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1.  HIV-1 RNA Levels and Antiretroviral Drug Resistance in Blood and Non-Blood Compartments from HIV-1–Infected Men and Women enrolled in AIDS Clinical Trials Group Study A5077 
PLoS ONE  2014;9(4):e93537.
Detectable HIV-1 in body compartments can lead to transmission and antiretroviral resistance. Although sex differences in viral shedding have been demonstrated, mechanisms and magnitude are unclear. We compared RNA levels in blood, genital-secretions and saliva; and drug resistance in plasma and genital-secretions of men and women starting/changing antiretroviral therapy (ART) in the AIDS Clinical Trials Group (ACTG) 5077 study.
Blood, saliva and genital-secretions (compartment fluids) were collected from HIV-infected adults (≥13 years) at 14 United-States sites, who were initiating or changing ART with plasma viral load (VL) ≥2,000 copies/mL. VL testing was performed on all compartment fluids and HIV resistance genotyping on plasma and genital-secretions. Spearman rank correlations were used to evaluate concordance and Fisher’s and McNemar’s exact tests to compare VL between sexes and among compartments.
Samples were available for 143 subjects; 36% treated (23 men, 29 women) and 64% ‘untreated’ (40 men, 51 women). RNA detection was significantly more frequent in plasma (100%) than genital-secretions (57%) and saliva (64%) (P<0.001). A higher proportion of men had genital shedding versus women (78% versus 41%), and RNA detection was more frequent in saliva versus genital-secretions in women when adjusted for censoring at the limit of assay detection. Inter-compartment fluid VL concordance was low in both sexes. In 22 (13 men, 9 women) paired plasma-genital-secretion genotypes from treated subjects, most had detectable resistance in both plasma (77%) and genital-secretions (68%). Resistance discordance was observed between compartments in 14% of subjects.
HIV shedding and drug resistance detection prior to initiation/change of ART in ACTG 5077 subjects differed among tissues and between sexes, making the gold standard blood-plasma compartment assessment not fully representative of HIV at other tissue sites. Mechanisms of potential sex-dependent tissue compartmentalization should be further characterized to aid in optimizing treatment and prevention of HIV transmission.
Trial Registration NCT00007488
PMCID: PMC3974754  PMID: 24699474
2.  Persistent Genital Tract HIV-1 RNA Shedding After Change in Treatment Regimens in Antiretroviral-Experienced Women with Detectable Plasma Viral Load 
Journal of Women's Health  2013;22(4):330-338.
To longitudinally assess the association between plasma viral load (PVL) and genital tract human immunodeficiency virus (GT HIV) RNA among HIV-1 infected women changing highly active antiretroviral therapy (HAART) because of detectable PVL on current treatment.
Women were eligible for the study if they had detectable PVL (defined as two consecutive samples with PVL>1000 copies/mL) and intended to change their current HAART regimen at the time of enrollment. Paired plasma and GT HIV-1 RNA were measured prospectively over 3 years. Longitudinal analyses examined rates of GT HIV-1 RNA shedding and the association with PVL.
Sixteen women were followed for a median of 11 visits contributing a total of 205 study visits. At study enrollment, all had detectable PVL and 69% had detectable GT HIV-1 RNA. Half of the women changed to a new HAART regimen with ≥3 active antiretroviral drugs. The probability of having detectable PVL ≥30 days after changing HAART was 0.56 (95% CI: 0.37 to 0.74). Fourteen women (88%) had detectable PVL on a follow-up visit ≥30 or 60 days after changing HAART; and 12 women (75%) had detectable GT HIV-1 RNA on a follow-up visit ≥30 or 60 days after changing HAART. When PVL was undetectable, GT shedding occurred at 11% of visits, and when PVL was detectable, GT shedding occurred at 47% of visits.
Some treatment-experienced HIV-infected women continue to have detectable virus in both the plasma and GT following a change in HAART, highlighting the difficulty of viral suppression in this patient population.
