VL) nonsuppression (VLN) rates, HIV drug resistance (HIVDR) prevalence, and associated factors among female sex workers (FSWs) in Ethiopia. METHODS: A cross-sectional biobehavioral survey was conducted among FSWs in 11 cities in Ethiopia in 2014. Whole blood was collected, and HIVDR genotyping was performed. Logistic regression analysis was performed to identify factors associated with VLN and HIVDR. RESULTS: Among 4900 participants, 1172 (23.9%) were HIV-positive and 1154 (98.5%) had a VL result. Participants were categorized into antiretroviral therapy (ART) (n = 239) and ART-naive (n = 915) groups based on self-report. From the 521 specimens (ART, 59; ART-naive, 462) with VL ≥1000 copies/mL, genotyping was successful for 420 (80.6%) and 92 (21.9%) had drug resistance mutations (DRMs). Pretreatment drug resistance (PDR) was detected in 16.5% (63/381) of the ART-naive participants. Nucleoside reverse transcriptase inhibitor (NRTI), non-NRTIs (NNRTIs), and dual-class DRMs were detected in 40 (10.5%), 55 (14.4%), and 35 (9.2%) of the participants, respectively. Among 239 participants on ART, 59 (24.7%) had VLN. Genotyping was successfully performed for 39 (66.1%). DRMs were detected in 29 (74.4%). All 29 had NNRTI, 23 (79.3%) had NRTI or dual-class DRMs. VLN was associated with age 35 years or older, CD4+ T-cell count <350 cells/mm3, and being forced into selling sex. PDR and acquired drug resistance were associated with CD4+ T-cell count <350 cells/mm3 (P < 0.001). CONCLUSIONS: The high VLN and HIVDR rates among FSWs underscore the need for targeted interventions to improve ART access and virologic monitoring to maximize the benefit of ART and limit the spread of HIV and HIVDR.

Resistance levels to non-nucleoside reverse transcriptase inhibitors among pregnant women with recent HIV infection in Malawi.

BACKGROUND: Information on HIV drug resistance (HIVDR) prevalence in people newly diagnosed with HIV is limited. We implemented a cross-sectional study to estimate HIVDR prevalence among pregnant women recently infected with HIV in Malawi. METHODS: The HIVDR study was nested within a routine antenatal clinic (ANC) sentinel surveillance survey. Dried blood spot samples were tested for recent infection using a limiting antigen antibody assay together with HIV viral load testing. HIV-1 protease and reverse transcriptase were sequenced using Sanger sequencing. Drug susceptibility was predicted using Stanford HIVdb algorithm (version 8.9). Weighted analysis was performed in Stata 15.1. RESULTS: Of the 21,642 pregnant women enrolled in the ANC survey, 8.4% (1826/21,642) tested HIV positive. Of these, 5.0% (92/1826) had recent HIV infection, and 90.2% (83/92) were tested by PCR. The amplification and sequencing success rate was 57.8% (48/83). The prevalence of any HIVDR was 14.6% (5/45) (95% CI: 4.7-36.8%), all of which indicated HIVDR to nonnucleoside reverse transcriptase inhibitors (NNRTIs). HIVDR to nucleoside reverse transcriptase inhibitors was 7.9% (2/45) (95% CI: 1.4-34.6%). Resistance to protease inhibitors currently in use in Malawi was not observed. CONCLUSIONS: Despite the low number of cases with presumed TDR, our study hints that resistance to NNRTIs was high, above the 10% target for regimen change. Further investigation is needed to establish the exact magnitude of presumed TDR among women recently infected with HIV. These findings support the transition to an integrase inhibitor-based first-line regimen for patients initiating or on ART.

High Levels of HIV-1 Drug Resistance in Children Who Acquired HIV Infection Through Mother to Child Transmission in the Era of Option B+, Haiti, 2013 to 2014.

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.

Detection of minority drug resistant mutations in Malawian HIV-1 subtype C-positive patients initiating and on first-line antiretroviral therapy.

