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The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)-1 variants persisted in women and infants 1 year or more after the administration of SD-NVP.We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 6-8 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT.ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 12-24 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay.K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.

BACKGROUND Injection opioid use plays a significant role in the transmission of HIV infection in many communities and several regions of the world. Access to evidence-based treatments for opioid use disorders is extremely limited. METHODS HIV Prevention Trials Network 058 (HPTN 058) was a randomized controlled trial designed to compare the impact of 2 medication-assisted treatment (MAT) strategies on HIV incidence or death among opioid-dependent people who inject drugs (PWID). HIV-negative opioid-dependent PWID were recruited from 4 communities in Thailand and China with historically high prevalence of HIV among PWID. A total of 1251 participants were randomly assigned to either (1) a 1-year intervention consisting of 2 opportunities for a 15-day detoxification with buprenorphine/naloxone (BUP/NX) combined with up to 21 sessions of behavioral drug and risk counseling [short-term medication-assisted treatment (ST-MAT)] or (2) thrice-weekly dosing for 48 weeks with BUP/NX and up to 21 counseling sessions [long-term medication-assisted treatment (LT-MAT)] followed by dose tapering. All participants were followed for 52 weeks after treatment completion to assess durability of impact. RESULTS Although the study was stopped early due to lower than expected occurrence of the primary end points, sufficient data were available to assess the impact of the interventions on drug use and injection-related risk behavior. At week 26, 22% of ST-MAT participants had negative urinalyses for opioids compared with 57% in the LT-MAT (P < 0.001). Differences disappeared in the year after treatment: at week 78, 35% in ST-MAT and 32% in the LT-MAT had negative urinalyses. Injection-related risk behaviors were significantly reduced in both groups after randomization. CONCLUSIONS Participants receiving BUP/NX 3 times weekly were more likely to reduce opioid injection while on active treatment. Both treatment strategies were considered safe and associated with reductions in injection-related risk behavior. These data support the use of thrice-weekly BUP/NX as a way to reduce exposure to HIV risk. Continued access to BUP/NX may be required to sustain reductions in opioid use.

Objective Maternal-to-child-transmission of HIV-1 infection remains a significant cause of HIV-1 infection despite successful prevention strategies. Testing protective HIV-1 vaccines remains a critical priority. The immunogenicity of ALVAC-HIV vCP1521 (ALVAC) in infants born to HIV-1-infected women in Uganda was evaluated in the first pediatric HIV-1 vaccine study in Africa. Design HIV Prevention Trials Network 027 was a randomized, double-blind, placebo-controlled phase I trial to evaluate the safety and immunogenicity of ALVAC in 60 infants born to HIV-1-infected mothers with CD4 counts of >500 cells per microliter, which were randomized to the ALVAC vaccine or placebo. ALVAC-HIV vCP1521 is an attenuated recombinant canarypox virus expressing HIV-1 clade E env, clade B gag, and protease gene products. Methods Infants were vaccinated at birth and 4, 8, and 12 weeks of age with ALVAC or placebo. Cellular and humoral immune responses were evaluated using interferon-γ enzyme-linked immunosorbent spot, carboxyfluorescein diacetate succinimidyl ester proliferation, intracellular cytokine staining, and binding and neutralizing antibody assays. Fisher exact test was used to compare positive responses between the study arms. Results Low levels of antigen-specific CD4 and CD8 T-cell responses (intracellular cytokine assay) were detected at 24 months (CD4-6/36 vaccine vs. 1/9 placebo; CD8-5/36 vaccine vs. 0/9 placebo) of age. There was a nonsignificant trend toward higher cellular immune response rates in vaccine recipients compared with placebo. There were minimal binding antibody responses and no neutralizing antibodies detected. Conclusions HIV-1-exposed infants are capable of generating low levels of cellular immune responses to ALVAC vaccine, similar to responses seen in adults.

HIV elite suppressors (ES) or controllers are individuals achieving control of viremia by their natural immunological mechanisms without highly active antiretroviral therapy (HAART). Study of the mechanisms responsible for the immunological suppression of viremia in ES may lead to the detection of individuals with ES and the effective control of HIV infection. We hypothesize that plasma glycoproteins play essential roles in the immune system of ES since plasma proteins are critical and highly relevant in anti-viral immunity and most plasma proteins are glycoproteins. To examine glycoproteins associated with ES, plasma samples from ES individuals (n=20), and from individuals on HAART (n=20), with AIDS (n=20), and no HIV infection (n=10) were analyzed by quantitative glycoproteomics. We found that a number of glycoproteins changed between ES versus HAART, AIDS and HIV- individuals. In sharp contrast, the level of plasma glycoproteins in the HAART cohort showed fewer changes compared with AIDS and HIV- individuals. These results showed that although both ES and HAART effectively suppress viremia, ES appeared to profoundly affect immunologically relevant glycoproteins in plasma as consequence of or support for anti-viral immunity. Bioinformatic analysis revealed that altered proteins in ES plasma were mainly associated with inflammation. This analysis suggests that overlapping, while distinguishable, glycoprotein profiles for inflammation and immune activation appeared to be present between ES and non-ES (HAART+AIDS) cohorts, indicating different triggers for inflammation and immune activation between natural and treatment-related viral suppression.

Background. In resource-limited settings, mothers infected with human immunodeficiency virus type 1 (HIV-1) face a difficult choice: breastfeed their infants but risk transmitting HIV-1 or not breastfeed their infants and risk the infants dying of other infectious diseases or malnutrition. Recent results from observational studies and randomized clinical trials indicate daily administration of nevirapine to the infant can prevent breast-milk HIV-1 transmission. Methods. Data from 5396 mother-infant pairs who participated in 5 randomized trials where the infant was HIV-1 negative at birth were pooled to estimate the efficacy of infant nevirapine prophylaxis to prevent breast-milk HIV-1 transmission. Four daily regimens were compared: nevirapine for 6 weeks, 14 weeks, or 28 weeks, or nevirapine plus zidovudine for 14 weeks. Results. The estimated 28-week risk of HIV-1 transmission was 5.8% (95% confidence interval [CI], 4.3%–7.9%) for the 6-week nevirapine regimen, 3.7% (95% CI, 2.5%–5.4%) for the 14-week nevirapine regimen, 4.8% (95% CI, 3.5%–6.7%) for the 14-week nevirapine plus zidovudine regimen, and 1.8% (95% CI, 1.0%–3.1%) for the 28-week nevirapine regimen (log-rank test for trend, P

