Characterization of nevirapine resistance mutations in women with subtype A vs. D HIV-1 6-8 weeks after single-dose nevirapine (HIVNET 012)

OBJECTIVE: To compare the number and type of nevirapine (NVP) resistance mutations detected in Ugandan women with subtype A vs. D HIV-1 infection after single-dose NVP prophylaxis. DESIGN: In the HIVNET 012 trial, a higher rate of NVP resistance (NVPR) was seen in women with subtype D than A after single-dose NVP. In this study, the number and type of NVPR mutations detected 6-8 weeks after NVP were compared in women with subtypes A vs. D. METHODS: Plasma samples were available for 282 (92%) of 306 women who received NVP in HIVNET 012. Samples were analyzed with the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Foster City, CA). Subtyping was performed by phylogenetic analysis of pol region sequences. RESULTS: Results were obtained for 279 women, including 147 with subtype A, 98 with subtype D, 6 with subtype C, and 28 with recombinant HIV-1. NVPR mutations were detected in 70 (25%) of 279 women. NVPR was more common in women with subtype D vs. A (35.7 vs. 19%, P = 0.0035). Complex patterns of NVPR mutations were detected in both subtypes. Among women with NVPR, 43% of women with subtype A and 46% of women with subtype D had >/=2 NVPR mutations. The mean number and pattern of NVPR mutations detected in women with subtypes A and D were similar. CONCLUSIONS: This study confirms a higher rate of NVPR in women with subtype D than A and further defines the pattern of NVPR mutations that emerge 6-8 weeks after single-dose NVP prophylaxis in these subtypes.     

Distinct patterns of emergence and fading of K103N and Y181C in women with subtype A vs. D after single-dose nevirapine: HIVNET 012

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.     

Comparison of mother-to-child transmission rates in Ugandan women with subtype A versus D HIV-1 who received single-dose nevirapine prophylaxis: HIV Network For Prevention Trials 012

OBJECTIVE: To compare the rate of mother-to-child transmission (MTCT) in women with subtype A versus D HIV-1 who received single-dose nevirapine (NVP). METHODS: The MTCT rates were compared in women with subtype A versus D at birth and at 8 weeks and 18 months of age of the infants. The rate of late MTCT (after 8 weeks of age) was also analyzed. RESULTS: HIV-1 subtypes were determined for 300 of 306 women who received NVP in the HIV Network for Prevention Trials 012 study (158 women with subtype A and 105 women with subtype D). Infant infection status was known for 297 women. The cumulative rate of MTCT at 18 months was 13.2% for subtype A and 18.3% for subtype D (P=0.34). The rate of late transmission was 3.8% for subtype A and 7.6% for subtype D (P=0.28). Maternal baseline viral load was a significant predictor of MTCT, but maternal baseline CD4 cell count and subtype were not. CONCLUSIONS: No significant difference was observed in the rate of MTCT in women with subtype A versus D. There was a trend toward a higher rate of MTCT among women with subtype D, however, which was also apparent among women whose infants were infected after 8 weeks of age.     

Quantitative analysis of HIV-1 variants with the K103N resistance mutation after single-dose nevirapine in women with HIV-1 subtypes A, C, and D

INTRODUCTION: 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. METHODS: Plasma for testing was collected 6 to 8 weeks postpartum from 301 women (144 subtype A, 63 subtype C, and 94 subtype D). RESULTS: 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%; P < 0.0001). K103N was rarely detected in pre-NVP samples. In a multivariate model, K103N detection was associated with HIV-1 subtype (C > A), 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 (C > A, P = 0.0001; D > A, P = 0.01; and C vs D, no difference), but not other factors. CONCLUSIONS: 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.     

Safety and trough concentrations of nevirapine prophylaxis given daily, twice weekly, or weekly in breast-feeding infants from birth to 6 months

