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1.  Bayesian network analyses of resistance pathways against efavirenz and nevirapine 
AIDS (London, England)  2008;22(16):2107-2115.
Objective
To clarify the role of novel mutations selected by treatment with efavirenz or nevirapine, and investigate the influence of HIV-1 subtype on nonnucleoside reverse transcriptase inhibitor (nNRTI) resistance pathways.
Design
By finding direct dependencies between treatment-selected mutations, the involvement of these mutations as minor or major resistance mutations against efavirenz, nevirapine, or coadministrated nucleoside analogue reverse transcriptase inhibitors (NRTIs) is hypothesized. In addition, direct dependencies were investigated between treatment-selected mutations and polymorphisms, some of which are linked with subtype, and between NRTI and nNRTI resistance pathways.
Methods
Sequences from a large collaborative database of various subtypes were jointly analyzed to detect mutations selected by treatment. Using Bayesian network learning, direct dependencies were investigated between treatment-selected mutations, NRTI and nNRTI treatment history, and known NRTI resistance mutations.
Results
Several novel minor resistance mutations were found: 28K and 196R (for resistance against efavirenz), 101H and 138Q (nevirapine), and 31L (lamivudine). Robust interactions between NRTI mutations (65R, 74V, 75I/M, and 184V) and nNRTI resistance mutations (100I, 181C, 190E and 230L) may affect resistance development to particular treatment combinations. For example, an interaction between 65R and 181C predicts that the nevirapine and tenofovir and lamivudine/emtricitabine combination should be more prone to failure than efavirenz and tenofovir and lamivudine/emtricitabine.
Conclusion
Bayesian networks were helpful in untangling the selection of mutations by NRTI versus nNRTI treatment, and in discovering interactions between resistance mutations within and between these two classes of inhibitors.
doi:10.1097/QAD.0b013e32830fe940
PMCID: PMC4788388  PMID: 18832874
Bayesian network; efavirenz; HIV-1 antiviral resistance; nevirapine
2.  HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing 
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-naïve 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-naïve 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.
doi:10.1371/journal.pone.0145772
PMCID: PMC4696791  PMID: 26717411
3.  Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings 
We describe drug resistance and levels of susceptibility after first-line virologic failure in patients with several subtypes. High-level nucleoside and nonnucleoside reverse transcriptase inhibitor resistance was noted. Associations between resistance patterns and screening viral load, CD4 cell count, and site were observed.
Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania.
Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests.
Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site.
Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance.
doi:10.1093/cid/ciu314
PMCID: PMC4148601  PMID: 24795328
HIV-1 drug resistance; nonsubtype B; first-line failure; resource limited setting
4.  Impact of Drug Resistance-Associated Amino Acid Changes in HIV-1 Subtype C on Susceptibility to Newer Nonnucleoside Reverse Transcriptase Inhibitors 
The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in an in vitro single-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases, <1%. The more common EFV/NVP resistance-associated substitutions, such as K103N, V106M, and G190A, had no major impact on ETR or RPV susceptibility. The low-level resistance to RPV and ETR conferred by E138K was not significantly enhanced in the presence of M184V/I, unlike for EFV and NVP. Among patient samples, 97% were resistant to EFV and/or NVP, while only 24% and 16% were resistant to ETR and RPV, respectively. Overall, only a few, relatively rare NNRTI resistance-associated amino acid substitutions caused resistance to ETR and/or RPV in an HIV-1 subtype C background, suggesting that these newer NNRTIs would be effective in NVP/EFV-experienced HIV-1 subtype C-infected patients.
