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1.  Low Concentrations of HIV-1 DNA at Birth Delays Diagnosis, Complicating Identification of Infants for Antiretroviral Therapy to Potentially Prevent the Establishment of Viral Reservoirs 
Among infants exposed to human immunodeficiency virus type 1 (HIV-1), detection of viral infection at birth was increased by 39% (95% confidence interval, 19%–47%) by increasing DNA input from dried blood spots into polymerase chain reaction. Infants with low concentrations of HIV-1 at birth may be the best target population to evaluate whether immediate antiretroviral therapy can prevent long-term infection.
PMCID: PMC3967830  PMID: 24501389
HIV-1; infant diagnosis; HIV-1 DNA; mother-to-child transmission; HIV-1 cure
3.  Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing 
Journal of Clinical Microbiology  2014;52(7):2320-2327.
Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique (n = 60) and Kenya (n = 51) were analyzed at 4 codons in each sample (n = 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P = 0.78) by pyrosequencing using a cutoff value of ≥2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ∼2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested (R2 > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at ≥2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.
PMCID: PMC4097683  PMID: 24740080
4.  Monitoring of HIV Type 1 DNA Load and Drug Resistance in Peripheral Blood Mononuclear Cells During Suppressive Antiretroviral Therapy Does Not Predict Virologic Failure 
Our objective was to determine whether monitoring HIV-1 DNA concentration or new resistance mutations in peripheral blood mononuclear cells (PBMCs) during effective antiretroviral therapy (ART) predicts virologic failure. A retrospective analysis used blood specimens and clinical data from three nevirapine containing arms of a four-arm, open-label, randomized trial comparing ART regimens in HIV-1-infected children who had failed mono- or dual-nucleoside therapy. Sensitive assays compared cell-associated HIV-1 DNA concentrations and nevirapine (NVP) and lamivudine (3TC) resistance mutations in children with plasma HIV-1 RNA <400 copies(c)/ml who did or did not experience subsequent virologic failure. Forty-six children were analyzed through the last available follow-up specimen, collected at 48 (n=16) or 96 (n=30) weeks of ART. Thirty-five (76%) had sustained viral suppression and 11 (24%) had plasma viral rebound to ≥400 c/ml (virologic failure detected at a median of 36 weeks). HIV-1 DNA levels at baseline, 24, 48, and 96 weeks of ART were similar in children who did vs. did not experience virologic failure (p=0.82). HIV-1 DNA levels did not increase prior to viral rebound. NVP resistance mutations were detected in 91% of subjects in the failure group vs. 3% in the suppressed group (p <0.0001). Among nine evaluable children, NVP mutations were first detected prior to virologic failure in two (22%), at viral rebound in five (56%), and after failure in two (22%) children. HIV-1 DNA concentrations did not predict virologic failure in this cohort. New drug resistance mutations were detected in the PBMCs of a minority of virologically suppressed children who subsequently failed ART.
PMCID: PMC3399556  PMID: 22081867
5.  Effects of Short-Course Zidovudine on the Selection of Nevirapine-Resistant HIV-1 in Women Taking Single-Dose Nevirapine 
The Journal of Infectious Diseases  2012;205(12):1811-1815.
Single-dose nevirapine (sdNVP) given to prevent mother-to-child-transmission of HIV-1 selects NVP-resistance. Short-course zidovudine (ZDV) was hypothesized to lower rates of NVP-resistance. HIV-1 infected pregnant women administered sdNVP with or without short-course ZDV were assessed for HIV-1 mutations (K103N, Y181C, G190A, and V106M) prior to delivery and postpartum. Postpartum NVP-resistance was lower among 31 taking ZDV+sdNVP compared to 33 taking only sdNVP (35.5% vs 72.7%; χ2 P = .003). NVP mutants decayed to <2% in 24/35 (68.6%) at a median 6 months postpartum, with no differences based on ZDV use (logrank P = .99). Short-course ZDV was associated with reduced NVP-resistance mutations among women taking sdNVP.