Background Buprenorphine/naloxone (BUP/NX) is not licenced for use in China or Thailand and there was little clinical experience with this drug combination in these countries at the inception of HIV Prevention Trial Network (HPTN) 058, a randomized trial comparing risk reduction counselling combined with either short-term or long-term medication assisted treatment with BUP/NX to prevent HIV infection and death amongst opioid-dependent injectors. Methods We conducted a safety phase that included the first 50 subjects enrolled at each of the three initial study sites ( N =150). Clinical and laboratory assessments were conducted at baseline and weekly for the first 4 weeks. Changes in laboratory parameters were estimated with random effects models. Results BUP/NX was well tolerated by study subjects and opioid withdrawal scores decreased substantially during the 3-day induction. Two participants experienced grade 3 clinical adverse events, which were categorized as probably not related to the study drug. Grade 2 or 3 increases in alanine aminotransferase (ALT) occurred in 25 (17%) subjects. The magnitude of ALT increase over 4-week follow-up was strongly associated with baseline ALT elevation. Conclusions In Chinese and Thai opioid-dependent injectors, we found BUP/NX to be effective in reducing opioid withdrawal symptoms and safe during short-term use. ALT increases were observed over 4-week-follow-up, which are consistent with reports from Western populations. Long-term safety and efficacy evaluations are indicated.

BACKGROUND This phase III, randomized, clinical trial compared single-dose nevirapine (sdNVP) plus HIV hyperimmune globulin (HIVIGLOB) with sdNVP alone for preventing maternal-to-child transmission of HIV. Primary objectives were to determine rates of HIV infection among infants and to assess the safety of HIVIGLOB in combination with sdNVP in HIV-infected Ugandan pregnant women and their infants. METHODS Mother-infant pairs were randomized to receive 200 mg of nevirapine to women in labor and 2 mg/kg NVP to newborns within 72 hours after birth (sdNVP arm) or to receive sdNVP plus a single intravenous 240-mL dose of HIVIGLOB given to women at 36- to 38-week gestation and a single intravenous 24-mL dose to newborns within 18 hours of birth (HIVIGLOB/sdNVP arm). Risk of HIV infection was determined using Kaplan-Meier and risk ratio estimates at birth, 2, 6, 14 weeks, 6, and 12 months of age. RESULTS Intent-to-treat analysis included 198 HIVIGLOB/sdNVP and 294 sdNVP mother-infant pairs. At 6 months of age, the primary endpoint, there was no statistically significant difference in HIV transmission in the HIVIGLOB/sdNVP arm vs. the sdNVP arm [18.7% vs. 15.0%; risk ratio = 1.240 (95% confidence interval: 0.833 to 1.846); P = 0.290]. Similarly, the proportion of serious adverse events in the HIVIGLOB/sdNVP and sdNVP arms, respectively, for mothers (18.9% vs. 19.3%; P = 0.91) and infants (62.6% vs. 59.5%; P = 0.51) was not significantly different. CONCLUSIONS Giving mother-infant pairs an infusion of peripartum HIV hyperimmune globulin in addition to sdNVP for preventing maternal-to-child transmission was as safe as sdNVP alone but was no more effective than sdNVP alone in preventing HIV transmission.

HIV viruses are usually genetically homogeneous shortly after infection, and become more heterogeneous over time. We developed a high-resolution melting (HRM) assay to analyze HIV diversity without sequencing. Plasma samples from the HIVNET 012 trial were obtained from nine Ugandan mother–infant pairs. DNA amplified from the HIV gag region was analyzed to determine the number of degrees over which the DNA melted (HRM score). HRM gag DNA was also cloned and sequenced (50 clones/mother; 20 clones/infant). The median HRM score for infants (4.3, range 4.2–5.3) was higher than that for control plasmids (3.4, range 3.2–3.8, p

In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of P= 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P< 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8+T-cell selective pressures.

To determine the utility of total lymphocyte count (TLC) in predicting the 12-month mortality in HIV-infected Ugandan children and to correlate TLC and CD4 cell %.This is a retrospective data analysis of clinical and laboratory data collected prospectively on 128 HIV-infected children in the HIV Network for Prevention Trials 012 trial.TLC and CD4 cell % measurements were obtained at birth, 14 weeks, and 12, 24, 36, 48, and 60 months of age and assessed with respect to risk of death within 12 months.Median TLC per microliter (CD4 cell %) was 4150 (41%) at birth, 4900 (24%) at 12 months, 4300 (19%) at 24 months, 4150 (19%) at 36 months, 4100 (18%) at 48 months, and 3800 (20%) at 60 months. The highest risk of mortality within 12 months was 34% - 37% at birth and declined to 13%-15% at 24 months regardless of TLC measurement. The correlation between CD4 cell % and TLC was extremely low overall (r = 0.01).The TLC did not predict a risk of progression to death within 12 months in HIV-infected Ugandan children. Therefore, TLC alone may not be a useful surrogate marker for determining those children at highest risk of death, who require antiretroviral therapy most urgently.

Background. Single-dose nevirapine (SD NVP) at birth plus NVP prophylaxis for the infant up to 6 weeks of age issuperiortoSDNVPaloneforpreventionofverticaltransmissionofhumanimmunodeficiencyvirus(HIV)through breastfeeding. We analyzed NVP resistance in HIV-infected Ugandan infants who received either SD NVP or extended NVP prophylaxis. Methods. We tested plasma HIV by using a genotyping assay (ViroSeq; Celera Diagnostics), a phenotypic resistance assay (PhenoSense; Monogram Biosciences), and sensitive point mutation assay (LigAmp, for K103N, Y181C, and G190A). Results. Wheninfantswere6weeksold,ViroSeqdetectedNVPresistanceinahigherproportionofinfantsinthe extended NVP arm than in the SD NVP arm (21 of 25 [84%] vs. 12 of 24 [50%]; P .01). Similar results were obtained with LigAmp and PhenoSense. In both study arms, infants who were HIV infected at birth frequently had NVP resistance detected. In contrast, infants in the extended NVP arm who were HIV infected after birth were more likely to have resistance detected at 6 weeks, compared with infants in the SD NVP arm. The use of extended NVP prophylaxiswasalsoassociatedwithdetectionofNVPresistancebyViroSeqat6months(7of7[100%]infantsinthe extended NVP arm had resistance detected, compared with 1 of 6 [16.7%] infants in the SD NVP arm; P .005). Conclusions. TheuseofextendedNVPprophylaxiswasassociatedwithincreasedselectionforandpersistenceof NVP resistance in HIV-infected Ugandan infants. Single-dose nevirapine (SD NVP) can reduce the risk of mother-to-child transmission (MTCT) of HIV [1, 2] but may lead to the emergence of resistant variants in infants who are HIV infected despite prophylaxis. After exposure to SD NVP, 46% of Ugandan infants [3] and 87%ofMalawianinfants[4]whowereHIVinfectedhad NVP-resistant variants detected in plasma samples ob