doi:10.1128/AAC.04215-14
PMCID: PMC4335849  PMID: 25421485
5.  Correction: 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, Soo-Yon | Blanco, Jose Luis | Jordan, Michael R. | Taylor, Jonathan | Lemey, Philippe | Varghese, Vici | Hamers, Raph L. | Bertagnolio, Silvia | de Wit, Tobias F. Rinke | Aghokeng, Avelin F. | Albert, Jan | Avi, Radko | Avila-Rios, Santiago | Bessong, Pascal O. | Brooks, James I. | Boucher, Charles A. B. | Brumme, Zabrina L. | Busch, Michael P. | Bussmann, Hermann | Chaix, Marie-Laure | Chin, Bum Sik | D’Aquin, Toni T. | De Gascun, Cillian F. | Derache, Anne | Descamps, Diane | Deshpande, Alaka K. | Djoko, Cyrille F. | Eshleman, Susan H. | Fleury, Herve | Frange, Pierre | Fujisaki, Seiichiro | Harrigan, P. Richard | Hattori, Junko | Holguin, Africa | Hunt, Gillian M. | Ichimura, Hiroshi | Kaleebu, Pontiano | Katzenstein, David | Kiertiburanakul, Sasisopin | Kim, Jerome H. | Kim, Sung Soon | Li, Yanpeng | Lutsar, Irja | Morris, Lynn | Ndembi, Nicaise | Kee, Peng NG | Paranjape, Ramesh S. | Peeters, Martine | Poljak, Mario | Price, Matt A. | Ragonnet-Cronin, Manon L. | Reyes-Terán, Gustavo | Rolland, Morgane | Sirivichayakul, Sunee | Smith, Davey M. | Soares, Marcelo A. | Soriano, Vincent V. | Ssemwanga, Deogratius | Stanojevic, Maja | Stefani, Mariane A. | Sugiura, Wataru | Sungkanuparph, Somnuek | Tanuri, Amilcar | Tee, Kok Keng | Truong, Hong-Ha M. | van de Vijver, David A. M. C. | Vidal, Nicole | Yang, Chunfu | Yang, Rongge | Yebra, Gonzalo | Ioannidis, John P. A. | Vandamme, Anne-Mieke | Shafer, Robert W.
PLoS Medicine  2015;12(6):e1001845.
doi:10.1371/journal.pmed.1001845
PMCID: PMC4452696  PMID: 26030872
6.  Quality of life, psychosocial health, and antiretroviral therapy among HIV-positive women in Zimbabwe 
AIDS care  2009;21(12):1517-1527.
Little is known about the psychosocial impact of antiretroviral therapy (ART) among women in sub-Saharan Africa. Therefore, we conducted a cross-sectional study in Zimbabwe to assess the impact of ART on HIV-positive women’s health-related quality of life, using the Medical Outcomes Study-HIV Quality of Life (QOL) questionnaire. Additionally, we assessed socio-demographics, reproductive and sexual health, HIV-related history, disclosure, social stigma, self-esteem, and depression. Structured interviews were conducted with 200 HIV-positive women and categorized into three groups by treatment: (1) Group 1 (n = 31) did not meet clinical or laboratory criteria to begin treatment; (2) Group 2 (n = 73) was eligible to begin treatment but awaiting initiation of treatment; and (3) Group 3 (n = 96) was on ART for a median of 13 months. The women had similar sociodemographic characteristics but varied significantly in clinical characteristics. Women on ART reported fewer AIDS-related symptoms in the last week and year and had higher current and lower baseline CD4 counts compared to women not on ART. On most QOL domains women on ART reported higher mean scores as compared to women not on ART ( p<0.01). Additionally, women on ART reported less depression compared to women not on ART ( p<0.001). Between the two groups of women not on ART, unexpectedly, there were no significant differences in their scores for QOL or depression. Thus, Zimbabwean women living with HIV experience better overall QOL and lower depression on ART. Altogether, our findings suggest that ART delivery in resource-poor communities can enhance overall QOL as well as psychosocial functioning, which has wide-ranging public health implications.
doi:10.1080/09540120902923055
PMCID: PMC4431539  PMID: 20024731
quality of life; depression; HIV treatment; sub-Saharan Africa; Zimbabwe
7.  Fertility desires and condom use among HIV-positive women at an antiretroviral roll-out program in Zimbabwe 
As access to anti-retroviral therapy (ART) increases in sub-Saharan Africa, fertility and contraception patterns are likely to change. Two hundred HIV-positive women at an ART roll-out site in Zimbabwe responded to a questionnaire on fertility desires and condom use. Ten women (5%) reported planning a pregnancy in the next year, comprising 0% of women not yet eligible for ART, 8.22% of women on the waitlist for ART, and 4.17% of women on ART. Younger age, fewer living children, and higher quality of life were individually associated with intended pregnancy in the next year; however in multivariate analysis only the association with higher quality of life remained significant. Reported ever use of condoms was relatively low (46.5%) and condom use varied by neither ART status nor by fertility desires. In conclusion, our data demonstrates fertility desires among HIV-positive women in Zimbabwe correlate with higher perceived quality of life.