PMCID: PMC3415891  PMID: 22492850
6.  Nevirapine Resistance by Timing of HIV Type 1 Infection in Infants Treated with Single-Dose Nevirapine 
In women, single-dose nevirapine for prophylaxis against mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) selects for nevirapine-resistant HIV-1, which subsequently decays rapidly. We hypothesized that the selection, acquisition, and decay of nevirapine-resistant HIV-1 differs in infants, varying by the timing of HIV-1 infection.
We conducted a prospective, observational study of 740 Mozambican infants receiving single-dose nevirapine prophylaxis and determined the timing of infection and concentrations of nevirapine-resistant HIV-1 over time.
Infants with established in utero infection had a high rate (87.0%) of selection of nevirapine-resistant HIV-1 mutants, which rapidly decayed to undetectable levels. The few without nevirapine resistance received zidovudine with single-dose nevirapine and/or their mothers took alternative antiretroviral drugs. Infants with acute in utero infection had a lower rate of nevirapine-resistant HIV-1 (33.3%; P =.006, compared with established in utero infection), but mutants persisted over time. Infants with peripartum infection also had a lower rate of nevirapine-resistant HIV-1 (38.1%; P =.001, compared with established in utero infection) but often acquired 100% mutant virus that persisted over time (P =.017, compared with established in utero infection).
The detection and persistence of nevirapine-resistant HIV-1 in infants after single-dose nevirapine therapy vary by the timing of infection and the antiretroviral regimen. In infants with persistent high-level nevirapine-resistant HIV-1, nevirapine-based antiretroviral therapy is unlikely to ever be efficacious because of concentrations in long-lived viral reservoirs. However, the absence or decay of nevirapine-resistant HIV-1 in many infants suggests that nevirapine antiretroviral therapy may be effective if testing can identify these individuals.
PMCID: PMC3038662  PMID: 20384494
7.  Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007–2013 
Antiviral therapy  2014;20(1):77-80.
U.S. guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to NRTIs, NNRTIs, and PIs. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA.
Persons with primary HIV-1 infection have enrolled in an observational cohort at the University of Washington Primary Infection Clinic since 1992. We performed a retrospective analysis of plasma specimens collected prospectively from the 82 antiretroviral-naive subjects who were enrolled from 2007–13, after FDA-approval of the first INSTI. Resistance testing was performed by consensus sequencing.
Specimens for analysis had been obtained a median of 24 (lQR 18–41, range 8–108) days after the estimated date of HIV-1 infection. All subjects were infected with HIV-1 subtype B except for one subject infected with subtype C. Consensus sequencing identified no subjects with major INSTI mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R, N155H). Using exact binomial confidence intervals, the upper bound of the 95% CI was 4.4%.
Although our sample size was small, this study does not support the need at this time to evaluate integrase mutations as part of routine consensus sequencing among persons newly diagnosed with HIV-1 infection. However, it is likely that the prevalence of transmitted INSTI mutations may increase with the recent commercial introduction of additional INSTIs and presumably greater INST1 use among persons living with HIV-1.
PMCID: PMC4312242  PMID: 24831260
8.  Increased mutations in env and pol suggest greater HIV-1 replication in sputum-compared to blood-derived viruses 
AIDS (London, England)  2009;23(8):923-928.
Low-level HIV-1 replication may occur during antiretroviral therapy (ART) that suppresses plasma HIV-1 RNA to <50c/mL (suppressive ART). Antiretroviral drugs appear less effective in macrophages and monocytes compared to lymphocytes, both in vitro, and as implied in vivo by greater viral evolution observed during suppressive ART. Our objective was to examine sputum, which is rich in macrophages for evidence of increased HIV-1 replication compared to that in the blood during suppressive ART.
Cross sectional study during suppressive ART. Comparison of HIV-1 DNA sequences derived from induced sputa and peripheral blood mononuclear cells (PBMC).
Multiple sequences encoding HIV-1 reverse transcriptase, protease, and envelope were generated using single-genome-sequencing. Reverse transcriptase and protease sequences were analyzed for genotypic drug resistance. The evolutionary distances of env sequences from the inferred most recent common ancestor of infection were calculated and CXCR4 co-receptor usage was predicted.
970 bidirectional sequences from 11 individuals were analyzed. HIV-1 env and pol derived from sputa had greater frequency of drug resistance mutations (P = 0.05), evolutionary divergence (P = 0.004) and tendency for CXCR4 usage (P = 0.1) compared to viruses derived from PBMC.