Human immunodeficiency virus type 1 (HIV-1) may utilize the CXCR4 coreceptor (X4 virus) the CCR5 coreceptor (R5 virus) or both (dual/mixed [DM] virus). We analyzed HIV-1 coreceptor tropism in Ugandan infants enrolled in the HIVNET (HIV Network for Prevention Trials) 012 trial. Plasma or serum was analyzed using a commercial coreceptor tropism assay. HIV env subtype was determined by phylogenetic methods. Tropism results were obtained for 57 samples from infants collected 6-14 weeks after birth. Fifty-two infants had only R5 virus and 5 had either X4 or DM virus. The mothers of those 5 infants also had X4 or DM virus. In infants subtype D infection was associated with high-level infectivity in CCR5-bearing cells and also with the detection of X4 or DM strains. High-level infectivity in CCR5-bearing cells was associated with decreased infant survival but infection with X4 or DM virus was not. HIV clones from infants with DM viral populations showed different patterns of coreceptor use. V3 loop sequence-based algorithms predicted the tropism of some but not all env clones. Complex patterns of HIV tropism were found in HIV-infected newborn infants. Subtype D infection was associated with X4 virus and with high-level replication in CCR5-bearing cells. High-level replication of R5 virus was associated with decreased infant survival. (authors)

The objective of this study is to estimate the number of infants alive and HIV-free at 6 to 8 weeks in sub-Saharan Africa due to intervention with antiretroviral prophylaxis to prevent mother-to-child transmission. The number of additional uninfected live infants at 6 to 8 weeks of age due to prophylaxis was calculated using prevalence and prophylaxis data from the World Health Organization and United Nations Children's Fund and data on the efficacy of nevirapine and birth and mortality rates among infants of infected mothers who received prophylaxis in sub-Saharan Africa. Approximately 31 474 African infants were spared from HIV infection or death by 6 to 8 weeks of age due to prophylaxis in 2004 and 2005. The number of infections and deaths averted per year increased from 13 710 in 2004 to 17 764 in 2005. The estimated number of infant HIV infections and deaths averted in 2004 and 2005 due to prophylaxis is encouraging.

Objective To determine costs for adverse event (AE) procedures for a large HIV perinatal trial by analyzing actual resource consumption using activity-based costing (ABC) in an international research setting. Methods The AE system for an ongoing clinical trial in Uganda was evaluated using ABC techniques to determine costs from the perspective of the study. Resources were organized into cost categories (eg, personnel, patient care expenses, laboratory testing, equipment). Cost drivers were quantified, and unit cost per AE was calculated. A subset of time and motion studies was performed prospectively to observe clinic personnel time required for AE identification. Results In 18 months, there were 9028 AEs, with 970 (11%) reported as serious adverse events. Unit cost per AE was $101.97. Overall, AE-related costs represented 32% ($920,581 of $2,834,692) of all study expenses. Personnel ($79.30) and patient care ($11.96) contributed the greatest proportion of component costs. Reported AEs were predominantly nonserious (mild or moderate severity) and unrelated to study drug(s) delivery. Conclusions Intensive identification and management of AEs to conduct clinical trials ethically and protect human subjects require expenditure of substantial human and financial resources. Better understanding of these resource requirements should improve planning and funding of international HIV-related clinical trials.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from ∼20% in early infection to ∼50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated “dual-R,” use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops (“dual-X”). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.

BACKGROUND The present study was undertaken to determine the risk and timing of late postnatal transmission (LPT) of human immunodeficiency virus type 1 (HIV-1). METHODS Breast-fed infants previously enrolled in 2 trials of antiretroviral prophylaxis were monitored in Malawi. Kaplan-Meier and proportional hazard models assessed cumulative incidence and association of factors with LPT. RESULTS Overall, 98 infants were HIV infected, and 1158 were uninfected. The cumulative risk of LPT at age 24 months was 9.68% (95% confidence interval, 7.80%-11.56%). The interval hazards at 1.5-6, 6-12, 12-18, and 18-24 months were 1.22%, 4.05%, 3.48%, and 1.27%, respectively. CONCLUSIONS The risk of LPT beyond 6 months is substantial. Weaning at 6 months could prevent >85% of LPT.

Summary Objective To determine HIV seroincidence, study participant retention rate, and baseline predictors of HIV incidence and study retention among high-risk injection drug users (IDUs) in Xinjiang, China. Methods A total of 508 eligible seronegative high-risk IDUs were enrolled. Study participants were tested for HIV-1 and counseled at the baseline, 6-month, and 12-month follow-up visits. Sociodemographic and behavioral data were collected during each study visit. The HIV-1 incidence rate and the retention rate were analyzed as a function of sociodemographic, behavioral, and recruitment variables. Results At 12 months of follow-up, the HIV-1 incidence rate was 8.8 per 100 person-years (95% CI 6.3–12.0%) and the participant retention rate was 93%. Marital status at baseline was the only predictor of HIV incidence. No baseline variables were predictive of study retention. Conclusions HIV incidence is high among IDUs in Xinjiang, China. Baseline predictors of incidence and retention were minimal. The participant retention rate in this study is promising for the undertaking of future HIV intervention studies.

A prospective observational study of HIV seroincidence among high-risk injection drug users (IDU) was carried out in Guangxi, China. The primary objectives of this study were (1) to estimate HIV seroincidence (2) to estimate participant retention rate and (3) to evaluate changes in drug use and sexual behavior over a one year period.Five hundred HIV seronegative IDU were enrolled. HIV-1 incidence and retention rates were analyzed as a function of sociodemographic, behavioral, and recruitment variables. Changes in drug use and sexual behavior were analyzed at the baseline, 6-month and 12-month follow- up visits.At 12 months of follow-up, the HIV-1 incidence rate was 3.1 per 100 person years, [95% CI: (1.6%; 5.2%)] and participant retention rate was 87%. Reported changes in high-risk behaviors over 12-months included significant decreases in the frequency of heroin injection, in direct or indirect sharing of injection equipment, and in the number of sexual partners.HIV incidence is high among IDU in Guangxi, China despite a self-reported decrease in some high risk behaviors over the course of the study.

