Last data update: Jun 11, 2024. (Total: 46992 publications since 2009)
Records 1-24 (of 24 Records) |
Query Trace: Chunfu Y [original query] |
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Evaluation of a dried blood and plasma collection device, SampleTanker(®), for HIV type 1 drug resistance genotyping in patients receiving antiretroviral therapy.
Diallo K , Lehotzky E , Zhang J , Zhou Z , de Rivera IL , Murillo WE , Nkengasong J , Sabatier J , Zhang G , Yang C . AIDS Res Hum Retroviruses 2014 30 (1) 67-73 Whatman 903 filter paper is the only filter paper that has been used for HIV drug resistance (HIVDR) genotyping in resource-limited settings. In this study, we evaluated another dried blood specimen collection device, termed SampleTanker(®) (ST), for HIVDR genotyping. Blood specimens from 123 antiretroviral therapy (ART)-experienced patients were used to prepare ST whole blood and ST plasma specimens; they were then stored at ambient temperature for 2 or 4 weeks. The remaining plasma specimens were stored at -80°C and used as frozen plasma controls. Frozen plasma viral load (VL) was determined using the Roche Amplicor HIV-1 Monitor test, v.1.5 and 50 specimens with VL ≥3.00 log10 copies/ml were genotyped using the broadly sensitive genotyping assay. The medium VL for the 50 frozen plasma specimens with VL ≥3.00 log10 was 3.58 log10 copies/ml (IQR: 3.32-4.11) and 96.0% (48/50) of them were genotyped. Comparing to frozen plasma specimens, significantly lower genotyping rates were obtained from ST whole blood (48.98% and 42.85%) and ST plasma specimens (36.0% and 36.0%) stored at ambient temperature for 2 and 4 weeks, respectively (p<0.001). Nucleotide sequence identity and resistance profile analyses between the matched frozen plasma and ST whole blood or ST plasma specimens revealed high nucleotide sequence identities and concordant resistance profiles (98.1% and 99.0%, and 96.6% and 98.9%, respectively). Our results indicate that with the current design, the ST may not be the ideal dried blood specimen collection device for HIVDR monitoring for ART patients in resource-limited settings. |
Prevalence of transmitted HIV-1 drug resistance among young adults attending HIV counselling and testing clinics in Kigali, Rwanda.
Mutagoma M , Ndahimana Jd , Kayirangwa E , Dahourou AG , Balisanga H , DeVos JR , McAlister D , Yang C , Bertagnolio S , Riedel DJ , Nsanzimana S . Antivir Ther 2016 21 (3) 247-51 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. |
Leveraging gains from African Center for Integrated Laboratory Training to combat HIV epidemic in sub-Saharan Africa.
Shrivastava R , Poxon R , Rottinghaus E , Essop L , Sanon V , Chipeta Z , van-Schalkwyk E , Sekwadi P , Murangandi P , Nguyen S , Devos J , Nesby-Odell S , Stevens T , Umaru F , Cox A , Kim A , Yang C , Parsons LM , Malope-Kgokong B , Nkengasong JN . BMC Health Serv Res 2021 21 (1) 22 BACKGROUND: In sub-Saharan Africa, there is dearth of trained laboratorians and strengthened laboratory systems to provide adequate and quality laboratory services for enhanced HIV control. In response to this challenge, in 2007, the African Centre for Integrated Laboratory Training (ACILT) was established in South Africa with a mission to train staffs from countries with high burdens of diseases in skills needed to strengthen sustainable laboratory systems. This study was undertaken to assess the transference of newly gained knowledge and skills to other laboratory staff, and to identify enabling and obstructive factors to their implementation. METHODS: We used Kirkpatrick model to determine training effectiveness by assessing the transference of newly gained knowledge and skills to participant's work environment, along with measuring enabling and obstructive factors. In addition to regular course evaluations at ACILT (pre and post training), in 2015 we sent e-questionnaires to 867 participants in 43 countries for course participation between 2008 and 2014. Diagnostics courses included Viral Load, and systems strengthening included strategic planning and Biosafety and Biosecurity. SAS v9.44 and Excel were used to analyze retrospective de-identified data collected at six months pre and post-training. RESULTS: Of the 867 participants, 203 (23.4%) responded and reported average improvements in accuracy and timeliness in Viral Load programs and to systems strengthening. For Viral Load testing, frequency of corrective action for unsatisfactory proficiency scores improved from 57 to 91%, testing error rates reduced from 12.9% to 4.9%; 88% responders contributed to the first national strategic plan development and 91% developed strategies to mitigate biosafety risks in their institutions. Key enabling factors were team and management support, and key obstructive factors included insufficient resources and staff's resistance to change. CONCLUSIONS: Training at ACILT had a documented positive impact on strengthening the laboratory capacity and laboratory workforce and substantial cost savings. ACILT's investment produced a multiplier effect whereby national laboratory systems, personnel and leadership reaped training benefits. This laboratory training centre with a global clientele contributed to improve existing laboratory services, systems and networks for the HIV epidemic and is now being leveraged for COVID-19 testing that has infected 41,332,899 people globally. |
High levels of HIV drug resistance among adults failing second-line antiretroviral therapy in Namibia.
