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1.  Estimating the Prevalence of Transmitted HIV Drug Resistance Using Pooled Samples 
Statistical methods in medical research  2013;10.1177/0962280212473514.
In many resource-poor countries, hiv-infected patients receive a standardized antiretroviral (art) cocktail. In these settings, population-level surveillance of drug resistance is needed to characterize the prevalence of resistance mutations and to enable art programs to select the optimal regimen for their local population. The surveillance strategy currently recommended by the World Health Organization is prohibitively expensive in some settings and may not provide a sufficiently precise rendering of the emergence of drug resistance. By using a novel assay on pooled sera samples, we decrease surveillance costs while simultaneously increasing the accuracy of drug resistance prevalence estimates for an important mutation that impacts first-line antiretroviral therapy. We present a Bayesian model for pooled-testing data that garners more information from each resistance assay conducted, compared with individual testing. We expand on previous pooling methods to account for uncertainty about the population distribution of within-subject resistance levels. In addition, our model accounts for measurement error of the resistance assay, and this added uncertainty naturally propagates through the Bayesian model to our inference on the prevalence parameter. We conduct a simulation study that informs our pool size recommendations and that shows that this model renders the prevalence parameter identifiable in instances when an existing non-model-based estimator fails.
doi:10.1177/0962280212473514
PMCID: PMC3994180  PMID: 23376965
pooling; HIV; primary drug resistance; Bayesian modelling; measurement error
2.  Abacavir alters the transcription of inflammatory cytokines in virologically suppressed, HIV-infected women 
Background
Abacavir (ABC) may be associated with a small, increased risk of myocardial infarction in HIV-infected adults, possibly related to cytokine-mediated inflammation.
Methods
To evaluate the induction of inflammatory cytokine transcription by ABC, we used samples from women randomized to receive zidovudine/lamivudine/ABC (Trizivir) or lopinavir/ritonavir and zidovudine/lamividine (Kaletra/Combivir) from the third trimester through six-months postpartum for the prevention of mother-to-child transmission (PMTCT). Women were matched by CD4 count and baseline HIV RNA. All women attained viral suppression (<50 copies/ml) by the time of sampling.
Results
Four cytokines showed a difference in expression between the treatment arms, all in a proinflammatory direction for the ABC arm: CD40LG 1.82-fold, (p=.027); IL-8 3.16-fold (p=.020); LTA 2.82-fold, (p=.008); and CCL5 −1.67-fold, (p=.035). At 12-months postpartum, 6-months after antiretroviral discontinuation, cytokine expression was similar by treatment arm.
Conclusions
We conclude that ABC may upregulate proinflammatory cytokines at the transcriptional level in this population.
doi:10.7448/IAS.15.2.17393
PMCID: PMC3499794  PMID: 22789611
HIV; abacavir; cytokine; transcription; antiretroviral; inflammation; AIDS
3.  Minor resistant variants in nevirapine-exposed infants may predict virologic failure on nevirapine-containing ART 
Background
Single-dose nevirapine (sdNVP) is widely used to prevent mother-to-child transmission (PMTCT) of HIV-1. This may result in NVP resistance in both mother and infant. The significance of low levels of NVP resistance mutations in infants treated with NVP-containing antiretroviral treatment (ART) is unknown.
Objectives
To determine the presence of pre-treatment NVP resistance in HIV-infected infants with and without prior NVP exposure.
Study Design
33 HIV-1-infected infants in a PMTCT trial received NVP-containing ART (26 infants with prior NVP exposure). Plasma and buffy coat samples obtained prior to ART initiation were evaluated for drug resistance by bulk sequencing and allele-specific PCR (ASPCR).
Results
ViroSeq™ identified NVP resistance in 3 of 33 infants; all failed first-line therapy. Pre-ART plasma NVP resistance by ASPCR was detected in 9 of 16 children experiencing virologic failure compared to 4 of 17 children without virologic failure (risk ratio 2.4, CI 0.94-7.8, p=0.08). Proviral resistance was not associated with virologic failure (risk ratio 1.2, CI 0.8-2.0, p= 0.40). In the nevirapine-exposed infants, those who started ART before 7 months had higher risk of virologic failure (RR 2.3; CI 0.96-9.2; p=0.11).