The greater frequency of HIV-1 drug resistance mutations and divergence of HIV-1 env in sputa- compared to PBMC-derived viruses suggests greater HIV-1 replication in the respiratory tract compared to the blood. Characterization of viral evolution over time and by cell-type could identify cells that provide a sanctuary for low-level viral replication in the respiratory tract during suppressive ART.
PMCID: PMC2677633  PMID: 19349849
drug resistance; highly active antiretroviral therapy; HIV-1; lung; macrophages; replication; sputum; virus
9.  Potential limitation of CCR5 antagonists: drug resistance more often linked to CXCR4-utilizing than to CCRS-utilizing HIV-1 
AIDS (London, England)  2008;22(17):2393-2395.
CCR5 antagonists are approved for treatment-experienced individuals, who are at risk of harbor­ing both drug-resistant and CXCR4-utlizing (X4) HIV-1. If CXCR4 usage and drug resistance are linked, CCR5 antagonists may select for CXCR4-utlizing viruses resistant to antiretrovirals. Analysis of 117 individual viruses found that 69% of CXCR4-utlizing viruses versus 48% of R5 viruses had drug resistance mutations (P = 0.025). Linkage of X4 and drug resistance may limit the effectiveness of CCR5 antagonists.
PMCID: PMC2585823  PMID: 18981780
10.  Optimization of the Oligonucleotide Ligation Assay, a Rapid and Inexpensive Test for Detection of HIV-1 Drug Resistance Mutations, for Non-North American Variants 
We evaluated the feasibility of the oligonucleotide ligation assay (OLA), a specific, sensitive, and economical ligase-based point mutation assay designed to detect HIV-1 drug–resistance mutations at 12 codons of HIV-1 subtype B pol, for potential use in resource-poor settings.
Specimens from HIV-1–infected individuals collected by 7 international laboratories, including subtypes A, B, C, D, F, G, J, and recombinants AE and AG, were tested by the OLA developed for HIV-1 subtype B. Common polymorphisms that interfered with reactivity of the OLA were identified and modified probes designed and evaluated.
92.5% (2410) of 2604 codons in specimens from 217 individuals were successfully genotyped by the subtype B OLA. A high rate (range 8.3%–31.2%) of indeterminate results (negative OLA reaction for both mutant and wild type) was observed for 5 codons. Modified probes at reverse transcriptase codons 151 and 184 and protease codon 90 increased the rate of valid OLA to 96.1%.
The OLA designed for HIV-1 subtype B genotyped most pol codons in non-B subtypes from Asia and Africa but was improved by addition of several modified probes. International laboratories experienced in molecular techniques were able to perform the OLA.
PMCID: PMC2583356  PMID: 18614915
HIV drug resistance; HIV-1 non-B subtypes; resistance testing; oligonucleotide ligation assay; minor genotypes; point mutation assay; dried blood spots
11.  Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection 
Science (New York, N.Y.)  2014;345(6196):570-573.
Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.
PMCID: PMC4230336  PMID: 25011556
12.  Simultaneous and Sensitive Detection of Human Immunodeficiency Virus Type 1 (HIV) Drug Resistant Genotypes by Multiplex Oligonucleotide Ligation Assay 
Journal of virological methods  2013;192(0):39-43.
Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2–5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral drugs (ARV) used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2–5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance.
PMCID: PMC3708483  PMID: 23660583
HIV; antiretroviral drug resistance; oligonucleotide ligation assay; minority genotypes
13.  Oral Acyclovir Suppression and Neurodevelopment after Neonatal Herpes 
The New England journal of medicine  2011;365(14):1284-1292.
Poor neurodevelopmental outcomes and recurrences of cutaneous lesions remain unacceptably frequent among survivors of neonatal herpes simplex virus (HSV) disease.
We enrolled neonates with HSV disease in two parallel, identical, double-blind, placebo-controlled studies. Neonates with central nervous system (CNS) involvement were enrolled in one study, and neonates with skin, eye, and mouth involvement only were enrolled in the other. After completing a regimen of 14 to 21 days of parenteral acyclovir, the infants were randomly assigned to immediate acyclovir suppression (300 mg per square meter of body-surface area per dose orally, three times daily for 6 months) or placebo. Cutaneous recurrences were treated with open-label episodic therapy.