Jordan MR , Hamunime N , Bikinesi L , Sawadogo S , Agolory S , Shiningavamwe AN , Negussie T , Fisher-Walker CL , Raizes EG , Mutenda N , Hunter CJ , Dean N , Steegen K , Kana V , Carmona S , Yang C , Tang AM , Parkin N , Hong SY . Medicine (Baltimore) 2020 99 (37) e21661 To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia. |
Low levels of HIV-1 drug resistance mutations in patients who achieved viral re-suppression without regimen switch: a retrospective study.
Onwuamah CK , Okpokwu J , Audu R , Imade G , Meloni ST , Okwuraiwe A , Chebu P , Musa AZ , Chaplin B , Dalhatu I , Agbaji O , Samuels J , Ezechi O , Ahmed M , Odaibo G , Olaleye DO , Okonkwo P , Salako BL , Raizes E , Yang C , Kanki PJ , Idigbe EO . BMC Microbiol 2020 20 (1) 17 BACKGROUND: We identified a HIV-positive cohort in virologic failure (VF) who re-suppressed without drug switch. We characterized their drug resistance mutations (DRM) and adherence profiles to learn how to better manage HIV drug resistance. A retrospective cohort study utilizing clinical data and stored samples. Patients received ART at three Nigerian treatment centres. Plasma samples stored when they were in VF were genotyped. RESULT: Of 126 patients with samples available, 57 were successfully genotyped. From ART initiation, the proportion of patients with adherence >/=90% increased steadily from 54% at first high viral load (VL) to 67% at confirmed VF, and 81% at time of re-suppressed VL. Sixteen (28%) patients had at least one DRM. Forty-six (81%) patients had full susceptibility to the three drugs in their first-line (1 L) regimen. Thirteen (23%) were resistant to at least one antiretroviral drug but three were resistant to drugs not used in Nigeria. Ten patients had resistance to their 1 L drug(s) and six were fully susceptible to the three drugs in the recommended second-line regimen. CONCLUSION: This cohort had little drug resistance mutations. We conclude that if adherence is not assured, patients could exhibit virologic failure without having developed mutations associated with drug resistance. |
Detection of minority drug resistant mutations in Malawian HIV-1 subtype C-positive patients initiating and on first-line antiretroviral therapy.