Conclusions
Low level drug resistance detected in plasma after NVP exposure prior to ART initiation may be associated with virologic failure on ART, while resistance in the DNA reservoir was not predictive of treatment outcome.
doi:10.1016/j.jcv.2010.03.017
PMCID: PMC2909836  PMID: 20427228
HIV; minor variant; drug resistance; nevirapine; PMTCT
4.  Ultrasensitive Detection of Minor Drug-Resistant Variants for HIV After Nevirapine Exposure Using Allele-Specific PCR: Clinical Significance 
Abstract
HIV-1 drug resistance mutations have been detected at low frequencies after single-dose nevirapine (sdNVP) for prevention of mother-to-child transmission (PMTCT). We investigated the relationship between these “minor variant” NVP-resistant viruses and clinical outcome with NVP-containing antiretroviral therapy (ART). An allele-specific quantitative PCR (ASPCR) assay was used to quantify the pre-ART frequency of K103N and Y181C in 26 women who had received sdNVP. The cohort was composed of 7 patients who experienced virologic failure and 19 control patients who maintained virologic suppression on NVP-containing ART; all were negative for resistance by standard genotyping. NVP resistance mutations were found in 17 of 26 (65%) patients using ASPCR. The frequency of NVP-resistant viruses ranged from 0.1% to 4.11%. Receiver operating characteristics (ROC) analysis identified a clinical threshold frequency of 0.19% for the ASPCR assay. Application of this threshold demonstrated minor variant resistance in 6 of 7 patients (86%) who failed treatment compared to 6 of 19 patients (32%) who were successful (OR = 13; 95% CI 1.27–133). ASPCR provides a means of detecting minor variant drug-resistant viruses that may impact subsequent treatment response. These data suggest a clinical role for highly sensitive assays to detect and quantify resistant viruses at low frequencies.
doi:10.1089/aid.2009.0082
PMCID: PMC2864062  PMID: 20334564
5.  Improvement in allele-specific PCR assay with the use of polymorphism-specific primers for the analysis of minor variant drug resistance in HIV-1 subtype C 
Journal of virological methods  2008;149(1):69-75.
Summary
In recent years, highly sensitive assays have been developed that detect HIV-1 drug resistance mutations when present at less than 1% of the viral population. These assays are powerful tools when attempting to determine the clinical implications of these low level resistant virions after the administration of single-dose nevirapine. This report demonstrates that non-drug resistant polymorphisms in the primer binding site for the allele specific PCR (ASPCR) assay impact primer binding resulting in significant discrepancies in the assay's performance. Specifically, the use of a “universal” set of ASPCR primers caused an overestimation of the K103N (ntAAC) mutation at position 103 of reverse transcriptase when primer binding site polymorphisms resided close to the 3′ end of the allele specific primer. Drug resistance was predicted at values ranging from 0.69–7.69% for a sample containing only 1% resistance mutations and 3.35–31.84% for a sample containing 5% mutations. Conversely, the use of polymorphism specific primers detected 1.15–1.36% and 5.20–5.71% resistance for the same 1% and 5% samples. The results demonstrate the need to account for sequence polymorphisms when designing and implementing this highly specific assay.
doi:10.1016/j.jviromet.2008.01.005
PMCID: PMC2927209  PMID: 18295909
HIV; drug resistance; minor variant; allele specific PCR; nevirapine; subtype C
6.  Reduced Viral Replication Capacity of Human Immunodeficiency Virus Type 1 Subtype C Caused by Cytotoxic-T-Lymphocyte Escape Mutations in HLA-B57 Epitopes of Capsid Protein▿  
Journal of Virology  2008;83(6):2460-2468.
Cytotoxic-T-lymphocyte (CTL) escape mutations in human immunodeficiency viruses encode amino acid substitutions in positions that disrupt CTL targeting, thereby increasing virus survival and conferring a relative fitness benefit. However, it is now clear that CTL escape mutations can also confer a fitness cost, and there is increasing evidence to suggest that in some cases, e.g., escape from HLA-B*57/B*5801-restricted responses, the costs to the escape virus may affect the clinical course of infection. To quantify the magnitude of the costs of HLA-B*57/B*5801 escape, a highly sensitive dual-infection assay that uses synonymous nucleotide sequence tags to quantify viral relative replication capacity (RRC) was developed. We then asked whether such CTL escape mutations had an impact equivalent to that seen for a benchmark mutation, the M184V antiretroviral drug resistance mutation of reverse transcriptase (RRCV184 = 0.86). To answer the question, the RRCs were quantified for escape mutations in three immunodominant HLA-B*57/B*5801 epitopes in capsid: A146P in IW9 (RRCP146 = 0.91), A163G in KF11 (RRCG163 = 0.89), and T242N in TW10 (RRCN242 = 0.86). Individually, the impact of the escape mutations on RRC was comparable to that of M184V, while coexpression of the mutations resulted in substantial further reductions, with the maximum impact observed for the triple mutant (RRCP146-G163-N242 = 0.62). By comparison to M184V, the magnitude of the reductions in RRC caused by the escape mutations, particularly when coexpressed, suggests that the costs of escape are sufficient to affect in vivo viral dynamics and may thus play a role in the protective effect associated with HLA-B*57/B*5801.
doi:10.1128/JVI.01970-08
PMCID: PMC2648284  PMID: 19109381

Results 1-6 (6)