PMCID: PMC4431572  PMID: 21243916
Antiretroviral therapy; fertility desires; condom use; quality of life
8.  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, Soo-Yon | Blanco, Jose Luis | Jordan, Michael R. | Taylor, Jonathan | Lemey, Philippe | Varghese, Vici | Hamers, Raph L. | Bertagnolio, Silvia | de Wit, Tobias F. Rinke | Aghokeng, Avelin F. | Albert, Jan | Avi, Radko | Avila-Rios, Santiago | Bessong, Pascal O. | Brooks, James I. | Boucher, Charles A. B. | Brumme, Zabrina L. | Busch, Michael P. | Bussmann, Hermann | Chaix, Marie-Laure | Chin, Bum Sik | D’Aquin, Toni T. | De Gascun, Cillian F. | Derache, Anne | Descamps, Diane | Deshpande, Alaka K. | Djoko, Cyrille F. | Eshleman, Susan H. | Fleury, Herve | Frange, Pierre | Fujisaki, Seiichiro | Harrigan, P. Richard | Hattori, Junko | Holguin, Africa | Hunt, Gillian M. | Ichimura, Hiroshi | Kaleebu, Pontiano | Katzenstein, David | Kiertiburanakul, Sasisopin | Kim, Jerome H. | Kim, Sung Soon | Li, Yanpeng | Lutsar, Irja | Morris, Lynn | Ndembi, Nicaise | NG, Kee Peng | Paranjape, Ramesh S. | Peeters, Martine | Poljak, Mario | Price, Matt A. | Ragonnet-Cronin, Manon L. | Reyes-Terán, Gustavo | Rolland, Morgane | Sirivichayakul, Sunee | Smith, Davey M. | Soares, Marcelo A. | Soriano, Vincent V. | Ssemwanga, Deogratius | Stanojevic, Maja | Stefani, Mariane A. | Sugiura, Wataru | Sungkanuparph, Somnuek | Tanuri, Amilcar | Tee, Kok Keng | Truong, Hong-Ha M. | van de Vijver, David A. M. C. | Vidal, Nicole | Yang, Chunfu | Yang, Rongge | Yebra, Gonzalo | Ioannidis, John P. A. | Vandamme, Anne-Mieke | Shafer, Robert W.
PLoS Medicine  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-naïve 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.
In this individual patient and sequence-level meta-analysis, Soo-Yon Rhee and colleagues measure regional trends in HIV-1 transmitted drug resistance prevalence and investigate the specific mutations responsible for TDR in different regions and in different virus subtypes.
Editors' Summary
Background
About 35 million people are currently infected with HIV, the virus that causes AIDS by destroying immune system cells and leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years of infection. Then, in 1996, effective antiretroviral (ARV) therapy—drug combinations that suppress HIV replication by inhibiting reverse transcriptase and other essential viral enzymes—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ARV therapy was expensive, HIV/AIDS remained fatal in low- and middle-income countries (LMICs). In 2003, the international community began to work towards achieving universal access to ARV therapy. Now, more than 10 million HIV-positive individuals in LMICs receive ARV therapy, usually as a fixed-dose combination of two nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir and lamivudine, plus a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as efavirenz or nevirapine.
Why Was This Study Done?
The global scale-up of ARV therapy has reduced deaths from HIV/AIDS and the incidence of HIV infection in LMICs, but the development of resistance to ARV therapy is threatening these advances. HIV rapidly accumulates genetic changes (mutations), some of which make HIV resistant to ARV therapy. Up to 30% of patients receiving a fixed-dose NRTI/NNRTI combination develop virological failure, and a high proportion of these patients develop mutations associated with resistance to the ARVs in their regimen. Moreover, the proportion of newly infected, ARV-naïve individuals with transmitted drug resistance (TDR) is also increasing. Organizations involved in HIV/AIDS control need to understand the regional and temporal mutational patterns of TDR to inform the development of guidelines for first-line ARV therapy and of inexpensive resistance mutation assays for use in LMICs. Here, using a statistical approach called meta-analysis to combine information from individual patients about the resistance mutations they carry, the researchers investigate the molecular epidemiology of TDR (the patterns of molecular changes underlying TDR in populations) and identify the HIV drug-resistance mutations most responsible for TDR in different world regions.