Summary Objective To determine the demographic characteristics and risk behaviors associated with HIV positive and negative injection drug users (IDUs) who were screened for inclusion in a prospective cohort study to estimate HIV incidence in Xinjiang, China. Methods Data analyses were performed on the demographic and risk assessment data collected at screening from high-risk IDUs, defined as anyone who reported injecting drugs at least three times per week in the last month or injected drugs after sharing equipment on at least three occasions in the last three months. Results HIV antibody prevalence among 781 IDUs at baseline survey was 29%. The following factors were significantly associated with an increase in risk for HIV infection: age ≥26years (OR=3.16, 95% CI [2.02;4.94]), lack of college education (OR=2.32, 95% CI [1.02;5.25]), frequency of heroin use (OR=1.23, 95% CI [1.02;1.47]) and sharing of rinse water (OR=1.47, 95% CI [1.18;1.84]). Female gender (OR=0.44, 95% CI [0.22;0.86]), being Han vs Uighur ethnicity (OR=0.18, 95% CI [0.11;0.27]), and other race vs Uighur ethnicity (OR=0.23, 95% CI [0.12;0.44]) were significantly associated with a lower risk of HIV infection. Conclusions HIV infection among high risk IDUs in Xinjiang, China is high and is associated with older male Uighur IDUs who lack college education, frequently inject heroin, and/or share rinse water. There is a great potential for HIV transmission from IDUs to the general population. Therefore, related risk behaviors in IDUs, especially their sexual behavior, should be closely monitored.

We used a sensitive point mutation assay, LigAmp, to detect and quantify K103N-containing variants in African women who received single-dose nevirapine (NVP) to prevent mother-to-child HIV-1 transmission.Plasma for testing was collected 6 to 8 weeks postpartum from 301 women (144 subtype A, 63 subtype C, and 94 subtype D).The portion of women with 0.5% or more K103N-containing variants was lowest for subtype A (60/144, 41.7%) and highest for subtype C (44/63, 69.8%; P0.0001). K103N was rarely detected in pre-NVP samples. In a multivariate model, K103N detection was associated with HIV-1 subtype (CA), after adjusting for log10 delivery viral load, the number of days between NVP dosing and sample collection, age, and parity. Among women with K103N detected: (1) the median %K103N was lower for subtype A (2.2%) than C (11.7%, P = 0.0001) or D (5.5%, P = 0.04), and (2) in a multivariate linear model, higher log10 (%K103N) was associated with HIV subtype (CA, P = 0.0001; DA, P = 0.01; and C vs D, no difference), but not other factors.After administration of single-dose NVP, K103N was detected more frequently and at higher levels in women with subtypes C and D than A. Further studies are needed to evaluate the clinical significance of NVP-resistant variants in this setting.

Background: The HIVNET 012 trial in Uganda demonstrated that single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. NVP resistance (NVPR) mutations were detected in 25% of women 6 to 8 weeks after NVP, with a higher rate of NVPR in women with subtype D than A. This study examined emergence and fading of specific NVPR mutations in women with these subtypes. Methods: Plasma HIV-1 was analyzed with the ViroSeq genotyping system (Celera Diagnostics, Alameda, CA). Genotypes were obtained from paired samples collected 7 days and 6 to 8 weeks after NVP from 140 women, 83 with subtype A and 57 with subtype D. Results: The rate of NVPR was similar in women with subtype A vs. D at 7 days but was higher in subtype D than A at 6 to 8 weeks. The higher rate of NVPR in subtype D was explained by at least 2 factors: Y181C faded from detection at a greater rate in women with subtype A (odds ratio = 3.06; 95% CI, 1.04, 8.90) and K103N accumulated at a greater rate in women with subtype D (odds ratio = 1.74; 95% CI, 0.62, 4.87). Conclusions: HIV-1 subtype influences selection and fading of HIV-1 variants with specific drug resistance mutations after antiretroviral drug exposure.

The HIVNET 012 trial in Uganda demonstrated that a regimen of single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. Previous studies show that HIV-1 with one or more NVP resistance (NVPR) mutations can be selected in many women as early as 7 days after single-dose NVP. We evaluated the genetic linkage of NVPR mutations in plasma from women in HIVNET 012 collected 7 days after single-dose NVP administration. The HIV-1 pol region was amplified and cloned from 20 plasma samples (16 with NVPR mutations detected by population sequencing and 4 with no NVPR mutations detected), and 10 clones from each sample were sequenced. Up to five different NVPR mutations were detected in clones from a single sample. K103N and Y181C were the most common mutations detected. Clones with two genetically linked mutations were detected in four samples. Different combinations of NVPR mutations were linked in individual clones, but none of the clones contained both K103N and Y181C. Further studies are needed to evaluate whether selection of minority variants with one or more NVPR mutations after single-dose NVP is clinically relevant.

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at −70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.

Objective: To evaluate the safety, tolerability, and trough levels of three pre-exposure prophylaxis regimens of nevirapine among HIV-1-uninfected subjects at high risk for HIV-1 infection. Methods: A phase I/II trial (HIVHOP 101) in which 33 such uninfected subjects received a 200 mg tablet of nevirapine once weekly (cohort A, n = 12), twice weekly (cohort B, n = 12), or every other day (cohort C, n = 9) for 12 weeks. Clinical signs/ symptoms, laboratory parameters, and nevirapine trough levels were assessed at entry and at 1, 2, 4, 6, 9, and 12 weeks, with a follow-up sample at 16 weeks. Results: No subject experienced clinical symptoms attributed to nevirapine, including rash. There were no significant changes in liver enzyme levels from baseline to week 12 in the three cohorts, except for glutamyl transpeptidase in cohort B. Median nevirapine trough levels at weeks 1 and 12 were 119 ng/ml (range, < 25-205) and 135 ng/ml (range, < 25-1065), respectively, for cohort A, 569 ng/ml (range, 135-2641) and 431 ng/ml (range, 42-2454) for cohort B, and 1942 ng/ml (range, 1214-2482) and 943 ng/ml (range, 262-5281) for cohort C. No subject became HIV-1 antibody positive by week 16. Conclusions: A single dose of nevirapine taken once weekly, twice weekly, or every other day for 12 weeks was safely tolerated by the subjects in this small study, and resulted in nevirapine levels well above the IC 50 (inhibitory concentration of 50%: 10 ng/ml) over the 12-week period in nearly all evaluable subjects.