BACKGROUND: Minority drug resistance mutations (DRMs) that are often missed by Sanger sequencing are clinically significant, as they can cause virologic failure in individuals treated with antiretroviral therapy (ART) drugs. OBJECTIVE: This study aimed to estimate the prevalence of minor DRMs among patients enrolled in a Malawi HIV drug resistance monitoring survey at baseline and at one year after initiation of ART. METHODS: Forty-one plasma specimens collected from HIV-1 subtype C-positive patients and seven clonal control samples were analysed using ultra-deep sequencing technology. RESULTS: Deep sequencing identified all 72 DRMs detected by Sanger sequencing at the level of ≥20% and 79 additional minority DRMs at the level of < 20% from the 41 Malawian clinical specimens. Overall, DRMs were detected in 85% of pre-ART and 90.5% of virologic failure patients by deep sequencing. Among pre-ART patients, deep sequencing identified a statistically significant higher prevalence of DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) compared with Sanger sequencing. The difference was mainly due to the high prevalence of minority K65R and M184I mutations. Most virologic failure patients harboured DRMs against both NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs). These minority DRMs contributed to the increased or enhanced virologic failures in these patients. CONCLUSION: The results revealed the presence of minority DRMs to NRTIs and NNRTIs in specimens collected at baseline and virologic failure time points. These minority DRMs not only increased resistance levels to NRTIs and NNRTIs for the prescribed ART, but also expanded resistance to additional major first-line ART drugs. This study suggested that drug resistance testing that uses more sensitive technologies, is needed in this setting.

Utilization of dried blood spot specimens can expedite nationwide surveillance of HIV drug resistance in resource-limited settings.

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.

High Prevalence of Abacavir-associated L74V/I Mutations in Kenyan Children Failing Antiretroviral Therapy.

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.

Prevalence of transmitted HIV-1 drug resistance among young adults attending HIV counselling and testing clinics in Kigali, Rwanda.

BACKGROUND: Scaling-up antiretroviral therapy (ART) in resource-limited settings has raised concerns of emerging HIV drug resistance (DR) and its transmission to newly infected individuals. To assess the prevalence of transmitted drug resistance (TDR) in recently HIV-infected individuals, a WHO TDR threshold survey was conducted among young adults in Kigali, Rwanda. METHODS: Between May and July 2011, HIV subtype and genotyping were performed on dried blood spots (DBS) prepared from blood specimens collected from newly HIV-diagnosed and ART-naive individuals aged 15 to 21 years in eight HIV voluntary counselling and testing (VCT) sites in Kigali. RESULTS: In total, 57 of the 68 DBS collected from eligible participants were successfully amplified. The median age of participants was 20 years and 86% were female. Most participants (96%) were infected with subtype A1 virus. Two participants (4%) had the K103N non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation and one (2%) had the M46L protease inhibitor (PI) mutation. The TDR prevalence was 3.5% (95% CI 0.4, 12.1) for NNRTI and 1.8% (95% CI 0.0, 9.4) for PI. CONCLUSIONS: The prevalence of HIV TDR in VCT attendees in Kigali was characterized as low (<5%) for all drug classes according to the WHO HIV DR threshold survey methodology. Despite a decade of widespread ART in Rwanda, TDR prevalence remains low, and so the current first-line ART regimens should continue to be effective. However, as scale-up of ART continues, frequent HIV DR surveillance is needed to monitor the effectiveness of available ART regimens at the population level.

Pre-treatment drug resistance among patients initiating antiretroviral therapy (ART) in Zimbabwe: 2008-2010.

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.

Molecular characterization of ambiguous mutations in HIV-1 polymerase gene: implications for monitoring HIV infection status and drug resistance.

Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1 pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10(-3) nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10(-3) nts/site/year) reported before. However, significantly higher index (14.41×10(-3) nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10(-2)3.48×10(-2)/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10(-2)/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.

Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.

UNLABELLED: 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.