PMCID: PMC3627435  PMID: 23531097
3.  Optimal Allocation of Gold Standard Testing under Constrained Availability: Application to Assessment of HIV Treatment Failure 
The World Health Organization (WHO) guidelines for monitoring the effectiveness of HIV treatment in resource-limited settings (RLS) are mostly based on clinical and immunological markers (e.g., CD4 cell counts). Recent research indicates that the guidelines are inadequate and can result in high error rates. Viral load (VL) is considered the “gold standard”, yet its widespread use is limited by cost and infrastructure. In this paper, we propose a diagnostic algorithm that uses information from routinely-collected clinical and immunological markers to guide a selective use of VL testing for diagnosing HIV treatment failure, under the assumption that VL testing is available only at a certain portion of patient visits. Our algorithm identifies the patient sub-population, such that the use of limited VL testing on them minimizes a pre-defined risk (e.g., misdiagnosis error rate). Diagnostic properties of our proposal algorithm are assessed by simulations. For illustration, data from the Miriam Hospital Immunology Clinic (RI, USA) are analyzed.
PMCID: PMC3963362  PMID: 24672142
Antiretroviral failure; constrained optimization; HIV/AIDS; resource limited; ROC; tripartite classification
4.  Darunavir Is a Good Third-Line Antiretroviral Agent for HIV Type 1-Infected Patients Failing Second-Line Protease Inhibitor-Based Regimens in South India 
Eleven protease mutations have been associated with reduced susceptibility to darunavir. In this study of 87 HIV-1-infected patients experiencing virological failure to second-line regimens containing protease inhibitors boosted with ritonavir (viral load >1,000 HIV RNA copies/ml), we observed a low prevalence (3%) of ≥3 darunavir resistance-associated mutations, indicating that this drug may be a good option for third-line antiretroviral therapy in southern India.
PMCID: PMC3581023  PMID: 23045961
6.  Antiretroviral Treatment Interruptions Induced by the Kenyan Postelection Crisis Are Associated With Virological Failure 
Antiretroviral treatment interruptions (TIs) cause suboptimal clinical outcomes. Data on TIs during social disruption are limited.
We determined effects of unplanned TIs after the 2007–2008 Kenyan postelection violence on virological failure, comparing viral load (VL) outcomes in HIV-infected adults with and without conflict-induced TI.
Two hundred and one patients were enrolled, median 2.2 years after conflict and 4.3 years on treatment. Eighty-eight patients experienced conflict-related TIs and 113 received continuous treatment. After adjusting for preconflict CD4, patients with TIs were more likely to have detectable VL, VL >5,000 and VL >10,000.
Unplanned conflict-related TIs are associated with increased likelihood of virological failure.
PMCID: PMC3920989  PMID: 24047971
HIV; treatment interruption; political crisis Kenya; virological failure; drug resistance
7.  Short Communication: New HIV Infections at Southern New England Academic Institutions: Implications for Prevention 
New HIV infections among younger men who have sex with men (MSM) in the United States are escalating. Data on HIV infections in college students are limited. In 2010, three MSM college students presented to our clinic with primary HIV infection (PHI) in a single month. To determine the number of college students among new HIV diagnoses, we reviewed clinical characteristics and molecular epidemiology of HIV-diagnosed individuals from January to December 2010 at the largest HIV clinic in Southern New England. PHI was defined as acute HIV infection or seroconversion within the last 6 months. Of 66 individuals diagnosed with HIV in 2010, 62% were MSM and 17% were academic students (12% college or university, 5% other). Seventy-three percent of students were MSM. Compared to nonstudents, students were more likely to be younger (24 versus 39 years), born in the United States (91% versus 56%), have another sexually transmitted disease (45% versus 11%), and present with PHI (73% versus 16%, all p-values<0.05). Thirty percent of individuals formed eight transmission clusters including four students. MSM were more likely to be part of clusters. Department of Health contact tracing of cluster participants allowed further identification of epidemiological linkages. Given these high rates of PHI in recently diagnosed students, institutions of higher education should be aware of acute HIV presentation and the need for rapid diagnosis. Prevention strategies should focus on younger MSM, specifically college-age students who may be at increased risk of HIV infection.
PMCID: PMC3537304  PMID: 22724920
8.  Unusual Insertion and Deletion at Codon 67 and 69 of HIV Type 1 Subtype C Reverse Transcriptase Among First-Line Highly Active Antiretroviral Treatment-Failing South Indian Patients: Association with Other Resistance Mutations 
AIDS Research and Human Retroviruses  2012;28(12):1763-1765.