Vitamin A is essential for immunity and growth. A controlled clinical that involved 697 human immunodeficiency virus (HIV)-infected pregnant women was conducted to determine whether vitamin A prevents anemia, low birth weight, growth failure, HIV transmission, and mortality. Women received daily doses of iron and folate, either alone or combined with vitamin A (3 mg retinol equivalent), from 18-28 weeks' gestation until delivery. In the vitamin A and control groups, respectively, the mean (± SE) birth weights were 2895 ± 31 g and 2805 ± 32 g (P = .05), the proportions of low-birth-weight infants were 14.0% and 21.1% (P = .03), the proportions of anemic infants at 6 weeks postpartum were 23.4% and 40.6% (P

BACKGROUND: An HIV-1 and HCV NAT blood screening assay (Procleix HIV-1/HCV, Gen-Probe, Inc.) simultaneously detecting HIV-1 and HCV RNA) has been implemented. Donor plasma samples reactive in the Procleix HIV-1/HCV assay are tested with the HIV-1 and HCV discriminatory assays to resolve whether HIV-1 RNA, HCV RNA, or both are present. STUDY DESIGN AND METHODS: To determine the specificity of the Procleix HIV-1/HCV assay, data were analyzed for samples from 192,288 donations, tested in 16-member pools. To determine sensitivity, data were analyzed for 2014 commercial samples known to contain HIV-1, HCV, or both, as well as 10 HIV-1 and 10 HCV commercial seroconversion panels. RESULTS: The specificity of the Procleix HIV-1/HCV assay was 99.7 percent. The HIV-1 and HCV discriminatory assays showed similar specificity. The sensitivity of the Procleix HIV-1/ HCV assay was 99.9, 99.6, and 100 percent, respectively, for samples containing HIV-1, HCV, or both. The Procleix discriminatory assays were comparably sensitive. The Procleix discriminatory assays detected all tested samples of known HIV-1 subtype or HCV genotype. Procleix HIV-1/HCV testing of seroconversion panels showed that the median times to a positive reaction for HIV-1 and HCV were reduced by 3 and 25 days, respectively, compared to serologic tests. CONCLUSION: These studies support the use of the Procleix HIV-1/HCV assay for routine blood donor screening.

Most HIV-1 infections in Uganda are caused by subtypes A and D. The prevalence of recombination and the sites of specific breakpoints between these subtypes have not been reported. HIV-1 pol sequences encoding protease (amino acids 1-99) and reverse transcriptase (amino acids 1-324) from 102 pregnant Ugandan women were analyzed by the Recombinant Identification Program, SimPlot, and examination of phylogenetically informative sites to identify sites of recombination between sequence segments belonging to different subtypes. Thirteen percent (13 of 102) of the pol sequences contained strong evidence of recombination between subtypes A and D. At least nine different patterns of recombination were observed. Five women infected with a recombinant virus transmitted the recombinant virus perinatally. In this population-based study, intersubtype recombinants were common. The large number of different types of pol recombinants identified suggests that recombination occurs readily in the pol region. Perinatal transmission of the recombinant viruses demonstrates their evolutionary stability.

The relative potency and tolerability of multidrug regimens used to treat infants and children infected with human immunodeficiency virus type 1 (HIV-1) are largely unknown. In Pediatric AIDS Clinical Trials Group (PACTG) Protocol 377, 181 infants and children were assigned to receive stavudine (d4T) plus nevirapine (NVP) and ritonavir (RTV); d4T plus lamivudine (3TC) and nelfinavir (NFV); d4T plus NVP and NFV; or d4T plus 3TC, NVP, and NFV. Eleven additional children received d4T and NVP plus NFV given twice daily. All subjects had not previously received protease inhibitors or nonnucleoside reverse-transcriptase inhibitors and all had been immunologically stable while receiving reverse-transcriptase inhibitor therapy. After 48 weeks of therapy, 17 (41%) of 41 subjects receiving d4T-NVP-RTV, 13 (30%) of 44 receiving d4T-NVP-NFV, 21 (42%) of 50 receiving d4T-3TC and NFV (3 times daily), and 22 (52%) of 42 receiving d4T-3TC-NVP-NFV were still receiving their assigned therapy and had HIV-1 RNA suppression to

Although anemia is a common finding among human immunodeficiency (HIV)-infected infants in sub-Saharan Africa, the factors contributing to the pathogenesis of anemia have not been well characterized. We sought to characterize the relative contribution of iron deficiency and chronic disease to the anemia among infants. Hemoglobin, ferritin, erythropoietin, tumor necrosis factor-alpha (TNF-alpha), neopterin, CD4(+) lymphocyte count and plasma HIV load were measured in 165 HIV-infected and 39 uninfected 9-mo-old infants seen in an outpatient pediatric clinic in Kampala, Uganda. Among HIV-infected and uninfected infants, the prevalence of anemia (hemoglobin110 g/L) was 90.9 and 76.9%, respectively (P = 0.015), and the prevalence of iron deficiency anemia (hemoglobin110 g/L and ferritin12 microg/L) was 44.3 and 45.4%, respectively (P = 0.92). The relatively higher prevalence of anemia among HIV-infected infants was attributed to the anemia of chronic disease. Among infants with and without iron deficiency, the fitted regression line was log(10) plasma erythropoietin = 2.86 - 0.016.hemoglobin, and log(10) plasma erythropoietin = 4.11 - 0.028.hemoglobin, respectively, with a difference in the slope of the regression lines between log(10) erythropoietin and hemoglobin among infants with and without iron deficiency (P = 0.049). Infants in Uganda have an extremely high prevalence of anemia, and nearly half of the anemia is due to iron deficiency. The erythropoietin response to anemia appears to be upregulated among infants with iron deficiency.