What Did the Researchers Do and Find?
The researchers identified 287 studies published between 2000 and 2013 from 111 countries that included the reverse transcriptase sequences of HIV viruses from 50,870 ARV-naïve, HIV-positive individuals. The researchers analyzed each virus sequence for the presence of 93 surveillance drug-resistance mutations (SDRMs) previously shown to be specific indicators of TDR. Meta-analysis of these data indicated that the average overall prevalence of TDR (the proportion of ARV-naïve, HIV-positive individuals infected with a virus carrying one or more SDRMs) ranged from 2.8% in sub-Saharan Africa to 11.5% in North America. In sub-Saharan Africa, the odds (chance) of TDR increased 1.09-fold per year following national ARV scale-up; this increase was attributable to an increase in NRTI- and NNRTI-associated resistance. By contrast, in LMICs in south/southeast Asia, the odds of TDR remained unchanged following ARV scale-up. In Latin America/Caribbean, North America, Europe, and upper-income Asian countries, the odds of TDR have increased by around 1.10-fold per year since 1995, mainly as a result of increased NNRTI resistance. Four NNRTI-associated and 16 NRTI-associated SDRMs accounted for most NNRTI- and NRTI-associated TDR, respectively, in all regions. Notably, in sub-Saharan Africa and south/southeast Asia, most of the NNRTI-associated SDRMs detected were associated with high-level resistance to nevirapine or efavirenz. Finally, the researchers report that 95% of TDR viruses in sub-Saharan Africa and south/southeast Asia were unrelated and had therefore arisen independently.
What Do These Findings Mean?
Because many drug-resistance mutations reduce HIV’s fitness and tend to be lost rapidly in individuals not exposed to ARV therapy, differences among the datasets used in this meta-analysis with respect to how long each ARV-naïve patient had been infected with HIV before virus sampling may limit the accuracy of these findings. Nevertheless, the finding that most of the TDR strains detected in sub-Saharan Africa and south/southeast Asia arose independently suggests that improved patient adherence to ARV therapy and the use of ARV regimens that contain drugs to which HIV rarely develops resistance (regimens with a high genetic barrier to resistance) should reduce the generation of new ARV-resistant strains and mitigate TDR increases. In addition, the finding that a few NNRTI-resistance mutations were responsible for most cases of transmitted high-level resistance suggests that an inexpensive assay that detects these specific mutations may be useful for pre-therapy screening in LMICs with high TDR levels.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001810.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, and personal stories about living with HIV/AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on antiretroviral drugs and on universal access to ARV therapy; Avert also provides personal stories about living with HIV/AIDS
The World Health Organization provides information on all aspects of HIV/AIDS (in several languages), including its guidelines on the use of antiretroviral therapy for treating and preventing HIV infection
The UNAIDS World AIDS Day Report 2014 provides up-to-date information about the AIDS epidemic and efforts to halt it, including progress towards universal access to antiretroviral therapy
The Stanford University HIV Drug Resistance Database includes information about surveillance drug-resistant mutations (SDRMs) and an interactive map displaying HIV drug resistance in ARV-naïve populations
doi:10.1371/journal.pmed.1001810
PMCID: PMC4388826  PMID: 25849352
9.  Outcomes by Sex Following Treatment Initiation With Atazanavir Plus Ritonavir or Efavirenz With Abacavir/Lamivudine or Tenofovir/Emtricitabine 