We report a high frequency of drug resistance mutations among patients with unusual insertions or deletions at the β3–β4 hairpin-loop-coding region of HIV-1 subtype C reverse transcriptase, during failure of first-line antiretroviral therapy containing only reverse transcriptase inhibitors in Chennai, India.
PMCID: PMC3505055  PMID: 22404052
9.  Genotypic HIV type-1 drug resistance among patients with immunological failure to first-line antiretroviral therapy in south India 
Antiviral therapy  2009;14(7):10.3851/IMP1411.
HIV type-1 (HIV-1) monitoring in resource limited settings relies on clinical and immunological assessment. The objective of this study was to study the frequency and pattern of reverse transcriptase (RT) drug resistance among patients with immunological failure (IF) to first-line therapy.
A cross-sectional study of 228 patients with IF was done, of which 126 were drug-naive (group A) when starting highly active antiretroviral therapy (HAART) and 102 were exposed to mono/dual therapy prior to HAART initiation (group B). A validated in-house genotyping method and Stanford interpretaion was used. Means, sd, median and frequencies (as percentages) were used to indicate the patient characteristics in each group. The χ2 test and Fisher's exact test were used to compare categorical variables as appropriate. All analyses were performed using SPSS software, version 13.0. P-values <0.05 were considered to be statistically significant.
RT drug resistance mutations were found in 92% and 96% of patients in groups A and B, respectively. Median (interquartile range) CD4+ T–cell count at failure was 181cells/ml (18–999) and time to failure was 40 months (2–100). M184V (80% versus 75%), thymidine analogue mutations (63% versus 74%), Y181C (39% versus 39%) and K103N (29% versus 39%) were predominant RT mutations in both groups. Extensive nucleoside reverse transcriptase inhibitor cross-resistance mutations were observed in 51% and 26%of patients in group B and A, respectively.
Alternative strategies for initial therapy and affordable viral load monitoring could reduce resistance accumulations and preserve available drugs for future options in resource-limited settings.
PMCID: PMC3816397  PMID: 19918105
10.  Effects of Political Conflict Induced Treatment Interruptions on HIV Drug Resistance 
AIDS reviews  2013;15(1):15-24.
34 million people worldwide were living with the Human Immunodeficiency Virus (HIV) by the end of 2010. Despite significant advances in antiretroviral therapy (ART), drug resistance remains a major deterrent to successful, enduring treatment. Unplanned interruptions in ART have negative effects on HIV treatment outcomes including increased morbidity and mortality, as well as development of drug resistance. Treatment interruptions due to political conflicts, not infrequent in resource-limited settings, result in disruptions in health care, infrastructure, or treatment facilities and patient displacement. Such circumstances are ideal bases for ART resistance development, however there is limited awareness of and data available on the association between political conflicts and the development of HIV drug resistance. In this review we identify and discuss this association and review how varying ART half-lives, genetic barriers, different HIV subtypes, and archived resistance can lead to lack of medication effectiveness upon post-conflict resumption of care. Optimized ART stopping strategies as well as infrastructural concerns and stable HIV treatment systems to ensure continuity of care and rapid resumption of care must be addressed in order to mitigate risks of HIV drug resistance development during and after political conflicts. Increased awareness of such associations by clinicians as well as politicians and stakeholders is essential.
PMCID: PMC3774601  PMID: 23449225
Treatment Interruption; Unplanned; Resistance; Political Crises; NNRTI Tail
12.  Sequence Quality Analysis Tool for HIV Type 1 Protease and Reverse Transcriptase 
Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1–2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.
PMCID: PMC3399557  PMID: 21916749
13.  Prediction of Treatment Failure Using 2010 World Health Organization Guidelines Is Associated With High Misclassification Rates and Drug Resistance Among HIV-Infected Cambodian Children 
The 2010 World Health Organization pediatric guidelines for nonvirological treatment monitoring have high misclassification rates and limited accuracy, even among children with extensive drug resistance. Affordable virological monitoring suitable for resource-limited settings is desperately needed.
