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1.  High Rates of HIV Seroconversion in Pregnant Women and Low Reported Levels of HIV Testing among Male Partners in Southern Mozambique: Results from a Mixed Methods Study 
PLoS ONE  2014;9(12):e115014.
Introduction
Prevention of acute HIV infections in pregnancy is required to achieve elimination of pediatric HIV. Identification and support for HIV negative pregnant women and their partners, particularly serodiscordant couples, are critical. A mixed method study done in Southern Mozambique estimated HIV incidence during pregnancy, associated risk factors and factors influencing partner's HIV testing.
Methods
Between April 2008 and November 2011, a prospective cohort of 1230 HIV negative pregnant women was followed during pregnancy. A structured questionnaire, HIV testing, and collection of dried blood spots were done at 2–3 scheduled visits. HIV incidence rates were calculated by repeat HIV testing and risk factors assessed by Poisson regression. A qualitative study including 37 individual interviews with men, women, and nurses and 11 focus group discussions (n = 94) with men, women and grandmothers explored motivators and barriers to uptake of male HIV testing.
Results
HIV incidence rate was estimated at 4.28/100 women-years (95%CI: 2.33–7.16). Significant risk factors for HIV acquisition were early sexual debut (RR 3.79, 95%CI: 1.04–13.78, p = 0.04) and living in Maputo Province (RR 4.35, 95%CI: 0.97–19.45, p = 0.05). Nineteen percent of women reported that their partner had tested for HIV (93% knew the result with 8/213 indicating an HIV positive partner), 56% said their partner had not tested and 19% did not know their partner test status. Of the 14 seroconversions, only one reported being in a serodiscordant relationship. Fear of discrimination or stigma was reported as a key barrier to male HIV testing, while knowing the importance of getting tested and receiving care was the main motivator.
Conclusions
HIV incidence during pregnancy is high in Southern Mozambique, but knowledge of partners' HIV status remains low. Knowledge of both partners' HIV status is critical for maximal effectiveness of prevention and treatment services to reach elimination of pediatric HIV/AIDS.
doi:10.1371/journal.pone.0115014
PMCID: PMC4277288  PMID: 25542035
2.  Long Term Follow-up of Children in the HIVNET 012 Perinatal HIV Prevention Trial: Five-Year Growth and Survival 
Objectives
To describe five 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 eighteen months of age were eligible for an extended follow-up study. Children whose families consented were enrolled and evaluated every six months from 24 to 60 months. At each visit, history, physical exam 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
528 study children were alive at age 18 months, and 491 (426 HIV uninfected; 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 to WHO norms. Five-year survival rates were 93% for uninfected children versus 43% for infected children. Long-term safety and growth outcomes in the two study arms were similar.
Conclusions
Both infected and uninfected children in the five-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. Likewise, the low five year survival among HIV infected children support the importance of early initiation of antiretroviral therapy. Both peripartum nevirapine and zidovudine were safe.
doi:10.1097/QAI.0000000000000015
PMCID: PMC4172334  PMID: 24121753
3.  Access to HIV prevention and care for HIV-exposed and HIV-infected children: a qualitative study in rural and urban Mozambique 
BMC Public Health  2014;14(1):1240.
Background
Follow-up of HIV-exposed children for the delivery of prevention of mother-to-child transmission services and for early diagnosis and treatment of HIV infection is critical to their survival. Despite efforts, uptake of postnatal care for these children remains low in many sub-Saharan African countries.
Methods
A qualitative study was conducted in three provinces in Mozambique to identify motivators and barriers to improve uptake of and retention in HIV prevention, care and treatment services for HIV-exposed and HIV-infected children. Participant recommendations were also gathered. Individual interviews (n = 79) and focus group discussions (n = 32) were conducted with parents/caregivers, grandmothers, community leaders and health care workers. Using a socioecological framework, the main themes identified were organized into multiple spheres of influence, specifically at the individual, interpersonal, institutional, community and policy levels.
Results
Study participants reported factors such as seeking care outside of the conventional health system and disbelief in test results as barriers to use of HIV services. Other key barriers included fear of disclosure at the interpersonal level and poor patient flow and long waiting time at the institutional level. Key facilitators for accessing care included having hope for children’s future, symptomatic illness in children, and the belief that health facilities were the appropriate places to get care.
