Comparison of nevirapine (NVP) resistance in Ugandan women 7 days vs. 6-8 weeks after single-dose nvp prophylaxis: HIVNET 012.

We compared nevirapine (NVP) resistance (NVPR) mutations in maternal plasma 7 days vs. 6-8 weeks after single-dose NVP prophylaxis. In the HIVNET 012 trial, Ugandan women received a single dose of NVP in labor for prevention of HIV-1 mother-to-child transmission. NVPR mutations were detected in 70 (25%) of 279 women 6-8 weeks after NVP. Samples collected 7 days after NVP were analyzed from a subset of those 279 women. Genotyping was performed with the ViroSeq HIV-1 Genotyping System. NVPR was analyzed using paired samples from 7 days and 6-8 weeks after NVP. Sixty-five women had genotyping results obtained for samples collected at both 7 days and 6-8 weeks post-NVP. Twenty-one (32%) of those women had NVPR mutations detected in one or both samples. This included three women with NVPR at 7 days only, seven with NVPR at 6-8 weeks only, and 11 with NVPR at both time points. Eight women had >1 NVPR mutation detected 7 days after NVP. Y181C was the most common NVPR mutation detected at 7 days, whereas K103N was the most common NVPR mutation detected at 6-8 weeks. We conclude that NVPR may be detected in women as early as 7 days after single-dose NVP. Complex patterns of NVPR are detected in some women. The Y181C NVPR mutation often fades from detection by 6-8 weeks. In contrast, the K103N mutation emerges more slowly, but often remains detectable 6-8 weeks after NVP.

Performance of Applied Biosystems ViroSeq HIV-1 Genotyping System for sequence-based analysis of non-subtype B human immunodeficiency virus type 1 from Uganda.

The Applied Biosystems ViroSeq HIV-1 Genotyping System is a commercially available, integrated system for sequence-based analysis of drug resistance mutations in human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for analysis of non-subtype B HIV-1 by analyzing plasma samples from Ugandan women and infants. Plasma samples were obtained from 105 women and 25 infants enrolled in a Ugandan clinical trial. HIV-1 analysis was performed with the ViroSeq system according to the manufacturer's instructions, except that the volume of plasma used for analysis was less than the recommended 0.5 ml for some samples. Viral loads ranged from 2,313 to 2,336,400 copies/ml. PCR products suitable for sequencing were amplified from all samples tested. Complete sequences for protease (amino acids 1 to 99) and RT (amino acids 1 to 320) were obtained for 102 of 105 (97%) of the maternal samples tested and all 25 of the infant samples tested. Complete double-stranded sequences were obtained for 90 of 105 (86%) of the maternal samples tested and 22 of 25 (88%) of the infant samples tested. The sequences obtained with this system were used for HIV-1 subtyping. The subtypes identified were A, C, D, and A/D recombinant HIV-1. The performances of the seven sequencing primers were similar for the subtypes examined. The ViroSeq system performs well for analysis of Ugandan plasma samples with subtypes A, C, D, and A/D recombinant HIV-1. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in countries where these subtypes are prevalent.

Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012).

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.

Impact of human immunodeficiency virus type 1 (hiv-1) subtype on women receiving single-dose nevirapine prophylaxis to prevent hiv-1 vertical transmission (hiv network for prevention trials 012 study).

In Uganda, the HIV Network for Prevention Trials (HIVNET) 012 study recently demonstrated that single-dose nevirapine (Nvp) prophylaxis is effective for preventing mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1). This exploratory study examines the relationship between HIV-1 subtype, MTCT, and the development of Nvp resistance (Nvp(R)) in women enrolled in HIVNET 012. For 102 women (32 whose infants were HIV-1 infected by age 6-8 weeks and 70 whose infants were uninfected), HIV-1 subtypes included 50 (49%) subtype A, 35 (34%) subtype D, 4 (4%) subtype C, 12 (12%) recombinant subtype, and 1 unclassified. There was no apparent difference in the rate of MTCT among women with subtype A versus D (adjusted odds ratio [OR], 1.24; 95% confidence interval [CI], 0.45-3.43). Nvp(R) mutations were detected more frequently at 6-8 weeks postpartum in women with subtype D than in women with subtype A (adjusted OR, 4.94; 95% CI, 1.21-20.22). Additional studies are needed to further define the relationship between HIV-1 subtype and Nvp(R) among women receiving Nvp prophylaxis.