Zhou Z , Tang K , Zhang G , Wadonda-Kabondo N , Moyo K , Rowe LA , DeVos JR , Wagar N , Zheng DP , Guo H , Nkengasong J , Frace M , Sammons S , Yang C . Afr J Lab Med 2018 7 (1) 708 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. |
Genotyping performance evaluation of commercially available HIV-1 drug resistance test
Rosemary A , Chika O , Jonathan O , Godwin I , Georgina O , Azuka O , Zaidat M , Philippe C , Oliver E , Oche A , David O , Jay S , Ibrahim D , Mukhtar A , Joshua D , Chunfu Y , Elliot R , Beth C , Phyllis K , Emmanuel I . PLoS One 2018 13 (6) e0198246 BACKGROUND: ATCC HIV-1 drug resistance test kit was designed to detect HIV-1 drug resistance (HIVDR) mutations in the protease and reverse transcriptase genes for all HIV-1 group M subtypes and circulating recombinant forms. The test has been validated for both plasma and dried blood spot specimen types with viral load (VL) of >/=1000 copies/ml. We performed an in-country assessment on the kit to determine the genotyping sensitivity and its accuracy in detecting HIVDR mutations using plasma samples stored under suboptimal conditions. METHODS: Among 572 samples with VL >/=1000 copies/ml that had been genotyped by ViroSeq assay, 183 were randomly selected, including 85 successful genotyped and 98 unsuccessful genotyped samples. They were tested with ATCC kits following the manufacturer's instructions. Sequence identity and HIVDR patterns were analysed with Stanford University HIV Drug Resistance HIVdb program. RESULTS: Of the 183 samples, 127 (69.4%) were successfully genotyped by either method. While ViroSeq system genotyped 85/183 (46.5%) with median VL of 32,971 (IQR: 11,150-96,506) copies/ml, ATCC genotyped 115/183 (62.8%) samples with median VL of 23,068 (IQR: 7,397-86,086) copies/ml. Of the 98 unsuccessful genotyped samples with ViroSeq assay, 42 (42.9%) samples with lower median VL of 13,906 (IQR: 6,122-72,329) copies/ml were successfully genotyped using ATCC. Sequence identity analysis revealed that the sequences generated by both methods were >98% identical and yielded similar HIVDR profiles at individual patient level. CONCLUSION: This study confirms that ATCC kit showed greater sensitivity in genotyping plasma samples stored in suboptimal conditions experiencing frequent and prolonged power outage. Thus, it is more sensitive particularly for subtypes A and A/G HIV-1 in resource-limited settings. |
Decoding noises in HIV computational genotyping.
Jia M , Shaw T , Zhang X , Liu D , Shen Y , Ezeamama AE , Yang C , Zhang M . Virology 2017 511 249-255 Lack of a consistent and reliable genotyping system can critically impede HIV genomic research on pathogenesis, fitness, virulence, drug resistance, and genomic-based healthcare and treatment. At present, mis-genotyping, i.e., background noises in molecular genotyping, and its impact on epidemic surveillance is unknown. For the first time, we present a comprehensive assessment of HIV genotyping quality. HIV sequence data were retrieved from worldwide published records, and subjected to a systematic genotyping assessment pipeline. Results showed that mis-genotyped cases occurred at 4.6% globally, with some regional and high-risk population heterogeneities. Results also revealed a consistent mis-genotyping pattern in gp120 in all studied populations except the group of men who have sex with men. Our study also suggests novel virus diversities in the mis-genotyped cases. Finally, this study reemphasizes the importance of implementing a standardized genotyping pipeline to avoid genotyping disparity and to advance our understanding of virus evolution in various epidemiological settings. |
Mutational Correlates of Virological Failure in Individuals Receiving a WHO-Recommended Tenofovir-Containing First-Line Regimen: An International Collaboration.
Rhee SY , Varghese V , Holmes SP , Van Zyl GU , Steegen K , Boyd MA , Cooper DA , Nsanzimana S , Saravanan S , Charpentier C , de Oliveira T , Etiebet MA , Garcia F , Goedhals D , Gomes P , Gunthard HF , Hamers RL , Hoffmann CJ , Hunt G , Jiamsakul A , Kaleebu P , Kanki P , Kantor R , Kerschberger B , Marconi VC , D'Amour Ndahimana J , Ndembi N , Ngo-Giang-Huong N , Rokx C , Santoro MM , Schapiro JM , Schmidt D , Seu L , Sigaloff KC , Sirivichayakul S , Skhosana L , Sunpath H , Tang M , Yang C , Carmona S , Gupta RK , Shafer RW . EBioMedicine 2017 18 225-235 Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naive individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 - A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F - were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen. |
Prevalence and dynamics of the K65R drug resistance mutation in HIV-1-infected infants exposed to maternal therapy with lamivudine, zidovudine and either nevirapine or nelfinavir in breast milk.