Conclusions
The results suggest that individual-level factors are critical drivers that influence the health-seeking behavior of caregivers of HIV-exposed and HIV-infected children in Mozambique. Noted strategies are to provide more information and awareness on the benefits of early pediatric testing and treatment with positive messages that incorporate success stories, to reach more pregnant women and mother-child pairs postpartum, and to provide counseling during tracing visits. Increasing uptake and retention may be achieved by improving patient flow at the institutional level at health facilities, by addressing concerns with family decision makers, and by working with community leaders to support the uptake of services for HIV-exposed children for essential preventive care.
doi:10.1186/1471-2458-14-1240
PMCID: PMC4265432  PMID: 25467030
Prevention of mother-to-child transmission; Barriers; Motivators; Pediatric HIV; Mozambique; HIV-exposed children; Infant HIV testing; Early infant diagnosis
4.  Feasibility and Safety of ALVAC-HIV vCP1521 Vaccine in HIV- exposed Infants in Uganda: Results from the First HIV Vaccine Trial in Infants in Africa 
Background
The development of a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) for prevention mother-to-child transmission of HIV would significantly advance the goal of eliminating HIV infection in children. Safety and feasibility results from Phase I, randomized, double blind, placebo-controlled trial of ALVAC-HIV vCP1521 in infants born to HIV-1-infected women in Uganda are reported.
Methods
HIV exposed infants were enrolled at birth and randomized (4:1) to receive vaccine or saline placebo intramuscular injections at birth, 4, 8 and 12 weeks of age. Vaccine reactogenicity was assessed at vaccination, and days 1 and 2 post-vaccination. Infants were followed until 24 months of age. HIV infection status was determined by HIV DNA PCR.
Findings
From October 2006 to May 2007, 60 infants (48 vaccine, 12 placebo) were enrolled with 98% retention at 24 months. One infant was withdrawn, but there were no missed visits or vaccinations among the 59 infants retained. Immune responses elicited by Diptheria, Polio, Hepatitis B and Heamophilus influenzae type B and measles vaccination were similar in the two arms. The vaccine was well tolerated with no severe or life-threatening reactogenicity events. Adverse events were equally distributed across both study arms. Four infants were diagnosed as HIV infected [3 at birth (2 vaccine, 1 placebo) and one in vaccine arm at 2 weeks of age].
Interpretation
The ALVAC-HIV vCP1521 vaccination was feasible and safe in infants born to HIV-infected women in Uganda. The conduct of high quality infant HIV vaccine trials is achievable in Africa.
doi:10.1097/QAI.0b013e31827f1c2d
PMCID: PMC3625520  PMID: 23221981
HIV vaccine; ALVAC; infants; Africa; breast milk transmission
5.  High HIV incidence in the postpartum period sustains vertical transmission in settings with generalized epidemics: a cohort study in Southern Mozambique 
Introduction
Acute infection with HIV in the postpartum period results in a high risk of vertical transmission through breastfeeding. A study was done to determine the HIV incidence rate and associated risk factors among postpartum women in Southern Mozambique, where HIV prevalence among pregnant women is 21%.
Methods
A prospective cohort study was conducted in six rural health facilities in Gaza and Maputo provinces from March 2008 to July 2011. A total of 1221 women who were HIV-negative on testing at delivery or within two months postpartum were recruited and followed until 18 months postpartum. HIV testing, collection of dried blood spot samples and administration of a structured questionnaire to women were performed every three months. Infant testing by DNA-PCR was done as soon as possible after identification of a new infection in women. HIV incidence was estimated, and potential risk factors at baseline were compared using Poisson regression.
Results
Data from 957 women were analyzed with follow-up after the enrolment visit, with a median follow-up of 18.2 months. The HIV incidence in postpartum women is estimated at 3.20/100 women-years (95% CI: 2.30–4.46), with the highest rate among 18- to 19-year-olds (4.92 per 100 women-years; 95% CI: 2.65–9.15). Of the new infections, 14 (34%) were identified during the first six months postpartum, 11 (27%) between 6 and 12 months and 16 (39%) between 12 and 18 months postpartum. Risk factors for incident HIV infection include young age, low number of children, higher education level of the woman's partner and having had sex with someone other than one's partner. The vertical transmission was 21% (95% CI: 5–36) among newly infected women.
Conclusions
Incidence of HIV is high among breastfeeding women in Southern Mozambique, contributing to increasing numbers of HIV-infected infants. Comprehensive primary prevention strategies targeting women of reproductive age, particularly pregnant and postpartum women and their partners, will be crucial for the elimination of paediatric AIDS in Africa.
doi:10.7448/IAS.17.1.18808
PMCID: PMC3946505  PMID: 24629842
PMTCT; breastfeeding; incidence; HIV; elimination paediatric HIV; Mozambique
6.  Efficacy and safety of an extended nevirapine regimen in infant children of breastfeeding mothers with HIV-1 infection for prevention of postnatal HIV-1 transmission (HPTN 046): a randomised, double-blind, placebo-controlled trial 
Lancet  2011;379(9812):221-228.