Smith, Kimberly Y. | Tierney, Camlin | Mollan, Katie | Venuto, Charles S. | Budhathoki, Chakra | Ma, Qing | Morse, Gene D. | Sax, Paul | Katzenstein, David | Godfrey, Catherine | Fischl, Margaret | Daar, Eric S. | Collier, Ann C. | Bolivar, Hector H. | Navarro, Sandra | Koletar, Susan L. | Gochnour, Diane | Seefried, Edward | Hoffman, Julie | Feinberg, Judith | Saemann, Michelle | Patterson, Kristine | Pittard, Donna | Currin, David | Upton, Kerry | Saag, Michael | Ray, Graham | Johnson, Steven | Santos, Bartolo | Funk, Connie A. | Morgan, Michael | Jackson, Brenda | Tebas, Pablo | Thomas, Aleshia | Kim, Ge-Youl | Klebert, Michael K. | Santana, Jorge L. | Marrero, Santiago | Norris, Jane | Valle, Sandra | Cox, Gary Matthew | Silberman, Martha | Shaik, Sadia | Lopez, Ruben | Vasquez, Margie | Daskalakis, Demetre | Megill, Christina | Shore, Jessica | Taiwo, Babafemi | Goldman, Mitchell | Boston, Molly | Lennox, Jeffrey | del Rio, Carlos | Lane, Timothy W. | Epperson, Kim | Luetkemeyer, Annie | Payne, Mary | Gripshover, Barbara | Antosh, Dawn | Reid, Jane | Adams, Mary | Storey, Sheryl S. | Dunaway, Shelia B. | Gallant, Joel | Wiggins, Ilene | Smith, Kimberly Y. | Swiatek, Joan A. | Timpone, Joseph | Kumar, Princy | Moe, Ardis | Palmer, Maria | Gothing, Jon | Delaney, Joanne | Whitely, Kim | Anderson, Ann Marie | Hammer, Scott M. | Yin, Michael T. | Jain, Mamta | Petersen, Tianna | Corales, Roberto | Hurley, Christine | Henry, Keith | Bordenave, Bette | Youmans, Amanda | Albrecht, Mary | Pollard, Richard B. | Olusanya, Abimbola | Skolnik, Paul R. | Adams, Betsy | Tashima, Karen T. | Patterson, Helen | Ukwu, Michelle | Rogers, Lauren | Balfour, Henry H. | Fox, Kathy A. | Swindells, Susan | Van Meter, Frances | Robbins, Gregory | Burgett-Yandow, Nicole | Davis, Charles E. | Boyce, Colleen | O'Brien, William A. | Casey, Gerianne | Morse, Gene D. | Hsaio, Chiu-Bin | Meier, Jeffrey L. | Stapleton, Jack T. | Mildvan, Donna | Revuelta, Manuel | Currin, David | El Sadr, Wafaa | Loquere, Avelino | El-Daher, Nyef | Johnson, Tina | Gross, Robert | Maffei, Kathyrn | Hughes, Valery | Sturge, Glenn | McMahon, Deborah | Rutecki, Barbara | Wulfsohn, Michael | Cheng, Andrew | Dix, Lynn | Liao, Qiming
This clinical trial identifies a significantly earlier time to virologic failure in women randomized to atazanavir/ritonavir compared to women randomized to efavirenz.
Background. We aimed to evaluate treatment responses to atazanavir plus ritonavir (ATV/r) or efavirenz (EFV) in initial antiretroviral regimens among women and men, and determine if treatment outcomes differ by sex.
Methods. We performed a randomized trial of open-label ATV/r or EFV combined with abacavir/lamivudine (ABC/3TC) or tenofovir/emtricitabine (TDF/FTC) in 1857 human immunodeficiency virus type 1–infected, treatment-naive persons enrolled between September 2005 and November 2007 at 59 sites in the United States and Puerto Rico. Associations of sex with 3 primary study endpoints of time to virologic failure, safety, and tolerability events were analyzed using Cox proportional hazards models. Model-based population pharmacokinetic analysis was performed using nonlinear mixed effects modeling (NONMEM version VII).
Results. Of 1857 participants, 322 were women. Women assigned to ATV/r had a higher risk of virologic failure with either nucleoside reverse transcriptase inhibitor backbone than women assigned to EFV, or men assigned to ATV/r. The effects of ATV/r and EFV upon safety and tolerability risk did not differ significantly by sex. With ABC/3TC, women had a significantly higher (32%) safety risk compared to men; with TDF/FTC, the safety risk was 20% larger for women compared to men, but not statistically significant. Women had slower ATV clearance and higher predose levels of ATV compared to men. Self-reported adherence did not differ significantly by sex.