Inzaule SC , Weidle PJ , Yang C , Ndiege K , Hamers RL , Rinke de Wit TF , Thomas T , Zeh C . J Antimicrob Chemother 2016 71 (6) 1619-26 BACKGROUND: K65R is a relatively rare drug resistance mutation (DRM) selected by the NRTIs tenofovir, didanosine, abacavir and stavudine and confers cross-resistance to all NRTIs except zidovudine. Selection by other NRTIs is uncommon. OBJECTIVES: In this study we investigated the frequency of emergence of the K65R mutation and factors associated with it in HIV-1-infected infants exposed to low doses of maternal lamivudine, zidovudine and either nevirapine or nelfinavir ingested through breast milk, using specimens collected from the Kisumu Breastfeeding Study. METHODS: Plasma specimens with viral load ≥1000 copies/mL collected from HIV-infected infants at 0-1, 2, 6, 14, 24 and 36 weeks of age and maternal samples at delivery were tested for HIV drug resistance using Sanger sequencing of the polymerase gene. Factors associated with K65R emergence were assessed using Fisher's exact test and the Wilcoxon rank-sum test. RESULTS: K65R was detected in samples from 6 of the 24 infants (25%) who acquired HIV-1 infection by the age of 6 months. K65R emerged in half of the infants by 6 weeks and in the rest by 14 weeks of age. None of the mothers at delivery or the infants with a positive genotype at first time of positivity had the K65R mutation. Infants with K65R had low baseline CD4 cell counts (P = 0.014), were more likely to have DRMs earlier (≤6 weeks versus ≥14 weeks, P = 0.007) and were more likely to have multiclass drug resistance (P = 0.035). M184V was the most common mutation associated with K65R emergence. K65R had reverted by 3 months after cessation of breastfeeding. CONCLUSIONS: A high rate of K65R emergence may suggest that ingesting low doses of lamivudine via breast milk could select for this mutation. The presence of this mutation may have a negative impact on future responses to NRTI-based ART. More in vitro studies are, however, needed to establish the molecular mechanism for this selection. |
HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing.
Rhee SY , Jordan MR , Raizes E , Chua A , Parkin N , Kantor R , Van Zyl GU , Mukui I , Hosseinipour MC , Frenkel LM , Ndembi N , Hamers RL , Rinke de Wit TF , Wallis CL , Gupta RK , Fokam J , Zeh C , Schapiro JM , Carmona S , Katzenstein D , Tang M , Aghokeng AF , De Oliveira T , Wensing AM , Gallant JE , Wainberg MA , Richman DD , Fitzgibbon JE , Schito M , Bertagnolio S , Yang C , Shafer RW . PLoS One 2015 10 (12) e0145772 The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naive individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naive individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy. |
Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-specific Assay.
Zhang G , Cai F , de Rivera IL , Zhou Z , Zhang J , Nkengasong J , Gao F , Yang C . J Clin Microbiol 2015 54 (1) 220-2 A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We now optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. |
Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum.
Guo Y , Tang K , Rowe LA , Li N , Roellig DM , Knipe K , Frace M , Yang C , Feng Y , Xiao L . BMC Genomics 2015 16 (1) 320 BACKGROUND: Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis-associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent years IaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome. RESULTS: Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45-767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5' and 3' ends of chromosome 6 and the gp60 region, largely the result of genetic recombination. CONCLUSIONS: The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum. |
Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.