A total of 74 neonates were enrolled — 45 with CNS involvement and 29 with skin, eye, and mouth disease. The Mental Development Index of the Bayley Scales of Infant Development (in which scores range from 50 to 150, with a mean of 100 and with higher scores indicating better neurodevelopmental outcomes) was assessed in 28 of the 45 infants with CNS involvement (62%) at 12 months of age. After adjustment for covariates, infants with CNS involvement who had been randomly assigned to acyclovir suppression had significantly higher mean Bayley mental-development scores at 12 months than did infants randomly assigned to placebo (88.24 vs. 68.12, P = 0.046). Overall, there was a trend toward more neutropenia in the acyclovir group than in the placebo group (P = 0.09).
Infants surviving neonatal HSV disease with CNS involvement had improved neurodevelopmental outcomes when they received suppressive therapy with oral acyclovir for 6 months. (Funded by the National Institute of Allergy and Infectious Diseases; CASG 103 and CASG 104 numbers, NCT00031460 and NCT00031447, respectively.)
PMCID: PMC3250992  PMID: 21991950
14.  Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B 
Insight concerning the switch in HIV-1 coreceptor use will lead to a better understanding of HIV-1 pathogenesis and host-virus dynamics. Predicting CXCR4 utilization by analyzing HIV-1 envelope consensus sequences is highly specific, but minority variants in the viral population are often missed resulting in low sensitivity. Commercial phenotypic assays are costly, and the development of sensitive in-house phenotypic assays to detect CXCR4-using HIV may not be feasible for some laboratories. A sensitive, inexpensive genotyping assay was developed to detect viral sequences associated with CXCR4-utilizing virus (X4). Codon-specific primer pairs were used to detect X4-associated codons at five positions in the HIV-1 envelope V3 loop (11, 13, 24, 25, and 32). Sixty plasma samples from HIV-1-infected individuals were analyzed by consensus sequencing and codon-specific PCR (CS-PCR). Forty-six of these were also phenotyped by Trofile or Enhanced Sensitivity Trofile (ESTA). CS-PCR detected X4 variants 17% more often than 11/24/25 consensus sequencing alone (n=60), 30% more often than Trofile (n=27), and in a limited data set, 16% more often than ESTA (n=19). CS-PCR combined with consensus sequencing had approximately 80% concordance with ESTA.
PMCID: PMC3636599  PMID: 23343425
15.  Human Immunodeficiency Viruses Appear Compartmentalized to the Female Genital Tract in Cross-Sectional Analyses but Genital Lineages Do Not Persist Over Time 
The Journal of Infectious Diseases  2013;207(8):1206-1215.
Background. Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood.
Methods. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests.
Results. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences.
Conclusions. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.
PMCID: PMC3603533  PMID: 23315326
HIV; female genital tract; compartmentalization; viral lineages; monotypic HIV; genetic distances; uterine cervix; phylogenetics
16.  Quantification of mitochondrial toxicity in HIV-infected individuals by quantitative PCR compared to flow cytometry 
Non-invasive diagnostic assays to evaluate mitochondrial toxicity could have significant clinical utility for HIV-infected individuals on antiretroviral therapy (ART).
This study compared the ratio of mitochondrial to nuclear DNA determined by quantitative PCR to the ratio of mitochondrial to nuclear-encoded proteins by flow cytometry, in peripheral blood mononuclear cells from 73 HIV-infected individuals with and without risk factors for mitochondrial toxicity.
PCR detected similar mitochondrial/nuclear DNA in HIV-infected individuals without a history of ART, and those receiving ART with lipodystrophy, lipoatrophy or a history of suspected lactic acidosis. However, the ratio was significantly greater in ART-untreated compared to those receiving either stavudine or didanosine. In contrast, flow cytometry did not detect any differences in mitochondrial/nuclear protein (1). There was no correlation between the assays (rho = −0.05, p = 0.65).