Objective To examine the emergence and fading of NVP resistance (NVP(R)) mutations in HIV-1-infected Ugandan women and infants who received single dose NVP to prevent HIV-1 vertical transmission. Design We examined NVP(R) in women and infants who received NVP in the HIVNET 012 clinical trial, including 41 out of 48 women with infected infants, 70 randomly-selected women with uninfected infants, and 33 out of 49 infected infants. Methods Plasma HIV-1 was analyzed using the Applied Biosystems ViroSeq HIV-1 Genotyping System. Results NVP(R) mutations were detected in 21 out of 111 (19%) women tested 6-8 weeks after delivery. The rate of NVP(R) was similar among women whose infants were or were not HIV-1 infected. K103N was the most common mutation detected. NVP(R) mutations faded from detection within 12-24 months in all 11 evaluable women. High baseline viral load and low baseline CD4 cell count were associated with development of NVP(R). NVP(R) mutations were detected in 11 out of 24 (46%) evaluable infants who were infected by 6-8 weeks of age. The most common NVP(R) mutation detected in infants was Y181C. Those mutations faded from detection by 12 months of age in all seven evaluable infants. Of nine evaluable infants with late HIV-1 infection, only one had evidence of NVP(R). Conclusions NVP(R) was detected more frequently in infants than women following NVP prophylaxis, and different patterns of NVP(R) mutations were detected in women versus infants. NVP(R) was detected infrequently in infants with late HIV-1 infection. NVP-resistant HIV-1 faded from detection in women and infants over time.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.

Objective: To evaluate the safety, tolerability, and anti-HIV activity of ritonavir-nelfinavir (RTV-NFV). Design: Single-site, open-label, nonrandomized, multiple-dose trial of RTV combined with two doses of NFV in protease inhibitor (PI)-naive, HIV-infected patients. Methods: Mean baseline HIV RNA was 39,500 copies/ml; mean baseline CD4 count was 323 cells/mm 3 . All patients received RTV at a dosage of 400 mg twice daily. Cohorts I (N = 10) and II (N = 10) received NFV at a dosage of 500 mg and 750 mg twice daily, respectively, for the initial 12 weeks of the study before allowing intensification with reverse transcriptase inhibitors. Results: The commonest effects of RTV-NFV therapy were study drug-related moderate-to-severe diarrhea (9 patients in cohorts I and II) and drug-related moderate-to-severe nausea (4 patients in cohorts I and II). HIV RNA was suppressed in a biphasic manner. At 48 weeks in cohort I, mean HIV RNA reduction was 2.82 log 10 0 copies/ml (standard error [SE] = .61; p = .001; N = 4); mean CD4 cell count increase was 236 cells/mm 3 (SE = 67.1; p = .006; N = 4). In cohort II, mean HIV RNA reduction at Week 48 was 2.21 log 10 copies/ml (SE = .430; p = .001; N = 8); mean CD4 cell count increase was 120 cells/mm 3 (SE = 47.5; p = .03; n = 8). In cohort I patients, 2 of 4 completing Week 48 had HIV RNA

The administration of single-dose nevirapine to women in labor and their infants can prevent HIV-1 mother-to-child transmission. We examined nevirapine resistance in infants who were HIV-1 infected despite single-dose nevirapine prophylaxis, including 18 Ugandan infants (HIVNET 012 trial, nine subtype A and nine subtype D) and 23 Malawian infants (NVAZ trial, all subtype C). Nevirapine resistance was more frequent in infants with subtype C than with subtypes A and D (87 versus 50%, P = 0.016).

HPTN 046 compared the efficacy and safety of infant nevirapine (NVP) among HIV-exposed breastfed infants randomized at 6 weeks to 6 months to t NVP or placebo to prevent postnatal infection: we report final 18-month outcomes.Randomized, placebo-controlled trial in 4 African countries. Infant diagnostic HIV testing was performed regularly from birth through 18 months. Kaplan-Meier analysis was used to assess 18-month cumulative infant HIV infection, HIV infection/or death, and mortality rates.Between 6 weeks and 6 months, postnatal HIV infection rates were significantly lower among infants receiving daily NVP from 6 weeks to 6 months 1.1% [95% confidence interval (CI): 0.2% to 1.8%], compared with placebo 2.4% (95% CI: 1.3% to 2.6%), P = 0.049, but not significantly lower thereafter. Eighteen-month postnatal infection rates were low: 2.2% (95% CI: 1.1% to 3.3%) versus 3.1% (95% CI: 1.9% to 4.4%), respectively, P = 0.28. Mortality and HIV infection/death did not differ between arms at any age. Infants of women receiving antiretroviral therapy (ART) for their own health had the lowest 18-month postnatal infection rates (0.5%, 95% CI: 0.0% to 1.1%). However, HIV infection/death rates at 18 months were not significantly different for infants of mothers on ART (3.7%, 95% CI: 1.9% to 5.5%), and infants of mothers with CD4 counts of ≥ 350 cells per cubic millimeter not receiving ART (4.8%, 95% CI: 2.7% to 6.8%; P = 0.46). There were no differences in adverse events between study arms.This trial demonstrated early but not late differences in postnatal HIV transmission among infants randomized at age 6 weeks to extended NVP or placebo, underscoring the importance of continued prophylaxis throughout breastfeeding.

OBJECTIVES To describe 5-year growth, survival, and long-term safety among children exposed to nevirapine or zidovudine in an African perinatal prevention trial, HIVNET 012. METHODS All study children who were alive at the age 18 months were eligible for an extended follow-up study. Children whose families consented were enrolled and evaluated every 6 months from 24 to 60 months. At each visit, history, physical examination, and growth measures were taken. From these measurements, Z scores based on World Health Organization (WHO) standards were computed. Serious adverse event data were collected. Data from the initial and extended follow-up cohorts were included in the analysis. RESULTS Five hundred twenty-eight study children were alive at the age 18 months, and 491 (426 HIV uninfected and 65 infected) were enrolled into the follow-up study. Both exposed but uninfected children and HIV-infected children were substantially below WHO growth standards for weight and height. Head circumference Z scores for uninfected children were comparable with WHO norms. Five-year survival rates were 93% for uninfected children versus 43% for infected children. Long-term safety and growth outcomes in the 2 study arms were similar. CONCLUSIONS Both infected and uninfected children in the 5-year HIVNET 012 follow-up showed poor height and weight growth outcomes, underscoring the need for early nutritional interventions to improve long-term growth of all infants born to HIV-infected women in resource-limited settings. Similarly, the low 5-year survival among HIV-infected children support the importance of early initiation of antiretroviral therapy. Both peripartum nevirapine and zidovudine were safe.