Rhee SY , Blanco JL , Jordan MR , Taylor J , Lemey P , Varghese V , Hamers RL , Bertagnolio S , de Wit TF , Aghokeng AF , Albert J , Avi R , Avila-Rios S , Bessong PO , Brooks JI , Boucher CA , Brumme ZL , Busch MP , Bussmann H , Chaix ML , Chin BS , D'Aquin TT , De Gascun CF , Derache A , Descamps D , Deshpande AK , Djoko CF , Eshleman SH , Fleury H , Frange P , Fujisaki S , Harrigan PR , Hattori J , Holguin A , Hunt GM , Ichimura H , Kaleebu P , Katzenstein D , Kiertiburanakul S , Kim JH , Kim SS , Li Y , Lutsar I , Morris L , Ndembi N , Ng KP , Paranjape RS , Peeters M , Poljak M , Price MA , Ragonnet-Cronin ML , Reyes-Teran G , Rolland M , Sirivichayakul S , Smith DM , Soares MA , Soriano VV , Ssemwanga D , Stanojevic M , Stefani MA , Sugiura W , Sungkanuparph S , Tanuri A , Tee KK , Truong HH , van de Vijver DA , Vidal N , Yang C , Yang R , Yebra G , Ioannidis JP , Vandamme AM , Shafer RW . PLoS Med 2015 12 (4) e1001810 BACKGROUND: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. METHODS AND FINDINGS: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naive individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions-a proxy for recent infection-yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs-K101E, K103N, Y181C, and G190A-accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. CONCLUSIONS: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen. |
Subtyping novel zoonotic pathogen Cryptosporidium chipmunk genotype I.
Guo Y , Cebelinski E , Matusevich C , Alderisio KA , Lebbad M , McEvoy J , Roellig DM , Yang C , Feng Y , Xiao L . J Clin Microbiol 2015 53 (5) 1648-54 Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and over 200-fold coverage. Based on the whole genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used in characterizing chipmunk genotype I in 25 human specimens from four US states and Sweden, one specimen each from an eastern grey squirrel, a chipmunk and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among animals, water and humans, the 15 subtypes identified differed mostly in the number of trinucleotide repeats (TCA, TCG or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the non-repeat region. Some geographic differences were found in subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the number of a 30-bp minisatellite repeat. Thus, Cryptosporidium chipmunk genotype I from humans and wildlife are genetically similar and zoonotic transmission could play a potential role in human infections. |
Viral Genetic Diversity and Polymorphisms in a Cohort of HIV-1-Infected Patients Eligible for Initiation of Antiretroviral Therapy in Abuja, Nigeria.
Yang C , Diallo K , Zheng DP , Rottinghaus EK , Bassey OO . AIDS Res Hum Retroviruses 2015 31 (5) 564-75 Studying the genetic diversity and natural polymorphisms of HIV-1 would benefit our understanding of HIV drug resistance (HIVDR) development and predict treatment outcomes. In this study, we have characterized the HIV-1 genetic diversity and natural polymorphisms at the 5' region of pol gene encompassing the protease (PR) and reverse transcriptase (RT) from 271 plasma specimens collected in 2008 from HIV-1-infected patients who were eligible for initiating antiretroviral therapy in Abuja (Nigeria). The analysis indicated that the predominant subtype was subtype G (31.0%), followed by CRF02-AG (19.2 %), CRF43-02G (18.5%), A/CRF36-cpx (11.4%) and the remaining (19.9%) were other subtypes and circulating (CRF) and unique (URF) recombinant forms. Recombinant viruses (68.6%) were the major viral strains in the region. Eighty-four subtype G sequences were further classified into two major and two minor clusters; sequences in the two major clusters were closely related to the HIV-1 strains in two of the three major subtype G clusters detected worldwide. Those in the two minor clusters appear to be new subtype G strains circulating only in Abuja. The pre-treatment DR prevalence was < 3%, however, numerous natural polymorphisms were present. Eleven polymorphic mutations (G16E, K20I, L23P, E35D, M36I, N37D/S/T, R57K, L63P, and V82I) were detected in the PR that were subtype or CRF specific while only 3 mutations (D123N, I135T and I135V) were identified in the RT. Overall, this study indicates an evolving HIV-1 epidemic in Abuja with recombinant viruses becoming the dominant strains and emergence of new subtype G strains; pre-treatment HIVDR was low and natural polymorphism occurrence in PR region was subtype or CRF dependent. |
Limited utility of dried-blood- and plasma spot-based screening for antiretroviral treatment failure with Cobas Ampliprep/TaqMan HIV-1 version 2.0.