From research to implementation: challenges in the prevention of mother to child HIV transmission in the developing world.

Scientific research has identified anti-retroviral regimens effective in the prevention of mother to child HIV transmission. Yet, 1800 HIV-infected infants are born every day. The majority of HIV-infected women live in developing countries where challenges to widespread implementation of these regimens have prevented decreases in pediatric HIV infection from being realized.

HIV-1 ICD p24 antigen detection in ugandan infants: use in early diagnosis of infection and as a marker of disease progression.

The objective of this study was to determine the use of immune-complex dissociated (ICD) p24 antigen detection for the diagnosis and prognosis of HIV-1 infection in Ugandan children. Plasma collected prospectively from children born to HIV-1 infected Ugandan women was stored and later analyzed for the presence of neutralizable HIV-1 p24 antigen using the Coulter ICD p24 antigen and neutralization kits. HIV-1 infection status, disease progression, and survival of the children were determined. Specimens from 311 children born to HIV-1 infected women, including 138 HIV-1 infected children, and 113 children born to negative women were tested. Sixty-nine (50%) infected children were p24 antigen positive at least once. For early HIV-1 diagnosis, the specificity and positive predictive value of the assay were consistently high (>95% and >83% respectively), but the sensitivity was low (6-53%), especially in the first months of life. The presence of p24 antigenemia in the first two years of life was associated with poor survival (20%) by 80 months of age compared with infected children without antigenemia (43%, P < 0.001). Early detection of p24 antigen (6 months, P < 0.001). The data suggest that ICD p24 antigen detection is not a sensitive method for the determination of infant HIV-1 status in our cohort of HIV-1 infected Ugandan children tested in the first two years of life. There was a strong correlation, however, between the presence and time of onset of p24 antigenemia and mortality among HIV-1 infected children.

Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission.

OBJECTIVE: A recent trial in Uganda demonstrated that a simple, inexpensive regimen of nevirapine (NVP) prophylaxis can dramatically reduce HIV-1 vertical transmission risk. In this regimen, women receive a single dose of NVP at the onset of labor and infants receive a single dose of NVP within 72 h of birth. The objective of this study was to determine whether HIV-1 variants with NVP resistance mutations were selected in Ugandan women who received this regimen in the Phase I/II trial HIVNET 006. METHODS: Reverse transcriptase (RT) sequences from plasma HIV-1 were analyzed from 15 women 6 weeks after NVP dosing. RT sequences from plasma collected prior to NVP dosing were also analyzed. RESULTS: The K103N NVP resistance mutation was detected 6 weeks after NVP administration in three (20%) out of 15 women (95% confidence interval, 0-40%). Pre-dose samples were available from two of the three women; both pre-dose samples lacked the mutation. Other NVP resistance mutations were absent from all 15 women. Women with the K103N mutation had a longer median NVP elimination half-life, decreased median oral clearance, and increased median area under the concentration time curve than those without the mutation. An evaluable sample was obtained from one of these three women 33 months after delivery; the K103N mutation was not detected in that sample. CONCLUSIONS: This preliminary study demonstrates that HIV-1 with the RT K103N mutation can be detected in some Ugandan women following a single dose of NVP. This suggests that non-nucleoside RT inhibitor resistance may be selected in some people by single dose NVP prophylaxis. Pharmacokinetic data suggested that a more prolonged exposure to NVP after dosing may favor selection of NVP-resistant HIV-1.

Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial.

BACKGROUND: The AIDS Clinical Trials Group protocol 076 zidovudine prophylaxis regimen for HIV-1-infected pregnant women and their babies has been associated with a significant decrease in vertical HIV-1 transmission in non-breastfeeding women in developed countries. We compared the safety and efficacy of short-course nevirapine or zidovudine during labour and the first week of life. METHODS: From November, 1997, to April, 1999, we enrolled 626 HIV-1-infected pregnant women at Mulago Hospital in Kampala, Uganda. We randomly assigned mothers nevirapine 200 mg orally at onset of labour and 2 mg/kg to babies within 72 h of birth, or zidovudine 600 mg orally to the mother at onset of labour and 300 mg every 3 h until delivery, and 4 mg/kg orally twice daily to babies for 7 days after birth. We tested babies for HIV-1 infection at birth, 6-8 weeks, and 14-16 weeks by HIV-1 RNA PCR. We assessed HIV-1 transmission and HIV-1-free survival with Kaplan-Meier analysis. FINDINGS: Nearly all babies (98.8%) were breastfed, and 95.6% were still breastfeeding at age 14-16 weeks. The estimated risks of HIV-1 transmission in the zidovudine and nevirapine groups were: 10.4% and 8.2% at birth (p=0.354); 21.3% and 11.9% by age 6-8 weeks (p=0.0027); and 25.1% and 13.1% by age 14-16 weeks (p=0.0006). The efficacy of nevirapine compared with zidovudine was 47% (95% CI 20-64) up to age 14-16 weeks. The two regimens were well tolerated and adverse events were similar in the two groups. INTERPRETATION: Nevirapine lowered the risk of HIV-1 transmission during the first 14-16 weeks of life by nearly 50% in a breastfeeding population. This simple and inexpensive regimen could decrease mother-to-child HIV-1 transmission in less-developed countries.