Background The level of viral diversity in an HIV-infected individual can change during the course of HIV infection, reflecting mutagenesis during viral replication and selection of viral variants by immune and other selective pressures. Differences in the level of viral diversity in HIV-infected infants may reflect differences in viral dynamics, immune responses, or other factors that may also influence HIV disease progression. We used a novel high resolution melting (HRM) assay to measure HIV diversity in Ugandan infants and examined the relationship between diversity and survival through 5 years of age. Methods Plasma samples were obtained from 31 HIV-infected infants (HIVNET 012 trial). The HRM assay was used to measure diversity in two regions in the gag gene (Gag1 and Gag2) and one region in the pol gene (Pol). Results HRM scores in all three regions increased with age from 6–8 weeks to 12–18 months (for Gag1: P = 0.005; for Gag2: P = 0.006; for Pol: P = 0.016). Higher HRM scores at 6–8 weeks of age (scores above the 75th percentile) were associated with an increased risk of death by 5 years of age (for Pol: P = 0.005; for Gag1/Gag2 (mean of two scores): P = 0.003; for Gag1/Gag2/Pol (mean of three scores): P = 0.002). We did not find an association between HRM scores and other clinical and laboratory variables. Conclusions Genetic diversity in HIV gag and pol measured using the HRM assay was typically low near birth and increased over time. Higher HIV diversity in these regions at 6–8 weeks of age was associated with a significantly increased risk of death by 5 years of age.

BACKGROUND:Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing. METHODS:We used the BED capture immunoassay (BED) and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later). Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer's instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent. RESULTS:During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n) obtained with the BED assay or in the avidity test results (%) when women were pregnant (n = 20 results) compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum). In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test). CONCLUSIONS:These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.

Objectives: To determine normal hematologic and selected blood chemistry values among healthy, full-term, non-HIV-exposed infants in Uganda and Malawi, and to determine the proportion of healthy babies with an apparent laboratory toxicity based on Division of AIDS toxicity tables. Design: This was a cross-sectional laboratory study of infants from birth to 6 months of age. Methods: Blood samples were collected from a total of 561 infants and analyzed according to age categories similar to those in the 2004 Division of AIDS toxicity tables. Select chemistry and hematology parameters were determined and values compared with those in the toxicity tables. Results: In the first 56 days of life, there were few graded toxicities except for neutropenia in 2 of 10 (20%) Ugandan and 13 of 45 (29%) Malawian infants at birth. After 7 days, about 20% of the infants in Uganda and Malawi would have been classified as having a neutropenia whereas 47% and 53% of those more than 2 months of age in Uganda and Malawi respectively, would have been reported as having an abnormal hemoglobin. Chemistry findings were not different from US norms. Conclusions: These findings underscore the importance of establishing relevant local laboratory norms for infants.

Objective: To assess serious gastroenteritis risk and mortality associated with early cessation of breastfeeding in infants enrolled in 2 prevention of maternal-to-child HIV-transmission trials in Uganda.Methods: We used hazard rates to evaluate serious gastroenteritis events by month of age and mortality among HIV-exposed uninfected infants enrolled in the HIV Network for Prevention Trials (HIVNET 012) (1997–2001) and HIV hyperimmune globulin (HIVIGLOB)/nevirapine (NVP) (2004–2007) trials. HIV-infected mothers were counseled using local infant feeding guidelines current at the time.Results: Breastfeeding cessation occurred earlier in HIVIGLOB/NVP compared with HIVNET 012 (median 4.0 versus 9.3 months,P,0.001). Rates of serious gastroenteritis were higher in HIVIGLOB/NVP (8.0/1000 child-months) than in HIVNET 012 (3.1/1000 child-months; P , 0.001). Serious gastroenteritis events also peaked earlier at 3–4 and 7–8 months (16.2/1000 and 15.0/1000 child-months,respectively) compared with HIVNET 012 at 9–10 months (20.8/1000 child-months). All cause infant mortality did not statistically differ between the HIVIGLOB/NVP and the HIVNET 012 trials [3.2/1000 versus 2.0/1000 child-months, respectively (P = 0.10)].Conclusions: Early breastfeeding cessation seen in the HIVIGLOB/NVP trial was associated with increased risk of serious gastroenteritis among HIV-exposed uninfected infants when compared with later breastfeeding cessation in the HIVNET 012 trial.Testing interventions, which could decrease HIV transmission through breastfeeding and allow safe

OBJECTIVE: To determine the predictors for early versus later (breastfeeding) transmission of HIV-1. METHODS: Secondary data analysis was performed on HIV Network for Prevention Trials 012 a completed randomized clinical trial assessing the relative efficacy of nevirapine (NVP) versus zidovudine in reducing mother-to-child transmission (MTCT) of HIV-1. We used Cox regression analysis to assess risk factors for MTCT. The ViroSeq HIV genotyping and a sensitive point mutation assay were used to detect NVP resistance mutations. RESULTS: In this subset analyses 122 of 610 infants were HIV infected of whom 99 (81.1%) were infected early (first positive polymerase chain reaction < or =56 days). Incidence of MTCT after 56 days was low [0.7% per month (95% confidence interval CI: 0.4 to 1.0)] but continued through 18 months. In multivariate analyses early MTCT "factors" included NVP versus zidovudine (hazard ratio (HR) = 0.57 95% CI: 0.38 to 0.86) pre-entry maternal viral load (VL HR = 1.76 95% CI: 1.28 to 2.41) and CD4 cell count (HR = 1.16 95% CI: 1.05 to 1.28). Maternal VL (6-8 weeks) was associated with late MTCT (HR = 3.66 95% CI: 1.78 to 7.50) whereas maternal NVP resistance (6-8 weeks) was not. CONCLUSIONS: Maternal VL was the best predictor of both early and late transmission. Maternal NVP resistance at 6-8 weeks did not predict late transmission.

Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.