Conclusions. This is the first randomized clinical trial to identify a significantly earlier time to virologic failure in women randomized to ATV/r compared to women randomized to EFV. This finding has important clinical implications given that boosted protease inhibitors are often favored over EFV in women of childbearing potential.
Clinical Trials Registration NCT00118898.
doi:10.1093/cid/cit747
PMCID: PMC3905755  PMID: 24253247
sex; atazanavir; efavirenz; abacavir; tenofovir
10.  Association between Latent Proviral Characteristics and Immune Activation in Antiretrovirus-Treated Human Immunodeficiency Virus Type 1-Infected Adults 
Journal of Virology  2014;88(15):8629-8639.
ABSTRACT
Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly for individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral therapy-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation.
IMPORTANCE Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment but were currently effectively treated did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong “legacy” impact.
doi:10.1128/JVI.01257-14
PMCID: PMC4135944  PMID: 24850730
11.  HIV Drug Resistance Mutations in Proviral DNA from a Community Treatment Program 
PLoS ONE  2015;10(1):e0117430.
Background
Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations.
Methods
The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu.
Results
Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively.
Conclusions
The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence.
doi:10.1371/journal.pone.0117430
PMCID: PMC4311981  PMID: 25635815
12.  HIV diversity and drug resistance from plasma and non-plasma analytes in a large treatment programme in western Kenya 
Introduction
Antiretroviral resistance leads to treatment failure and resistance transmission. Resistance data in western Kenya are limited. Collection of non-plasma analytes may provide additional resistance information.
Methods
We assessed HIV diversity using the REGA tool, transmitted resistance by the WHO mutation list and acquired resistance upon first-line failure by the IAS–USA mutation list, at the Academic Model Providing Access to Healthcare (AMPATH), a major treatment programme in western Kenya. Plasma and four non-plasma analytes, dried blood-spots (DBS), dried plasma-spots (DPS), ViveSTTM-plasma (STP) and ViveST-blood (STB), were compared to identify diversity and evaluate sequence concordance.
Results
Among 122 patients, 62 were treatment-naïve and 60 treatment-experienced; 61% were female, median age 35 years, median CD4 182 cells/µL, median viral-load 4.6 log10 copies/mL. One hundred and ninety-six sequences were available for 107/122 (88%) patients, 58/62 (94%) treatment-naïve and 49/60 (82%) treated; 100/122 (82%) plasma, 37/78 (47%) attempted DBS, 16/45 (36%) attempted DPS, 14/44 (32%) attempted STP from fresh plasma and 23/34 (68%) from frozen plasma, and 5/42 (12%) attempted STB. Plasma and DBS genotyping success increased at higher VL and shorter shipment-to-genotyping time. Main subtypes were A (62%), D (15%) and C (6%). Transmitted resistance was found in 1.8% of plasma sequences, and 7% combining analytes. Plasma resistance mutations were identified in 91% of treated patients, 76% NRTI, 91% NNRTI; 76% dual-class; 60% with intermediate-high predicted resistance to future treatment options; with novel mutation co-occurrence patterns. Nearly 88% of plasma mutations were identified in DBS, 89% in DPS and 94% in STP. Of 23 discordant mutations, 92% in plasma and 60% in non-plasma analytes were mixtures. Mean whole-sequence discordance from frozen plasma reference was 1.1% for plasma-DBS, 1.2% plasma-DPS, 2.0% plasma-STP and 2.3% plasma-STB. Of 23 plasma-STP discordances, one mutation was identified in plasma and 22 in STP (p<0.05). Discordance was inversely significantly related to VL for DBS.