Background. Antiretroviral therapy (ART) in resource-limited settings (RLSs) is monitored clinically and immunologically, according to World Health Organization (WHO) or national guidelines. Revised WHO pediatric guidelines were published in 2010, but their ability to accurately identify virological failure is unclear.
Methods. We evaluated performance of WHO 2010 guidelines and compared them with WHO 2006 and Cambodia 2011 guidelines among children on ≥6 months of first-line ART at Angkor Hospital for Children between January 2005 and September 2010. We determined sensitivity, specificity, positive and negative predictive values, and accuracy using bootstrap resampling to account for multiple tests per child. Human immunodeficiency virus (HIV) resistance was compared between those correctly and incorrectly identified by each guideline.
Results. Among 457 children with 1079 viral loads (VLs), 20% had >400 copies/mL. For children with WHO stage 1/2 HIV, misclassification as failure (met CD4 failure criteria, but VL undetectable) was 64% for WHO 2006 guidelines, 33% for WHO 2010 guidelines, and 81% for Cambodia 2011 guidelines; misclassification as success (did not meet CD4 failure, but VL detectable) was 11%, 12%, and 12%, respectively. For children with WHO stage 3/4 HIV, misclassification as failure was 35% for WHO 2006 guidelines, 40% for WHO 2010 guidelines, and 43% for Cambodia 2011 guidelines; misclassification as success was 13%, 24%, and 21%, respectively. Compared with WHO 2006 guidelines, WHO 2010 guidelines significantly increased the risk of misclassification as success in stage 3/4 HIV (P < .05). The WHO 2010 guidelines failed to identify 98% of children with extensive reverse-transcriptase resistance.
Conclusions. In our cohort, lack of virological monitoring would result in unacceptable treatment failure misclassification, leading to premature ART switch and resistance accumulation. Affordable virological monitoring suitable for use in RLSs is desperately needed.
PMCID: PMC3491779  PMID: 22539664
14.  Capacity building and predictors of success for HIV-1 drug resistance testing in the Asia-Pacific region and Africa 
The TREAT Asia Quality Assessment Scheme (TAQAS) was developed as a quality assessment programme through expert education and training, for laboratories in the Asia-Pacific and Africa that perform HIV drug-resistance (HIVDR) genotyping. We evaluated the programme performance and factors associated with high-quality HIVDR genotyping.
Laboratories used their standard protocols to test panels of human immunodeficiency virus (HIV)-positive plasma samples or electropherograms. Protocols were documented and performance was evaluated according to a newly developed scoring system, agreement with panel-specific consensus sequence, and detection of drug-resistance mutations (DRMs) and mixtures of wild-type and resistant virus (mixtures). High-quality performance was defined as detection of ≥95% DRMs.
Over 4.5 years, 23 participating laboratories in 13 countries tested 45 samples (30 HIV-1 subtype B; 15 non-B subtypes) in nine panels. Median detection of DRMs was 88–98% in plasma panels and 90–97% in electropherogram panels. Laboratories were supported to amend and improve their test outcomes as appropriate. Three laboratories that detected <80% DRMs in early panels demonstrated subsequent improvement. Sample complexity factors – number of DRMs (p<0.001) and number of DRMs as mixtures (p<0.001); and laboratory performance factors – detection of mixtures (p<0.001) and agreement with consensus sequence (p<0.001), were associated with high performance; sample format (plasma or electropherogram), subtype and genotyping protocol were not.
High-quality HIVDR genotyping was achieved in the TAQAS collaborative laboratory network. Sample complexity and detection of mixtures were associated with performance quality. Laboratories conducting HIVDR genotyping are encouraged to participate in quality assessment programmes.
PMCID: PMC3709369  PMID: 23845227
HIV; drug resistance; genotyping; quality assessment
15.  Viremia and HIV-1 Drug Resistance Mutations Among Patients Receiving Second-Line Highly Active Antiretroviral Therapy in Chennai, Southern India 
Analysis of human immunodeficiency virus type 1 pol gene sequences from 107 patients receiving second-line antiretroviral therapy (ART) revealed that a high prevalence of resistance mutations among second-line ART-experienced patients limits the ART-sequencing options, suggesting darunavir as the third-line drug in India.