Sawadogo S , Shiningavamwe A , Chang J , Maher AD , Zhang G , Yang C , Gaeb E , Kaura H , Ellenberger D , Lowrance DW . J Clin Microbiol 2014 52 (11) 3878-83 The 2013 WHO antiretroviral therapy (ART) guidelines recommend dried blood spots (DBS) as an alternative specimen type for viral load (VL) monitoring. We assessed the programmatic utility of screening for ARV treatment failure (TF) at 5,000 and 1,000 copies/mL using DBS and dried plasma spots (DPS) with a commonly used VL assay, the Roche COBAS Ampliprep/COBAS TaqManV.2.0 (CAP/CTM). Plasma, DBS, and DPS were prepared from 839 whole-blood specimens collected from patients on ART ≥ six months at three public facilities in Namibia. VL was measured in plasma, DBS and DPS using the CAP/CTM and results were compared using plasma VL as the reference standard. The clinical sensitivity, specificity, Positive and Negative Predictive Value, (PPV and NPV) of DBS were 0.99, 0.55, 0.33 and 0.99, and 0.99, 0.26, 0.29 and 0.99 at ARV TF diagnostic thresholds of 5,000 copies/mL and 1,000 copies/mL, respectively; for DPS, they were 0.88, 0.98, 0.92 and 0.97, and 0.91, 0.96, 0.89, and 0.97 at TF diagnostic thresholds of 5,000 copies/mL and 1,000 copies/mL, respectively. TF prevalence in DBS was overestimated by 33% and 57% at the two thresholds, respectively. A high rate of false-positive results would occur if the CAP/CTM with DBS were used to screen for ARV TF. WHO recommendations for DBS-based VL monitoring should be specific to VL assay version and type. Despite the higher performance of DPS, the programmatic utility for TF screening may be limited by requirements for processing the whole blood at the collection site. |
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.
Zhang G , Cai F , Zhou Z , Devos J , Wagar N , Diallo K , Zulu I , Wadonda-Kabondo N , Stringer JS , Weidle PJ , Ndongmo CB , Sikazwe I , Sarr A , Kagoli M , Nkengasong J , Gao F , Yang C . J Clin Microbiol 2013 51 (11) 3666-74 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. |
Molecular characterization of ambiguous mutations in HIV-1 polymerase gene: implications for monitoring HIV infection status and drug resistance.
Zheng DP , Rodrigues M , Bile E , Nguyen DB , Diallo K , Devos JR , Nkengasong JN , Yang C . PLoS One 2013 8 (10) e77649 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 2.04x10(-3) nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2x10(-3) nts/site/year) reported before. However, significantly higher index (14.41x10(-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 (0.54x10(-2)3.48x10(-2)/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89x10(-2)/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naive 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. |
Field evaluation of a broadly sensitive HIV-1 in-house genotyping assay for use with both plasma and dried blood spot specimens in a resource-limited country.
Inzaule S , Yang C , Kasembeli A , Nafisa L , Okonji J , Oyaro B , Lando R , Mills LA , Laserson K , Thomas T , Nkengasong J , Zeh C . J Clin Microbiol 2013 51 (2) 529-39 HIV-1 drug resistance (HIVDR) assays are important tools in clinical management of HIV-infected patients on antiretroviral therapy (ART) and surveillance of drug-resistant variants at population levels. The high cost associated with commercial assays hinders their use in resource-limited settings. We adopted and validated a low-cost in-house assay using 68 matched plasma and dried blood spot (DBS) samples with a median viral load (VL) of 58,187 copies/ml, ranging from 253 to 3,264,850 against the commercial assay ViroSeq. Results indicated that the in-house assay not only had a higher plasma genotyping rate than did ViroSeq (94% versus 78%) but also was able to genotype 89.5% (51/57) of the matched DBS samples with VLs of ≥1,000 copies/ml. The sensitivity in detecting DR mutations by the in-house assay was 98.29% (95% confidence interval [CI], 97.86 to 98.72) on plasma and 96.54 (95% CI, 95.93 to 97.15) on DBS, and the specificity was 99.97% (95% CI, 99.91 to 100.00) for both sample types compared to ViroSeq. The minor DR mutation differences detected by the in-house assay against ViroSeq did not result in clinical significance. In addition, cost analysis showed that the in-house assay could reduce the genotyping cost by about 60% for both plasma and DBS compared to ViroSeq. This field condition evaluation highlights the potential utility of a cost-effective, subtype-independent, in-house genotyping assay using both plasma and DBS specimens for HIVDR clinical monitoring and population-based surveillance in resource-limited settings. |
Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.