PIP: A study was conducted to assess the safety and efficacy of short-course nevirapine compared with zidovudine given to women during labor and to neonates during the first week of life. 626 HIV-1 infected pregnant women attending the antenatal clinic from November 1997 to April 1999 at Mulago Hospital in Kampala, Uganda, were randomly given nevirapine or zidovudine. Infants were tested for HIV-1 infection at birth, at 6-8 weeks, and at 14-16 weeks. Findings revealed that the estimated risk of HIV-1 transmission in the zidovudine and nevirapine groups was 10.4% and 8.2%, respectively, at birth; 21.3% and 11.9%, by 6-8 weeks; and 25.1% and 13.1%, by 14-16 weeks. There was a 47% relative efficacy rate of the nevirapine regimen at 14-16 weeks compared to zidovudine. Based on the findings, nevirapine lowers the risk of HIV-1 transmission by nearly 50% during the first 14-16 weeks of life in breast-fed infants.

A phase I/II study of the safety and pharmacokinetics of nevirapine in HIV-1-infected pregnant Ugandan women and their neonates (HIVNET 006).

OBJECTIVE: To determine the safety, pharmacokinetics, tolerance, antiretroviral activity, and infant HIV infection status after giving a single dose of nevirapine to HIV-1-infected pregnant women during labor and their newborns during the first week of life. DESIGN: An open label phase I/II study. SETTING: Tertiary care hospital, Kampala, Uganda. PATIENTS AND INTERVENTIONS: Nevirapine, 200 mg, was given as a single dose during labor to 21 HIV-1-infected pregnant Ugandan women. In cohort 1, eight infants did not receive nevirapine whereas in cohort 2, 13 infants received a single dose of nevirapine, 2 mg/kg, at 72 h of age. OUTCOMES: The number and type of adverse events; nevirapine concentrations in the plasma and breast milk; maternal plasma HIV-1 RNA copy number before and up to 6 weeks after delivery; and HIV-1 infection status of the infants were monitored. RESULTS: Nevirapine was well tolerated by women and infants; no serious adverse events that were related to nevirapine were observed. Median nevirapine concentration in the women at delivery was 1623 ng/ml (range 238-2356 ng/ml); median cord/maternal blood ratio of 0.75 (0.37-0.93). The median half-life in women was 61.3 h (27-90 h) and the transplacental nevirapine half-life in infants who did not receive a neonatal dose was 54 h. The median half-life after a single dose at 72 h in infants was 46.5 h. During the first week of life, the median colostrum/breast milk to maternal plasma nevirapine concentration was 60.5% (25-122%). The median nevirapine concentration in breast milk 1 week after delivery was 103 ng/ml (25-309 ng/ml). Plasma nevirapine concentrations were above 100 ng/ml in all infants from both cohorts tested at age 7 days. Maternal HIV-1 RNA levels decreased by a median of 1.3 logs at 1 week postpartum, and returned to baseline by 6 weeks postpartum. Detectable plasma HIV-1 RNA was observed in one out of 22 (4.5%) infants at birth; three out of 21 (14%) at 6 weeks; and four out of 21 (19%) at 6 months of age. CONCLUSION: The administration of a single dose of nevirapine to women during labor and to their newborns at 72 h was well tolerated and showed potent antiretroviral activity in the women at 1 week after dosing without rebound above baseline 6 weeks after a single dose. The nevirapine concentration was maintained above the target of 100 ng/ml in infants at age 7 days, even in those infants not receiving a neonatal dose. This regimen has promise as prophylaxis against intrapartum and early breast milk transmission in a breastfeeding population.