Background. A cross-sectional study among individuals receiving second-line antiretroviral treatment was conducted to report on the level of detectable viremia and the types of drug resistance mutations among those with detectable human immunodeficiency virus (HIV) type 1 plasma viral loads (PVLs).
Methods. PVLs were measured using Abbott m2000rt real-time polymerase chain reaction, and genotyping was performed with the ViroSeq genotyping system, version 2.0, and ViroSeq analysis software, version 2.8.
Results. Of 107 patient plasma specimens consecutively analyzed, 30 (28%) had undetectable PVLs (<150 copies/mL), and 77 (72%) were viremic with a median PVL of 5450 copies/mL (interquartile range, 169–1 997 967). Sequencing was done for 107 samples with PVLs >2000 copies/mL: 33 patients (73%) had 1 of the protease (PR) inhibitor mutations; 41 (91%) had nucleoside reverse-transcriptase inhibitor (NRTI) mutations; 33 (73%) had non-NRTI (NNRTI) mutations; and 30 (66.7%) had both NRTI and NNRTI mutations. Triple-class resistance to NRTIs, NNRTIs, and PR inhibitors was observed in 24 (53%) patients. Based on the mutational profiles observed, all 45 sequences were susceptible to darunavir and tipranavir, whereas 47% showed resistance to lopinavir, 58% showed resistance to atazanavir, and >60% showed resistance to saquinavir, indinavir, nelfinavir, and fosamprenavir.
Conclusions. The results of the study showed that the majority of patients receiving second-line antiretroviral therapy started to accumulate PR resistance mutations, and the mutation profiles suggest that darunavir might be the drug of choice for third-line regimens in India.
PMCID: PMC3571716  PMID: 22323567
16.  Global analysis of sequence diversity within HIV-1 subtypes across geographic regions 
Future virology  2012;7(5):505-517.
HIV-1 sequence diversity can affect host immune responses and phenotypic characteristics such as antiretroviral drug resistance. Current HIV-1 sequence diversity classification uses phylogeny-based methods to identify subtypes and recombinants, which may overlook distinct subpopulations within subtypes. While local epidemic studies have characterized sequence-level clustering within subtypes using phylogeny, identification of new genotype – phenotype associations are based on mutational correlations at individual sequence positions. We perform a systematic, global analysis of position-specific pol gene sequence variation across geographic regions within HIV-1 subtypes to characterize subpopulation differences that may be missed by standard subtyping methods and sequence-level phylogenetic clustering analyses.
Materials & methods
Analysis was performed on a large, globally diverse, cross-sectional pol sequence dataset. Sequences were partitioned into subtypes and geographic subpopulations within subtypes. For each subtype, we identified positions that varied according to geography using VESPA (viral epidemiology signature pattern analysis) to identify sequence signature differences and a likelihood ratio test adjusted for multiple comparisons to characterize differences in amino acid (AA) frequencies, including minority mutations. Synonymous nonsynonymous analysis program (SNAP) was used to explore the role of evolutionary selection witihin subtype C.
In 7693 protease (PR) and reverse transcriptase (RT) sequences from untreated patients in multiple geographic regions, 11 PR and 11 RT positions exhibited sequence signature differences within subtypes. Thirty six PR and 80 RT positions exhibited within-subtype geography-dependent differences in AA distributions, including minority mutations, at both conserved and variable loci. Among subtype C samples from India and South Africa, nine PR and nine RT positions had significantly different AA distributions, including one PR and five RT positions that differed in consensus AA between regions. A selection analysis of subtype C using SNAP demonstrated that estimated rates of nonsynonymous and synonymous mutations are consistent with the possibility of positive selection across geographic subpopulations within subtypes.
We characterized systematic genotypic pol differences across geographic regions within subtypes that are not captured by the subtyping nomenclature. Awareness of such differences may improve the interpretation of future studies determining the phenotypic consequences of genetic backgrounds.