Summary
Background
Nevirapine given once-daily for the first 6, 14, or 28 weeks of life to infants exposed to HIV-1via breastfeeding reduces transmission through this route compared with single-dose nevirapine at birth or neonatally. We aimed to assess incremental safety and efficacy of extension of such prophylaxis to 6 months.
Methods
In our phase 3, randomised, double-blind, placebo-controlled HPTN 046 trial, we assessed the incremental benefit of extension of once-daily infant nevirapine from age 6 weeks to 6 months. We enrolled breastfeeding infants born to mothers with HIV-1 in four African countries within 7 days of birth. Following receipt of nevirapine from birth to 6 weeks, infants without HIV infection were randomly allocated (by use of a computer-generated permuted block algorithm with random block sizes and stratified by site and maternal antiretroviral treatment status) to receive extended nevirapine prophylaxis or placebo until 6 months or until breastfeeding cessation, whichever came first. The primaryefficacy endpoint was HIV-1 infection in infants at 6 months and safety endpoints were adverse reactions in both groups. We used Kaplan-Meier analyses to compare differences in the primary outcome between groups. This study is registered with ClinicalTrials.gov, number NCT00074412.
Findings
Between June 19, 2008, and March 12, 2010, we randomly allocated 1527 infants (762 nevirapine and 765 placebo); five of whom had HIV-1 infection at randomisation and were excluded from the primary analyses. In Kaplan-Meier analysis, 1.1% (95% CI 0.3–1.8) of infants who received extended nevirapine developed HIV-1 between 6 weeks and 6 months compared with 2.4% (1.3–3.6) of controls (difference 1.3%, 95% CI 0–2.6), equating to a 54% reduction in transmission (p=0.049). However, mortality (1.2% for nevirapine vs 1.1% for placebo; p=0.81) and combined HIV infection and mortality rates (2.3% vs 3.2%; p=0.27) did not differ between groups at 6 months. 125 (16%) of 758 infants given extended nevirapine and 116 (15%) of 761 controls had serious adverse events, but frequency of adverse events, serious adverse events, and deaths did not differ significantly between treatment groups.
Interpretation
Nevirapine prophylaxis can safely be used to provide protection from mother-to-child transmission of HIV-1 via breastfeeding for infants up to 6 months of age.
Funding
US National Institutes of Health.
doi:10.1016/S0140-6736(11)61653-X
PMCID: PMC3539769  PMID: 22196945
7.  Safety and Efficacy of HIV Hyperimmune Globulin (HIVIGLOB) for Prevention of Mother-to-Child HIV Transmission in HIV-1 infected Pregnant Women and their Infants in Kampala, Uganda (HIVIGLOB/NVP STUDY) 
Background
This phase III randomized clinical trial compared single dose nevirapine (sdNVP) plus HIV immunoglobulin (HIVIGLOB) to sdNVP alone for preventing maternal-to-child transmission (PMTCT) 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 200mg of NVP to women in labor and 2mg/kg NVP to newborns within 72 hours after birth (sdNVP arm) or to receive sdNVP plus a single intravenous 240ml dose of HIVIGLOB given to women at 36-38 weeks gestation and a single intravenous 24ml 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 versus the sdNVP arm (18.7% vs.15.0%; RR =1.240 [95% CI: 0.833-1.846]; 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), were not significantly different.
Conclusion
Giving mother-infant pairs an infusion of peripartum HIV hyperimmunoglobulin in addition to sdNVP for PMTCT was as safe as sdNVP alone, but was no more effective than sdNVP alone in preventing HIV transmission.
doi:10.1097/QAI.0b013e31822f8914
PMCID: PMC3204156  PMID: 21826009
HIV; HIVIGLOB; sdNVP; breastfeeding; PMTCT; Uganda
8.  Analysis of HIV tropism in Ugandan infants 
Current HIV research  2010;8(7):498-503.