Rottinghaus E , Bile E , Modukanele M , Maruping M , Mine M , Nkengasong J , Yang C . J Clin Microbiol 2012 51 (1) 55-60 BACKGROUND: Dried blood spots (DBS) collected on filter paper have eased the difficulty of blood collection in resource-limited settings. Currently Whatman 903 (W-903) is the only filter paper that has been used for HIV viral load (VL) and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN) for VL and HIVDR genotyping using W-903 as a comparison group. METHODS: DBS specimens were generated from 344 adult ART-patients in Botswana. VL was measured with NucliSENS EasyQ HIV-1 v2.0 and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log10 copies/ml) using an in-house method. RESULTS: Bland-Altman analysis revealed strong concordance in quantitative VL analysis between W-903 and A-226 (bias= -0.034 +/- 0.246 log10 copies/ml [Mean difference +/- SD]) and M-TFN (bias = -0.028+/- 0.186 log10 copies/ml) while qualitative VL analysis for virological failure determination, defined as VL ≥ 3.00 log10 copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) when compared to W-903. DBS collected on M-TFN had the highest genotyping efficiency (100%) compared to W-903 and A-226 (91.7%), and appear more sensitive in detecting major HIVDR mutations. CONCLUSIONS: DBS collected on A-226 and M-TFN filter papers performed similarly to W-903 for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN as specimen collection devices for HIVDR monitoring surveys. |
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.
Rottinghaus EK , Ugbena R , Diallo K , Bassey O , Azeez A , Devos J , Zhang G , Aberle-Grasse J , Nkengasong J , Yang C . Clin Infect Dis 2012 54 (8) 1187-95 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 (kappa = 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. |
Comparison of HIV-1 resistance profiles in plasma RNA versus PBMC DNA in heavily treated patients in Honduras, a resource-limited country.
Diallo K , Murillo WE , de Rivera IL , Albert J , Zhou Z , Nkengasong J , Zhang G , Sabatier JF , Yang C . Int J Mol Epidemiol Genet 2012 3 (1) 56-65 The World Health Organization currently does not recommend the use of dried blood spot specimens for drug resistance testing in patients undergoing antiretroviral therapy (ART). Therefore, HIV-1 resistance testing using peripheral blood mononuclear cells (PBMCs) may be of value in resource-limited settings. We compared genotypic resistance profiles in plasma and PBMCs from patients failing ART in two cities of Honduras (Tegucigalpa and San Pedro Sula), a resource-limited country. One hundred patients failing ART were randomly selected from a longitudinal patient monitoring cohort. Plasma and PBMC samples without patient identifier were used for genotypic resistance testing. Sequence data were analyzed, resistance profiles were determined and compared using Stanford HIV Drug Resistance Database algorithm. Specimens with concordant resistance profiles between the two compartments were 88% (95% CI: 80.3% - 94.5 %). Nine specimens (12%, 95% CI: 6.5% - 21.3%) had discordant resistance profiles of clinical significance. Logistic regression analyses indicated that patients on triple therapy were 17.24 times more likely to have concordant drug resistance profile than those on non-triple therapies (OR=17.24, 95% CI: 3.48, 83.33), while patients with increasing number of regimens and years on ART have a decreased rate of concordance (OR = 0.59, 95% CI: 0.32, 1.09 and OR = 0.62, 95% CI: 0.43, 0.88), respectively, than those with less number of regimens and years on ART. Our results show high level of concordance between plasma and PBMC resistance profiles, indicating the possibility of using PBMCs for drug resistance testing in resources-limited settings. |
Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.
Zhou Z , Wagar N , Devos JR , Rottinghaus E , Diallo K , Nguyen DB , Bassey O , Ugbena R , Wadonda-Kabondo N , McConnell MS , Zulu I , Chilima B , Nkengasong J , Yang C . 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(R) and ViroSeq(R), 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(R) and ViroSeq(R) 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(R) and ViroSeq(R) 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. |
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