PMCID: PMC3400699  PMID: 22822410
geography; HIV-1; pol gene sequences; protease; reverse transcriptase; subtyping
17.  Reverse Transcriptase (RT) Substitution at Codons 208 and 228 Among Treatment-experienced HIV-1 Subtype-C Infected Indian Patients Is Strongly Associated with Thymidine Analogue Mutations (TAMs) 
In HIV-1 subtype C infected populations in south India, we searched for novel mutations associated with failing antiretroviral therapy that included nucleoside reverse transcriptase (RT) inhibitors. HIV-1 RT sequences were generated from treated and untreated groups and each nucleotide position was analysed with appropriate corrections for multiple testing. We found that nonsynonymous mutations at positions 208 and 228 were strongly associated with the presence of thymidine analogue mutations in the treated group, and were not present at all in the naïve group. The role of these substitutions on treatment outcomes and the evolution of drug resistance in HIV-1 subtype-C infected populations warrant further investigation.
PMCID: PMC3259528  PMID: 22245718
HIV-1 Drug Resistance Mutation; HIV-1 Subtype C; HIV in India; Thymidine Analogue Mutation (TAMs); HIV-1 RT mutation at codon 208; 228; HIV Drug Resistance in India
18.  Comparison of predicted susceptibility between genotype and virtual phenotype HIV drug resistance interpretation systems among treatment-naive HIV-infected patients in Asia: TASER-M cohort analysis 
BMC Research Notes  2012;5:582.
Accurate interpretation of HIV drug resistance (HIVDR) testing is challenging, yet important for patient care. We compared genotyping interpretation, based on the Stanford University HIV Drug Resistance Database (Stanford HIVdb), and virtual phenotyping, based on the Janssen Diagnostics BVBA’s vircoTYPE™ HIV-1, and investigated their level of agreement in antiretroviral (ARV) naive patients in Asia, where non-B subtypes predominate.
Sequences from 1301 ARV-naive patients enrolled in the TREAT Asia Studies to Evaluate Resistance – Monitoring Study (TASER-M) were analysed by both interpreting systems. Interpretations from both Stanford HIVdb and vircoTYPE™ HIV-1 were initially grouped into 2 levels: susceptible and non-susceptible. Discrepancy was defined as a discordant result between the susceptible and non-susceptible interpretations from the two systems for the same ARV. Further analysis was performed when interpretations from both systems were categorised into 3 levels: susceptible, intermediate and resistant; whereby discrepancies could be categorised as major discrepancies and minor discrepancies. Major discrepancy was defined as having a susceptible result from one system and resistant from the other. Minor discrepancy corresponded to having an intermediate interpretation in one system, with a susceptible or resistant result in the other. The level of agreement was analysed using the prevalence adjusted bias adjusted kappa (PABAK).
Overall, the agreement was high, with each ARV being in “almost perfect agreement”, using Landis and Koch’s categorisation. Highest discordance was observed for efavirenz (75/1301, 5.8%), all arising from susceptible Stanford HIVdb versus non-susceptible vircoTYPE™ HIV-1 predictions. Protease Inhibitors had highest level of concordance with PABAKs all above 0.99, followed by Nucleoside Reverse Transcriptase Inhibitors with PABAKs above 0.97 and non-NRTIs with the lowest PABAK of 0.88. The 68/75 patients with discordant efavirenz results harboured the V179D/E mutations compared to 7/1226 with no efavirenz discrepancy (p-value <0.001). In the 3-level comparison, all but one of the discrepancies was minor.
The two systems agreed well with lowest concordance observed for efavirenz. When interpreting HIVDR, especially in non-B subtypes, clinical correlation is crucial, in particular when efavirenz resistance is interpreted based on V179D/E.
PMCID: PMC3505153  PMID: 23095645
Asia; HIV; Resistance; Interpretation; Algorithm
19.  Low prevalence of transmitted K65R and other tenofovir resistance mutations across different HIV-1 subtypes: implications for pre-exposure prophylaxis 
Tenofovir-containing regimens have demonstrated potential efficacy as pre-exposure prophylaxis (PrEP) in preventing HIV-1 infection. Transmitted drug resistance mutations associated with tenofovir, specifically the reverse transcriptase (RT) mutation K65R, may impact the effectiveness of PrEP. The worldwide prevalence of transmitted tenofovir resistance in different HIV-1 subtypes is unknown.