Conclusions
In a large treatment programme in western Kenya, we report high HIV-1 subtype diversity; low plasma transmitted resistance, increasing when multiple analytes were combined; and high-acquired resistance with unique mutation patterns. Resistance surveillance may be augmented by using non-plasma analytes for lower-cost genotyping in resource-limited settings.
doi:10.7448/IAS.17.1.19262
PMCID: PMC4238965  PMID: 25413893
HIV; drug resistance; subtype; diversity; Kenya; analyte; AMPATH
13.  Early virologic response to abacavir/lamivudine and tenofovir/emtricitabine during ACTG A5202 
HIV clinical trials  2013;14(6):284-291.
Background
ACTG A5202 randomized treatment-naive individuals to tenofovir-emtricitabine (TDF/FTC) or abacavir-lamivudine (ABC/3TC) combined with efavirenz (EFV) or atazanavir/ritonavir (ATV/r). Individuals in the high screening viral load (VL) stratum (≥100,000 copies/mL) had increased rates of virologic failure with ABC/3TC.
Objective
Compare regimen-specific early virologic response.
Methods
Using Wilcoxon rank-sum tests, we compared regimen-specific VL changes from entry to week 4 in A5202 subjects (n=1813) and from entry to week 1, 2 and 4 in a 179-patient substudy. We evaluated associations between week 4 VL change and time to virologic failure with Cox proportional-hazards models.
Results
TDF/FTC- and ABC/3TC produced similar Week 4 viral load declines in the entire study population and in the high VL stratum. EFV produced greater VL declines from baseline at week 4 than ATV/r (median −2.1 vs. −1.9 log10 copies/mL; p<0.001). In the substudy of subjects with week 1, 2 and 4 VL data, there was no difference in viral load decline in those randomized to TDF/FTC versus ABC/3TC, but EFV resulted in greater VL decline from entry at each of these timepoints than ATV/r. Smaller Week 4 viral load decline was associated with increased risk of virologic failure.
Conclusions
Within all treatment arms, a less robust week 4 virologic response was associated with higher risk for subsequent virologic failure. However, between-regimen differences in week 4 VL declines did not parallel the previously reported differences in longer term virologic efficacy in A5202, suggesting that between-regimen differences in responses were not due to intrinsic differences in antiviral activity.
doi:10.1310/hct1406-284
PMCID: PMC4060613  PMID: 24334181
Anti-HIV Agents; HIV Infections/drug therapy/*virology; Treatment Outcome
14.  Nucleoside Reverse Transcriptase Inhibitor Resistance Mutations Associated with First-Line Stavudine-Containing Antiretroviral Therapy: Programmatic Implications for Countries Phasing Out Stavudine 
The Journal of Infectious Diseases  2013;207(Suppl 2):S70-S77.
Background
The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure.
Methods
We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance.
Results
Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ≥two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M.
Conclusions
Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.
doi:10.1093/infdis/jit114
PMCID: PMC3657117  PMID: 23687292
HIV-1; drug resistance; mutations; nucleoside reverse transcriptase inhibitor; NRTI; stavudine; d4T; zidovudine; AZT; tenofovir; TDF; subtypes
15.  An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings 
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
doi:10.3791/51242
PMCID: PMC4024245  PMID: 24747156
Medicine; Issue 85; Biomedical Technology; HIV-1; HIV Infections; Viremia; Nucleic Acids; genetics; antiretroviral therapy; drug resistance; genotyping; affordable
16.  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.
Background
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.
Methods
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.
Results
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.
Conclusions
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
ClinicalTrials.gov NCT00007488
doi:10.1371/journal.pone.0093537
PMCID: PMC3974754  PMID: 24699474
17.  HIV-1 Amino Acid Changes Among Participants With Virologic Failure: Associations With First-line Efavirenz or Atazanavir Plus Ritonavir and Disease Status 
The Journal of Infectious Diseases  2012;206(12):1920-1930.
Background. Although specific human immunodeficiency virus type 1 (HIV-1) drug resistance mutations are well studied, little is known about cumulative amino acid changes, or how regimen and participant characteristics influence these changes.
Methods. In the AIDS Clinical Trials Group randomized study A5202 of treatment-naive HIV-infected participants, cumulative HIV-1 amino acid changes from pretreatment to virologic failure were evaluated in protease and reverse transcriptase (RT) gene sequences.