We previously reported the existence of CXCR4-using HIV-1 in 6-14 week-old Ugandan infants. Whether these viruses were transmitted from the mother perinatally or evolved after transmission is not known. In the current study, we investigated the origin of the CXCR4-using viruses in these infants by comparing HIV-1 envelope clones from the infants to those from their mothers at or near the time of delivery.Envelope clones were isolated from five Ugandan infant plasma samples that harbored CXCR4-using viruses, collected at the time of HIV diagnosis (four at birth, one at week 6), and from their mothers at delivery. Coreceptor usage and phylogenetic relatedness of HIV-1 populations in mother-infant pairs were analyzed in detail using the Trofile assay and sequence analysis of envelope clones, respectively.X4-tropic clones were identified in two mother-infant pairs and dual-tropic clones were found in three pairs, either alone or in combination with R5-tropic viruses. Dual-tropic clones varied in their ability to infect CXCR4-expressing cells. In each mother-infant pair, X4-tropic or dual-tropic clones shared similar phenotypic profiles and V3 sequence patterns; gp160 sequences of X4-tropic and dual-tropic clones from infants were phylogenetically indistinguishable from those of their mothers. The virus populations were phylogenetically homogenous in three infants and segregated according to coreceptor tropism in the remaining two infants.This study demonstrates that X4-tropic and dual-tropic HIV-1 can be transmitted from mother to infant, before, during or shortly after delivery, and establishes vertical transmission as an important source of CXCR4-using viruses in infants.

We analyzed HIV gp41 from 195 men in the United States who were HIV-1 infected between 1999 and 2002, before enfuvirtide (ENF) was approved for clinical use in the United States. gp41 genotyping results were obtained for 175 samples. None of the samples had major ENF resistance mutations. Six (3.4%) samples had minor ENF resistance mutations in the HR1 region (V38G, N43K, L44M, L45M). Twenty-eight (16%) samples had the N42S polymorphism, which is associated with ENF hypersusceptibility. Accessory mutations in the HR2 region were identified in some samples (E137K, S138A). Five of the six samples with HR1 resistance mutations were analyzed with a phenotypic assay; one sample had reduced ENF susceptibility (a sample with N42S + L44M + E137K). Prior to the availability of ENF, some men in the United States were infected with HIV that contained mutations associated with ENF resistance or hypersusceptibility. However, most of the mutations were not associated with phenotypic ENF resistance.

Two non-isotopic polymerase chain reaction (PCR) methods were evaluated by testing blood from 41 HIV-1-seropositive and 16 HIV-1-seronegative Ugandan mothers and 56 of their children (aged 0.5-15.0 months). Amplification of HIV-1 sequences was performed in duplicate using a biotinylated primer pair to the gag region (SK 462-431) and nested primer pairs (JA 17-20) to the pol region of HIV-1. gag sequences were hybridized using a microtiter plate coated with the SK 102 probe followed by colorimetric detection using an avidin-horseradish peroxidase conjugate and tetramethylbenzidine/peroxide substrate. pol sequences were detected on agarose gel stained with ethidium bromide. Results of HIV-1 PCR analysis showed that 40 out of 41 (98%) seropositive mothers and 10 out of 29 (34%) seropositive children had detectable HIV-1 gag and pol sequences. None of the 16 seronegative mothers nor 27 seronegative or Western blot-indeterminate children had detectable HIV-1 sequences. Our results suggest that non-isotopic PCR methods are sensitive, specific, and potentially useful in the early diagnosis of HIV-1 infection in developed and developing countries.

UNICEF/WHO recommends that infants born to HIV-infected mothers who do not have access to acceptable, feasible, affordable, sustainable, and safe replacement feeding should be exclusively breastfed for at least 6 months. The aim of three trials in Ethiopia, India, and Uganda was to assess whether daily nevirapine given to breastfed infants through 6 weeks of age can decrease HIV transmission via breastfeeding.HIV-infected women breastfeeding their infants were eligible for participation. Participants were randomly assigned to receive either single-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborns after birth) or 6 week extended-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborn babies after birth plus nevirapine 5 mg daily from days 8-42 for the infant). The randomisation sequences were generated by computer at a central data coordinating centre. The primary endpoint was HIV infection at 6 months of age in infants who were HIV PCR negative at birth. Analyses were by modified intention to treat, excluding infants with missing specimens and those with indeterminate or confirmed HIV infection at birth. These studies are registered with ClinicalTrials.gov, numbers NCT00074399, NCT00061321, and NCT00639938.2024 liveborn infants randomised in the study had at least one specimen tested before 6 months of age (1047 infants in the single-dose group and 977 infants in the extended-dose group). The modified intention-to-treat population included 986 infants in the single-dose group and 901 in the extended-dose group. At 6 months, 87 children in the single-dose group and 62 in the extended-dose group were infected with HIV (relative risk 0.80, 95% CI 0.58-1.10; p=0.16). At 6 weeks of age, 54 children in the single-dose group and 25 in the extended-dose group were HIV positive (0.54, 0.34-0.85; p=0.009). 393 infants in the single-dose group and 346 in the extended-dose group experienced grade 3 or 4 serious adverse events during the study (p=0.54).Although a 6-week regimen of daily nevirapine might be associated with a reduction in the risk of HIV transmission at 6 weeks of age, the lack of a significant reduction in the primary endpoint-risk of HIV transmission at 6 months-suggests that a longer course of daily infant nevirapine to prevent HIV transmission via breast milk might be more effective where access to affordable and safe replacement feeding is not yet available and where the risks of replacement feeding are high.US National Institutes of Health; US National Institute of Allergy and Infectious Diseases; Fogarty International Center.

The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 μl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 μl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.

Use of single dose nevirapine (SD NVP) for prevention of HIV-1 mother-to-child transmission (pMTCT) is associated with selection of K103N-containing HIV variants. Repeat use of SD NVP for pMTCT may influence emergence and persistence of NVP-resistant variants. K103N-containing variants were studied in 48 Ugandan women who received SD NVP in the HIVNET 012 trial and were re-exposed to SD NVP in one (n=44) or two (n=4) subsequent pregnancies during a 5-year follow-up study. Methods: Samples were analyzed using the LigAmp assay (assay cutoff: 0.5% K103N). Among 44 women who were re-exposed to SD NVP in one subsequent pregnancy 37.8% had K103N detected within 1 year of SD-NVP re-exposure. Detection of K103N was independently associated with detection of K103N 6-8 weeks after the first SD NVP exposure and with pre-NVP viral load. The portion of women with undetectable K103N by 2 years after SD NVP administration was similar after first versus second use of SD NVP for pMTCT. K103N was undetectable in 93.2% of evaluable women by 3 years of re-exposure. Only two of four women who received SD NVP in two pregnancies during the follow-up study had K103N detected after the last SD NVP exposure. K103N was detected in some women within 1 year of SD NVP reexposure but faded from detection in most women by 3 years after re-exposure. Detection of K103N by 1 year after SD NVP re-exposure was associated with prior selection of K103N-containing variants and with pre-NVP viral load. (authors)