Sequences from treatment-naïve studies and databases were aggregated and analyzed by Stanford Database tools and as per the International AIDS Society (IAS-USA) resistance criteria. RT sequences were collected from GenBank, the Stanford HIV Sequence Database and the Los Alamos HIV Sequence Database. Sequences underwent rigorous quality control measures. Tenofovir-associated resistance mutations included K65R, K70E, T69-insertion and ≥3 thymidine analogue mutations (TAMs), inclusive of M41L or L210W.
A total of 19,823 sequences were evaluated across diverse HIV-1 subtypes (Subtype A: 1549 sequences, B: 9783, C: 3198, D: 483, F: 372, G: 594, H: 41, J: 69, K: 239, CRF01_AE: 1797 and CRF02_AG: 1698). Overall, tenofovir resistance prevalence was 0.4% (n=77/19,823, 95% confidence interval or CI: 0.3 to 0.5). K65R was found in 20 sequences (0.1%, 95% CI: 0.06 to 0.15). Differences in the prevalence of K65R between HIV-1 subtypes were not statistically significant. K70E and ≥3 TAMs were found in 0.015% (95% CI: 0.004 to 0.04) and 0.27% (95% CI: 0.2 to 0.4) of sequences, respectively.
Prevalence of transmitted K65R and other tenofovir resistance mutations across diverse HIV-1 subtypes and recombinants is low, suggesting minimal effect on tenofovir-containing PrEP regimens.
PMCID: PMC3494163  PMID: 23305651
HIV; drug resistance; PrEP; tenofovir; K65R; non-B subtypes
21.  Short Communication: Transmitted Drug Resistance and Molecular Epidemiology in Antiretroviral Naive HIV Type 1-Infected Patients in Rhode Island 
Transmission of HIV-1 drug resistance has important clinical and epidemiological consequences including earlier treatment failure and forward transmission of resistance strains in high-risk groups. To evaluate the prevalence and molecular epidemiology of transmitted drug resistance in Rhode Island, we collected genotypic, demographic, clinical, and laboratory data from treatment-naive individuals presenting to the largest outpatient HIV clinic in the state from January 2007 to November 2007. Sequences from 35 treatment-naive individuals were available, 83% of whom were men who had sex with men (MSM). All sequences were HIV-1 subtype B. Drug resistance mutations were identified in 7/35 [20%; 95% confidence interval (CI), 0.08–0.37] patients, six of whom had K103N. Two phylogenetic transmission clusters were found, involving 17% (6/35) of individuals, three in each cluster. We did not find an association between belonging to a cluster and age, gender, AIDS-defining illness, CD4 cell count, or viral load. Drug resistance mutations were more commonly observed in transmission clusters (p = 0.08). Individuals in one cluster all had K103N and were MSM who had attended local bathhouses. Individuals forming clusters were significantly more likely to have visited a bathhouse compared to nonclusters (p = 0.02). The prevalence of transmitted drug resistance in Rhode Island is high, further justifying genotypic testing on presentation to care and prior to treatment initiation. Molecular epidemiological analysis and association of resistance with phylogenetic networks using data obtained for clinical purposes may serve as useful tools for the prevention of drug resistance transmission and for contact tracing.
PMCID: PMC3048815  PMID: 20954831
22.  Drug resistance patterns and virus re-suppression among HIV-1 subtype C infected patients receiving non-nucleoside reverse transcriptase inhibitors in South Africa 
Journal of AIDS & clinical research  2011;2(117):1000117.
Emergence of HIV-1 drug resistance is at times an inevitable and anticipated consequence of antiretroviral therapy (ART) failure. We examined drug resistance patterns and virus re-suppression among subtype C-infected South African patients receiving first-line ART.
Treatment records of 431 patients on NNRTI-containing regimens for a median of 45 months were analyzed. Patients with viral load (VL) >400 copies/mL were followed and drug resistance mutations (DRM) were re-assessed. Associations between clinical/demographic measures and drug resistance/virologic outcomes were examined using Fisher exact and ordinal and logistic regression.