HIV-infected infants may have CXCR4-using (X4-tropic) HIV, CCR5-using (R5-tropic) HIV, or a mixture of R5-tropic and X4-tropic HIV (dual/mixed, DM HIV). The level of infectivity for R5 virus (R5-RLU) varies among HIV-infected infants. HIV tropism and R5-RLU were measured in samples from HIV-infected Ugandan infants using a commercial assay. DM HIV was detected in 7/72 (9.7%) infants at the time of HIV diagnosis (birth or 6–8 weeks of age, 4/15 (26.7%) with subtype D, 3/57 (5.3 %) with other subtypes, P=0.013). A transition from R5-tropic to DM HIV was observed in only two (6.7%) of 30 infants over 6–12 months. Six (85.7%) of seven infants with DM HIV died, compared to 21/67 (31.3%) infants with R5-tropic HIV (p=0.09). Higher R5-RLU at 6–8 weeks was not associated with decreased survival. Infants with in utero infection had a higher median R5-RLU than infants who were HIV-uninfected at birth (p=0.025).
PMCID: PMC3075545  PMID: 21073438
CCR5; CXCR4; HIV-1; infant; survival; transmission; tropism
9.  Analysis of HIV Diversity Using a High-Resolution Melting Assay 
Abstract
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 < 0.001) and lower than that for mothers (5.7, range 4.4–7.7, p = 0.005, exact Wilcoxon rank sum test). The intraclass correlation coefficient reflecting assay reproducibility was 94% (95% CI: 89–98%). HRM scores were also compared to sequenced-based measures of HIV diversity; higher HRM scores were associated with higher genetic diversity (p < 0.001), complexity (p = 0.009), and Shannon entropy (p = 0.022), but not with length variation (p = 0.111). The HRM assay provides a novel, rapid method for assessing HIV diversity without sequencing. This assay could be applied to any region of the HIV genome or to other genetic systems that exhibit DNA diversity.
doi:10.1089/aid.2009.0259
PMCID: PMC2920076  PMID: 20666583
10.  Early Weaning of HIV-Exposed Uninfected Infants and Risk of Serious Gastroenteritis: Findings from Two Perinatal HIV Prevention Trials in Kampala, Uganda 
Journal of acquired immune deficiency syndromes (1999)  2009;10.1097/QAI.0b013e3181bdf68e.
Objective
To assess serious gastroenteritis risk and mortality associated with early cessation of breastfeeding in infants enrolled in two 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 HIVNET 012 (1997-2001) and HIVIGLOB/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 to HIVNET 012 (median 4.0 vs. 9.3 months, p<0.001). Rates of serious gastroenteritis were higher in HIVIGLOB/NVP (8.0/1000 child-months) compared to 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 to HIVNET 012 at 9 to10 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)]
Conclusion
Early breastfeeding cessation seen in the HIVIGLOB/NVP trial was associated with increased risk of serious gastroenteritis among HIV-exposed uninfected infants when compared to later breastfeeding cessation in the HIVNET 012 trial. Testing interventions which could decrease HIV transmission through breastfeeding and allow safe breastfeeding into the second year of life are urgently needed.
doi:10.1097/QAI.0b013e3181bdf68e
PMCID: PMC2888913  PMID: 19779355
HIV; infants; breastfeeding cessation; serious gastroenteritis; mortality; Uganda
11.  Considerations in Using US-Based Laboratory Toxicity Tables to Evaluate Laboratory Toxicities Among Healthy Malawian and Ugandan Infants 
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.
doi:10.1097/QAI.0b013e3181db059d
PMCID: PMC3033212  PMID: 20588184
Division of AIDS; normal laboratory reference values; non–HIV-exposed; toxicity tables; Ugandan; Malawian; infants
12.  Pregnancy Does Not Affect HIV Incidence Test Results Obtained Using the BED Capture Enzyme Immunoassay or an Antibody Avidity Assay 
PLoS ONE  2010;5(10):e13259.
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.
doi:10.1371/journal.pone.0013259
PMCID: PMC2952593  PMID: 20949006
13.  Short Communication: In Utero HIV Infection Is Associated with an Increased Risk of Nevirapine Resistance in Ugandan Infants Who Were Exposed to Perinatal Single Dose Nevirapine 
Use of single dose nevirapine (sdNVP) to prevent HIV mother-to-child transmission is associated with the emergence of NVP resistance in many infants who are HIV infected despite prophylaxis. We combined results from four clinical trials to analyze predictors of NVP resistance in sdNVP-exposed Ugandan infants. Samples were tested with the ViroSeq HIV Genotyping System and a sensitive point mutation assay (LigAmp, for detection of K103N, Y181C, and G190A). NVP resistance was detected at 6–8 weeks in 36 (45.0%) of 80 infants using ViroSeq and 33 (45.8%) of 72 infants using LigAmp. NVP resistance was more frequent among infants who were infected in utero than among infants who were diagnosed with HIV infection after birth by 6–8 weeks of age. Detection of NVP resistance at 6–8 weeks was not associated with HIV subtype (A vs. D), pre-NVP maternal viral load or CD4 cell count, infant viral load at 6–8 weeks, or infant sex. NVP resistance was still detected in some infants 6–12 months after sdNVP exposure. In this study, in utero HIV infection was the only factor associated with detection of NVP resistance in infants 6–8 weeks after sdNVP exposure.