Mediation of entry of human immunodeficiency virus-1 into alveolar macrophages by CD4 without facilitation by surfactant-associated protein-A.

The mechanism of uptake of human immunodeficiency virus-1 (HIV-1) into alveolar macrophages (AM), freshly isolated blood monocytes (MN), and cultured MN (CM) was investigated focusing on the role of CD4 and of surfactant-associated protein A (SP-A). By radioimmunoassay which obviated the problems of auto- and nonspecific fluorescence of more differentiated macrophages, each of the macrophage populations studied expressed CD4. Semiquantitative polymerase chain reaction was performed to assess uptake of HIV-1(JR-FL) into cells. OKT4a (directed against CD4) blocked uptake of HIV-1 into CM, AM, and MN by 67 to 100%. OKT4 (directed against another epitope of CD4) had a smaller and less consistent effect (0-90%), and control antibodies showed minimal effects and only at supersaturating concentrations. SP-A had no effect on uptake of HIV-1 into AM. SP-A also had no consistent effect on production of HIV-1(JR-FL) by AM infected in vitro (p24 antigen ELISA). Thus CD4 is the major receptor for HIV-1 in mononuclear phagocytes, including AM, and SP-A does not modulate entry.

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and p24 antigen in breast milk of HIV-1-infected Ugandan women and vertical transmission.

OBJECTIVE: To determine the correlation between the detection of human immunodeficiency virus type 1 (HIV-1) in breast milk, the duration of breastfeeding, and vertical transmission of HIV-1 infection in Ugandan women. METHODS: A prospective study of HIV-1 infection in pregnant Ugandan women and their infants has been ongoing since 1990 with follow-up of mother-infant pairs for at least 2 years. Expressed breast milk specimens were collected from 201 HIV-1-seropositive and 86 HIV-1-seronegative Ugandan women approximately 6 weeks after delivery. The presence of HIV-1 DNA in the cellular fraction of the breast milk was detected by polymerase chain reaction (PCR), and HIV-1 p24 antigen was detected in the cell-free breast milk supernatant using p24 antigen enzyme immunoassay (EIA) after immune complex dissociation (ICD). The duration of breastfeeding and the clinical status of the mothers and their children were recorded. HIV-1 EIA, Western blot, PCR, or p24 antigen detection were used for the determination of the HIV-1 infection status of the children. RESULTS: Of the 201 HIV-1-infected women studied, 47 had HIV-1-infected children, 143 had children who seroreverted, and 11 had children of indeterminate status. Breast milk supernatants were available for ICD p24 antigen testing from 188 of the HIV-1-infected women and none had detectable p24 antigen. Breast milk cell pellets were available and contained amplifiable DNA in 125 of the HIV-1-infected women (20 transmitters, 104 nontransmitters, 1 indeterminate). HIV-1 DNA was detected by PCR in 72% (75/104) of nontransmitters and 80% (16/20) of the transmitters. The duration of breastfeeding by transmitter mothers (15.8 months) was not significantly different from nontransmitter mothers (14.4 months). CONCLUSIONS: No correlation was found between the detection of HIV-1 in breast milk or the duration of breastfeeding and transmission of HIV-1 infection in this study of Ugandan women.

Enhanced production of human immunodeficiency virus type 1 by in vitro-infected alveolar macrophages from otherwise healthy cigarette smokers.

Since cellular activation is required for replication of human immunodeficiency virus type 1 (HIV-1), the capacity of alveolar macrophages (AM) from smokers, which are relatively activated, and nonsmokers to support the production of HIV-1JR-FL was examined. Peak HIV-1 p24 antigen level in culture supernatants of infected AM from 13 smokers was significantly higher than that of 13 nonsmokers: 31,394 +/- 8295 versus 7037 +/- 2550 pg/mL (mean +/- SE; P < .002). This difference could not be explained on the basis of viral entry, extent of reverse transcription, or release of monokines, including tumor necrosis factor-alpha, interleukin-1 beta or -6, and granulocyte-macrophage colony-stimulating factor. HIV-1 production by blood monocytes from smokers and nonsmokers infected in vitro was negligible. Thus, cigarette smoking selectively increases the susceptibility of AM to productive infection with HIV-1. This finding provides a biologic plausibility to observations that smoking may enhance the progression of AIDS.