Ten percent of patients (43/431) were viremic at enrollment (98% previously suppressed); sequences were obtained from 38/43. Of those, 82% had 1–7 DRM. In bivariate analysis remote exposure to single-dose nevirapine or prior ART; higher CD4 counts; lower VL; and >6 months of virologic failure were significantly associated with number of DRM. Of 25 viremic patients followed for a median of 8 months on a continued first-line regimen, 12 (48%) re-suppressed, six with K103N and three with M184V. Thirteen (52%) had continued virologic failure which was significantly associated with detectable VL>6 months prior to enrollment and number of DRM.
Among these HIV-1 subtype C-infected patients, DRM numbers and patterns were associated with prior exposure to sub-optimal ART, adherence and duration of virologic failure. Viral re-suppression in the presence of K103N and M184V challenges assumptions about drug resistance. In resource-limited settings, where genotyping and alternative drug options are unavailable, continuing first-line treatment, reinforcing adherence and regular virologic monitoring may be effective even after virologic failure.
PMCID: PMC3174802  PMID: 21927716
HIV-1; Subtype C; drug resistance; mutations; NNRTI; first-line ART; South Africa; ART experienced
23.  Evaluation of two human immunodeficiency virus-1 genotyping systems: ViroSeq™ 2.0 and an in-house method 
Journal of virological methods  2009;159(2):211-216.
Commercial HIV-1 genotypic resistance assays are very expensive, particularly for use in resource-constrained settings like India. Hence a cost effective in-house assay for drug resistance was validated against the standard ViroSeq™ HIV-1 Genotyping System 2.0 (Celera Diagnostics, CA, USA). A total of 50 samples were used for this evaluation (21 proficiency panels and 29 clinical isolates). Known resistance positions within HIV-1 protease (PR) region (1–99 codons) and HIV-1 reverse-transcriptase (RT) region (1–240 codons) were included. The results were analysed for each codon as follows: (i) concordant; (ii) partially concordant; (iii) indeterminate and (iv) discordant. A total of 2750 codons (55 codons per patient sample × 50 samples) associated with drug resistance (1050 PR and 1700 RT) were analysed. For PR, 99% of the codon results were concordant and 1% were partially concordant. For RT, 99% of the codon results were concordant, 0.9% were partially concordant and 0.1% were discordant. No indeterminate results were observed and the results were reproducible. Overall, the in-house assay provided comparable results to those of US FDA approved ViroSeq™, which costs about a half of the commercial assay ($ 100 vs. $ 230), making it suitable for resource-limited settings.
PMCID: PMC2923210  PMID: 19490976
ViroSeq™ HIV-1 genotyping; In-house HIV-1 drug resistance assay; Concordance; Mixtures; Indeterminate rate; HIV-1 genotyping evaluation
24.  Transmitted drug resistance in nonsubtype B HIV-1 infection 
HIV therapy  2009;3(5):447-465.
HIV-1 nonsubtype B variants account for the majority of HIV infections worldwide. Drug resistance in individuals who have never undergone antiretroviral therapy can lead to early failure and limited treatment options and, therefore, is an important concern. Evaluation of reported transmitted drug resistance (TDR) is challenging owing to varying definitions and study designs, and is further complicated by HIV-1 subtype diversity. In this article, we discuss the importance of various mutation lists for TDR definition, summarize TDR in nonsubtype B HIV-1 and highlight TDR reporting and interpreting challenges in the context of HIV-1 diversity. When examined carefully, TDR in HIV-1 non-B protease and reverse transcriptase is still relatively low in most regions. Whether it will increase with time and therapy access, as observed in subtype-B-predominant regions, remains to be determined.
PMCID: PMC2800377  PMID: 20161523
diversity; drug resistance; HIV-1; nonsubtype B; resistance transmission; transmitted drug resistance; treatment-naive
25.  TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories 
Journal of virological methods  2009;159(2):185-193.
The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme – designated TAQAS – is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratory’s result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0–83%) and a significant correlation with the detection of DRMs (p < 0.01). Interpretation of antiretroviral resistance showed ~70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories.
PMCID: PMC2863146  PMID: 19490972
HIV; Drug resistance; TAQAS; Quality assessment; Genotyping

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