Assessment of the mitochondrial/nuclear DNA ratio by quantitative PCR performed better than the mitochondrial/nuclear-encoded protein ratio by flow cytometry to detect adverse effects of nucleoside analogues on mitochondria.
PMCID: PMC3966180  PMID: 23044657
HIV; Antiretroviral Therapy; Mitochondrial Toxicity; Flow Cytometry; Quantitative PCR; Diagnostics
17.  Associations between genital tract infections, genital tract inflammation and cervical cytobrush HIV-1 DNA in US versus Kenyan women 
Cervical shedding of HIV-1 DNA may influence HIV-1 sexual transmission. HIV-1 DNA was detected in 250/316 (80%) and 207/259 (79%) cervical cytobrush specimens from 56 United States (US) and 80 Kenyan women, respectively. Plasma HIV-1 RNA concentration was associated with increased HIV-1 DNA shedding among US and Kenyan women. Kenyan women had higher cervicovaginal concentrations of pro-inflammatory interleukins (IL)-1β, IL-6, IL-8 and anti-inflammatory secretory leukocyte protease inhibitor (SLPI) compared to US women (all p < 0.01). HIV-1 DNA shedding was associated with increased concentrations of IL-1β and IL-6 and lower SLPI among US women, but not Kenyan women.
PMCID: PMC3549039  PMID: 23018377
18.  Interaction Between Lactobacilli, Bacterial Vaginosis-Associated Bacteria, and HIV Type 1 RNA and DNA Genital Shedding in U.S. and Kenyan Women 
Bacterial vaginosis has been associated with genital HIV-1 shedding; however, the effect of specific vaginal bacterial species has not been assessed. We tested cervicovaginal lavage from HIV-1-seropositive women for common Lactobacillus species: L. crispatus, L. jensenii, and seven BV-associated species: BVAB1, BVAB2, BVAB3, Leptotrichia, Sneathia, Megasphaera, and Atopobium spp. using quantitative PCR. We used linear and Poisson regression to evaluate associations between vaginal bacteria and genital HIV-1 RNA and DNA. Specimens from 54 U.S. (310 visits) and 50 Kenyan women (137 visits) were evaluated. Controlling for plasma viral load, U.S. and Kenyan women had similar rates of HIV-1 RNA (19% of visits vs. 24%; IRR=0.95; 95% CI 0.61, 1.49) and DNA shedding (79% vs. 76%; IRR=0.90; 0.78, 1.05). At visits during antiretroviral therapy (ART), the likelihood of detection of HIV-1 RNA shedding was greater with BVAB3 (IRR=3.16; 95% CI 1.36, 7.32), Leptotrichia, or Sneathia (IRR=2.13; 1.02, 4.72), and less with L. jensenii (IRR=0.39; 0.18, 0.84). At visits without ART, only L. crispatus was associated with a lower likelihood of HIV-1 RNA detection (IRR=0.6; 0.40, 0.91). Vaginal Lactobacillus species were associated with lower risk of genital HIV-1 shedding, while the presence of certain BV-associated species may increase that risk.
PMCID: PMC3537306  PMID: 23020644
19.  Improved Detection of Rare HIV-1 Variants using 454 Pyrosequencing 
PLoS ONE  2013;8(10):e76502.
454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
PMCID: PMC3788733  PMID: 24098517
20.  Nevirapine-Resistant HIV-1 DNA in Breast Milk After Single-Dose Nevirapine With or Without Zidovudine for Prevention of Mother-to-Child Transmission 
Among 30 human immunodeficiency virus type 1 (HIV-1)–infected women who received single-dose nevirapine (NVP), 17 (57%) had NVP-resistant HIV-1 detected in breast milk. NVP resistance in breast milk persisted for at least 8 months postpartum and was apparently transmitted to at least 1 infant. NVP resistance was detected less often in women who also received zidovudine.
PMCID: PMC3656540  PMID: 23687579
21.  An Increasing Proportion of Monotypic HIV-1 DNA Sequences during Antiretroviral Treatment Suggests Proliferation of HIV-Infected Cells 
Journal of Virology  2013;87(3):1770-1778.
Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656 env and pol sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%; P < 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4+ T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.