doi:10.1089/aid.2009.0003
PMCID: PMC2752753  PMID: 19552593
14.  Comparison of Laboratory Methods for Analysis of Non-nucleoside Reverse Transcriptase Inhibitor Resistance in Ugandan Infants 
Abstract
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.
doi:10.1089/aid.2008.0235
PMCID: PMC2799186  PMID: 19621988
15.  Short Communication: In Utero HIV Infection Is Associated with an Increased Risk of Nevirapine Resistance in Ugandan Infants Who Were Exposed to Perinatal Single Dose Nevirapine 
Abstract
Use of single dose nevirapine (sdNVP) to prevent HIV mother-to-child transmission is associated with the emergence of NVP resistance in many infants who are HIV infected despite prophylaxis. We combined results from four clinical trials to analyze predictors of NVP resistance in sdNVP-exposed Ugandan infants. Samples were tested with the ViroSeq HIV Genotyping System and a sensitive point mutation assay (LigAmp, for detection of K103N, Y181C, and G190A). NVP resistance was detected at 6–8 weeks in 36 (45.0%) of 80 infants using ViroSeq and 33 (45.8%) of 72 infants using LigAmp. NVP resistance was more frequent among infants who were infected in utero than among infants who were diagnosed with HIV infection after birth by 6–8 weeks of age. Detection of NVP resistance at 6–8 weeks was not associated with HIV subtype (A vs. D), pre-NVP maternal viral load or CD4 cell count, infant viral load at 6–8 weeks, or infant sex. NVP resistance was still detected in some infants 6–12 months after sdNVP exposure. In this study, in utero HIV infection was the only factor associated with detection of NVP resistance in infants 6–8 weeks after sdNVP exposure.
doi:10.1089/aid.2009.0003
PMCID: PMC2752753  PMID: 19552593
16.  Comparison of Laboratory Methods for Analysis of Non-nucleoside Reverse Transcriptase Inhibitor Resistance in Ugandan Infants 
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.
doi:10.1089/aid.2008.0235
PMCID: PMC2799186  PMID: 19621988
17.  Predictors of Early and Late Mother-to-Child Transmission of HIV in a Breastfeeding Population: HIV Network for Prevention Trials 012 Experience, Kampala, Uganda 
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 ≤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.
doi:10.1097/QAI.0b013e3181afd352
PMCID: PMC2767188  PMID: 19617849
early/late postnatal; MTCT; HIV-1
18.  Vertical transmission of X4-tropic and dual-tropic HIV-1 in five Ugandan mother–infant pairs 
AIDS (London, England)  2009;23(14):1903-1908.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1097/QAD.0b013e32832f1802
PMCID: PMC2764460  PMID: 19593079
coreceptor tropism; CXCR4; HIV; mother-to-child; transmission; X4
19.  Total Lymphocyte Count: not a surrogate marker for risk of death in HIV infected Ugandan children 
Objectives
To determine the utility of Total Lymphocyte Count (TLC) in predicting the 12 month mortality in HIV infected Ugandan children; to correlate TLC and CD4 cell %.
Design
This is a retrospective data analysis of clinical and laboratory data collected prospectively on 128 HIV infected children in the HIVNET 012 trial.
Methods
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.
Results
Median TLC/ul (CD4 cell %) were 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).
Conclusion
The TLC did not predict a risk of progression to death within 12 months and therefore TLC alone may not be a useful surrogate marker for determining those children in greatest need for antiretroviral therapy in HIV infected Ugandan children.
doi:10.1097/QAI.0b013e318183a92a
PMCID: PMC2721476  PMID: 18769352
Total Lymphocyte Count; HIV; Africa; children
20.  Analysis of nevirapine (NVP) resistance in Ugandan infants who were HIV-infected despite receiving single dose (SD) nevirapine (NVP) vs. SD NVP plus daily NVP up to 6-weeks of age to prevent HIV vertical transmission 
The Journal of infectious diseases  2008;198(7):1075-1082.