Results. Among 265 participants with virologic failure, those assigned atazanavir plus ritonavir (ATV/r) did not have significantly more protease changes compared with those assigned efavirenz (EFV) (P ≥ .13). In contrast, participants with virologic failure assigned EFV had more RT changes, including and excluding known resistance codons (P < .001). At pretreatment, lower CD4 cell count, major resistance, more amino acid mixtures (all P < .001), hepatitis C antibody negativity (P = .05), and black race/ethnicity (P = .02) were associated with more HIV-1 amino acid changes.
Conclusions. Virologic failure following EFV-containing treatment was associated with more HIV-1 amino acid changes compared to failure of ATV/r-containing treatment. Furthermore, we show that non–drug resistance mutations occurred more frequently among those failing EFV, the clinical relevance of which warrants further investigation. Pretreatment immunologic status may play a role in viral evolution during treatment, as evidenced by increased amino acid changes among those with lower pretreatment CD4 count.
Clinical Trials Registration. NCT00118898.
doi:10.1093/infdis/jis613
PMCID: PMC3502379  PMID: 23148287
18.  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.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.3851/IMP1411
PMCID: PMC3816397  PMID: 19918105
19.  Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1 
The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses.
doi:10.1128/AAC.00614-13
PMCID: PMC3754322  PMID: 23796938
20.  Capacity building and predictors of success for HIV-1 drug resistance testing in the Asia-Pacific region and Africa 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.7448/IAS.16.1.18580
PMCID: PMC3709369  PMID: 23845227
HIV; drug resistance; genotyping; quality assessment
21.  In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis 
PLoS ONE  2013;8(4):e61102.
Background
Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.
Methods
Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.
Results
Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.
Conclusion
Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.
doi:10.1371/journal.pone.0061102
PMCID: PMC3629221  PMID: 23613794
22.  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.
doi:10.1093/cid/cir967
PMCID: PMC3571716  PMID: 22323567
23.  HIV disclosure patterns, predictors, and psychosocial correlates among HIV-positive women in Zimbabwe 
AIDS Care  2011;24(3):358-368.
Disclosure of positive HIV status in Sub-Saharan Africa has been associated with safer sexual practices and better antiretroviral therapy (ART) adherence, but associations with psychosocial function are unclear. We examined patterns and psychosocial correlates of disclosure in a Zimbabwean community. Two hundred HIV positive women at different stages of initiating ART participated in a cross-sectional study examining actual disclosures, disclosure beliefs, perceived stigma, self-esteem, depression, and quality of life. Ninety-seven percent of the women disclosed to at least one person, 78% disclosed to their current husband/partner, with an average disclosure of 4.0 persons per woman. The majority (85–98%) of disclosures occurred in a positive manner and 72–95% of the individuals reacted positively. Factors significantly correlated with HIV disclosure to partners included being married, later age at menses, longer duration of HIV since diagnosis, being on ART, being more symptomatic at baseline, ever having used condoms, and greater number of partners in the last year. In multivariate analysis, being married and age at menses predicted disclosure to partners. Positive disclosure beliefs, but not the total number of disclosures, significantly correlated with lower perceived stigma (rho=0.44 for personalized subscale and rho=0.51 for public subscale, both p<0.0001), higher self-esteem (rho=0.15, p=0.04), and fewer depressive symptoms (rho=−0.14, p=0.05). In conclusion, disclosure of positive HIV status among Zimbabwean women is common and is frequently met with positive reactions. Moreover, positive disclosure beliefs correlate significantly with psychosocial measures, including lower perceived stigma, higher self-esteem, and lower depression.
doi:10.1080/09540121.2011.608786
PMCID: PMC3243809  PMID: 21902570
disclosure; stigma; depression; HIV; antiretroviral therapy; Zimbabwe; Sub-Saharan Africa
24.  Global analysis of sequence diversity within HIV-1 subtypes across geographic regions 
Future virology  2012;7(5):505-517.
Aims
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.
Results
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.
Conclusion
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.
doi:10.2217/fvl.12.37
PMCID: PMC3400699  PMID: 22822410
geography; HIV-1; pol gene sequences; protease; reverse transcriptase; subtyping
25.  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) 
Summary
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.
doi:10.1097/QAI.0b013e31823e2d2b
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

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