PMCID: PMC3554159  PMID: 23175380
22.  Selection of HIV Resistance associated with Antiretroviral Therapy (ART) Initiated due to Pregnancy and Suspended Postpartum 
Compare the risk of HIV-drug-resistance in women stopping suppressive nelfinavir-(NFV) or nevirapine (NVP)-based-ART after pregnancy.
Specimens collected after stopping ART were tested for drug-resistance by an oligonucleotide ligation assay (OLA) and consensus sequencing. When postpartum drug-resistance was detected, specimens obtained at study entry and during ART were evaluated.
16/38 women with ART-induced suppression of viral replication suspended ART postpartum. Resistance mutations were detected in 75% who stopped NFV- and 50% who stopped NVP-ART. M184V, associated with 3TC-resistance, was more frequent among those randomized to NFV- compared to NVP-ART (6/8 vs. 1/8; P=0.04), and NNRTI-resistance was detected in 4/8 stopping NVP-ART.
HIV-drug-resistance was frequently observed among women who stopped suppressive NVP- or NFV-ART postpartum. This suggests that NFV-ART may have suboptimal potency; that staggering discontinuation of NVP-ART may be warranted; and/or ART-adherence may be lax in women who choose to stop ART postpartum.
PMCID: PMC3196829  PMID: 21765365
HIV-1; Antiretroviral drug resistance; Antiretroviral discontinuation; Pregnancy; Oligonucleotide ligation assay (OLA); Minority genotypes; Nelfinavir; Nevirapine; Lamivudine; Zidovudine
23.  CD4+ cell count and risk for antiretroviral drug resistance among women using peripartum nevirapine for perinatal HIV prevention 
To determine the association between antenatal CD4+ cell count and development of viral drug resistance following use of peripartum nevirapine (NVP) for perinatal HIV prevention.
Secondary analysis of data from previously conducted randomized control trial.
Lusaka, Zambia.
HIV positive pregnant women.
We analyzed data from a clinical trial of single-dose tenofovir/emtricitabine (TDF/FTC) to reduce viral drug resistance associated with peripartum NVP. The trial population was categorized according to antenatal CD4+ cell count (200–350 cells/uL, 351–500 cells/uL, and >500 cells/uL).
Main outcome measures
Relative risk for acquiring drug resistance—determined by consensus sequencing and oligonucleotide ligation assay (OLA)—was estimated using multivariable logistic regression.
Of 397 study participants, 119 (30%) had a CD4+ count of 200–350 cells/uL, 135 (34%) had a CD4+ count of 351–500 cells/uL, and 143 (36%) had a CD4+ count >500 cells/uL. Among women receiving no intervention, risk for drug resistance appeared to increase as CD4+ cell count decreased. Participants with CD4+ cell counts of 200–350 cells/uL randomized to the study arm had the lowest risk, suggesting higher efficacy of the intervention within this stratum. These results were consistent at two and six weeks, regardless of how drug resistance was measured.
Women with CD4+ cell counts of 200–350 cells/uL may be at increased risk for viral drug resistance following use of peripartum NVP. Given the high prevalence of NVP resistance and the clear benefits of treatment, antiretroviral therapy should be initiated among pregnant women with CD4+ cell counts ≤ 350 cells/uL.
PMCID: PMC3076308  PMID: 21199294
HIV; antiretroviral therapy; non-nucleoside reverse transcriptase inhibitor; resistance; prevention of mother-to-child transmission; CD4+ cell count
24.  Herpes simplex virus type 2 suppressive therapy with acyclovir or valacyclovir does not select for specific HIV-1 resistance in HIV-1/HSV-2 dually-infected persons 
The Journal of infectious diseases  2011;203(1):117-121.
Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1 infected persons from Botswana, Kenya, Peru, and the US taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval: 0-2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance.
PMCID: PMC3024584  PMID: 21148504
HSV-2; HIV-1 resistance; V75I
25.  Herpes Simplex Virus Type 2 Suppressive Therapy with Acyclovir or Valacyclovir Does Not Select for Specific HIV-1 Resistance in HIV-1/HSV-2 Dually Infected Persons 
The Journal of Infectious Diseases  2011;203(1):117-121.
Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1–infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%–2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance.
PMCID: PMC3024584  PMID: 21148504

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