Background
Single dose (SD) nevirapine (NVP) at birth plus NVP to the infant up to 6 weeks of age is superior to SD NVP alone for prevention of HIV vertical transmission 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 using a genotyping assay (ViroSeq), a phenotypic resistance assay (PhenoSense), and sensitive point mutation assay (LigAmp, for K103N, Y181C, G190A).
Results
At 6 weeks, NVP resistance was detected by ViroSeq in a higher proportion of infants in the extended NVP arm than in the SD NVP arm (21/25=84% vs. 12/24=50%, p=0.01). Similar results were obtained with LigAmp and PhenoSense. Infants who were HIV-infected at birth had high rates of resistance in both study arms. In contrast, infants who were HIV-infected after birth were more likely to have resistance detected at 6 weeks in the extended NVP arm. Use of extended NVP prophylaxis was also associated with detection of NVP resistance by ViroSeq at 6 months (7/7=100% extended NVP arm vs. 1/6=16.7% SD NVP arm, p=0.005).
Conclusions
Use of extended NVP prophylaxis was associated with increased selection and persistence of NVP resistance in HIV-infected Ugandan infants.
doi:10.1086/591503
PMCID: PMC2587235  PMID: 18684096
HIV-1; infant; mother-to-child transmission; nevirapine; resistance
21.  Associations of Chemokine Receptor Polymorphisms With HIV-1 Mother-to-Child Transmission in Sub-Saharan Africa: Possible Modulation of Genetic Effects by Antiretrovirals 
Background
HIV-1 mother-to-child transmission (MTCT) remains an important route of infection in sub-Saharan Africa.
Methods
Genetic variants in CCR5 promoter, CCR2, CX3CR1, and Stromal cell-derived factor-1 (SDF-1) genes were determined in 980 infants from sub-Saharan Africa using real-time polymerase chain reaction to determine association with MTCT.
Results
In antiretroviral-naive mother–infant pairs (n = 637), CCR5 promoter polymorphisms at positions 59029: A allele vs. G/G [odds ratio (OR): 1.61, 95% confidence interval (CI): 1.04 to 2.48; P = 0.032] and 59356: T allele vs. C/C (OR: 0.63, 95% CI: 0.41 to 0.96; P = 0.033) and CCR2-180: G allele vs. A/A (OR: 3.32, 95% CI: 1.13 to 9.73; P = 0.029) were associated with risk of MTCT. Treatment of HIV-1–infected mothers and infants with single-dose nevirapine or perinatal zidovudine altered but did not eliminate the association of genetic variants with MTCT.
Conclusions
CCR5 promoter, CCR2, and CX3CR1 polymorphisms were associated with risk of MTCT likely through their role as an HIV-1 coreceptor or by modulating the early immune response. Host genetics may continue to alter MTCT when short-course interventions that only partially suppress virus are used. These findings will need to be confirmed in validation cohorts with a large number of infected infants.
doi:10.1097/QAI.0b013e318186eaa4
PMCID: PMC2748918  PMID: 18845960
mother-to-child transmission; HIV-1; chemokine/chemokine receptor genotypes; antiretrovirals
22.  Analysis of nevirapine resistance mutations in cloned HIV-1 variants from HIV-infected Ugandan infants using a single step amplification-sequencing method (AmpliSeq) 
AIDS research and human retroviruses  2008;24(9):1209-1213.
We analyzed genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV-infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically-linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
doi:10.1089/aid.2008.0109
PMCID: PMC2562759  PMID: 18788912
23.  Analysis of Nevirapine Resistance Mutations in Cloned HIV Type 1 Variants from HIV-Infected Ugandan Infants Using a Single-Step Amplification-Sequencing Method (AmpliSeq) 
AIDS Research and Human Retroviruses  2008;24(9):1209-1213.
Abstract
We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
doi:10.1089/aid.2008.0109
PMCID: PMC2562759  PMID: 18788912
24.  Identification of Ugandan HIV Type 1 Variants with Unique Patterns of Recombination in pol Involving Subtypes A and D 
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.
doi:10.1089/088922202317406655
PMCID: PMC2573392  PMID: 12015904
25.  Coreceptor Tropism in Human Immunodeficiency Virus Type 1 Subtype D: High Prevalence of CXCR4 Tropism and Heterogeneous Composition of Viral Populations▿  
Journal of Virology  2007;81(15):7885-7893.
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.
doi:10.1128/JVI.00218-07
PMCID: PMC1951291  PMID: 17507467

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