Despite the success of antiretroviral prophylaxis in reducing mother-to-child HIV-1 transmission, postpartum transmission through breast milk remains a problem. Antiretroviral administration to the infant during the period of breast-feeding could protect against postnatal transmission. An open-label phase 1/2 study was designed to assess the safety and trough concentrations of nevirapine (NVP) given once weekly (OW), twice weekly (TW), or once daily (OD) to HIV-exposed breast-feeding infants for 24 weeks. Following maternal dosing with 200 mg NVP orally at onset of labor, breast-feeding infants were randomized within 48 hours of birth to 1 of 3 regimens: arm 1, NVP given OW (4 mg/kg from birth to 14 days, upward arrow to 8 mg/kg from 15 days to 24 weeks), arm 2, NVP given TW (4 mg/kg from birth to 14 days, upward arrow to 8 mg/kg from 15 days to 24 weeks), and arm 3, NVP given OD (2 mg/kg from birth to 14 days, upward arrow to 4 mg/kg from 15 days to 24 weeks). Trough NVP concentrations and clinical and laboratory abnormalities were monitored. Of the 75 infants randomized (26 to OW, 25 to TW, and 24 to OD dosing), 63 completed the 32-week follow-up visit. No severe skin, hepatic, or renal toxicity related to NVP was observed. Neutropenia occurred in 8 infants. Trough NVP levels were lower than the therapeutic target (100 ng/mL) in 48 of 75 (64.0%) samples from infants in the OW arm, 3 of 65 (4.6%) samples in the TW arm, and 0 of 72 samples in the OD arm. Median (range) trough NVP concentrations were 64 ng/mL (range: <25-1519 ng/mL) with OW dosing; 459 (range: <25-1386 ng/mL) with TW dosing; and 1348 (range: 108-4843 ng/ml) with OD dosing. Our data indicate that NVP prophylaxis for 6 months was safe and well tolerated in infants. OD NVP dosing resulted in all infants with trough concentration greater than the therapeutic target and maintenance of high drug concentrations. A phase 3 study is planned to assess the efficacy of OD infant NVP regimen to prevent breast-feeding HIV-1 transmission.     

In vivo effects of HIV-1 exposure in the presence and absence of single-dose nevirapine on cellular plasma activation markers of infants born to HIV-1-seropositive mothers

Short-course antiretroviral drug regimens reduce the risk of mother-to-child transmission of HIV-1, but mechanisms affording protection of such interventions remain poorly defined. Because T-cell activation is an important factor in productive HIV-1 infection, we tested the hypothesis that single-dose nevirapine (NVP) reduces immune activation, which in turn reduces the likelihood of transmission. We compared concentrations of cord and maternal blood plasma immune activation markers, neopterin, beta2-microglobulin, and soluble l-selectin, in 2 groups of HIV-1-exposed newborns whose mothers either received NVP at the onset of labor or who only received NVP as postexposure prophylaxis within 72 hours of birth and among HIV-unexposed controls. In utero exposure of the infant to HIV-1, regardless of NVP exposure, led to demonstrable increases in immune activation markers, this being most notable in the presence of preexisting infection. Contrary to what was hypothesized, immune activation was increased by prebirth exposure to single-dose NVP, with this effect being enhanced in infants already infected at birth. Our data suggest that reductions in immune activation do not explain transmission prevention effects of single-dose NVP. Our data also suggest a biological explanation for why HIV-1-infected infants exposed perinatally to antiretroviral drugs might experience hastened disease progression, namely, in some HIV-1-infected individuals, NVP may synergize with HIV-1 to enhance an environment that favors increased HIV-1 replication.     

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.     

Analysis of HIV-1 sequences vertically transmitted to infants in Kisumu, Kenya.

BACKGROUND: HIV-1 prevalence in Kenya among women aged between 15-19 years is approximately 23%. These women are prospective mothers and therefore can play an important role in mother-to-child transmission of HIV. The risk of a seropositve mother transmitting the virus to her infant is 25-35% in developing countries, such as Kenya, where antiretroviral drugs are not readily available. OBJECTIVES: This study was undertaken to evaluate the molecular nature of HIV-1 strains, assess recombination and it's relevance in mother-to-child transmission in Kenya. STUDY DESIGN: HIV-1 gag and gp120 sequences were derived from peripheral blood mononuclear cells (PBMC) of 16 infected mothers and infants, from Kisumu, Kenya. PCR, cloning, and phylogenetic analyses were conducted to examine any sequence differences between HIV-1 strains derived from mother-infant pairs. RESULTS: The cohort consisted of seven pairs harboring possible subtype A/D recombinants, eight pairs with apparent pure A or D strains and one possible dual infection. This dual infection comprised of a pure subtype A region and an A/D recombinant, and was detected in one of the mother's sample. Interestingly, only the recombinant virus was detected in the paired baby sample. CONCLUSIONS: This study shows that HIV-1 inter-subtype recombinants can be effectively transmitted vertically to infants, and could possibly be favored in this setting where multiple subtypes infect women. Together, dual infections and the co-existence of multiple HIV-1 subtypes is encouraging the emergence of recombinant HIV strains and their rapid dispersal.     

Independent effects of nevirapine prophylaxis and HIV-1 RNA suppression in breast milk on early perinatal HIV-1 transmission

BACKGROUND: The mechanism of action of single-dose nevirapine on reducing mother-to-child transmission of HIV-1 may involve reduction of maternal HIV-1 or prophylaxis of infants. METHODS: In a study that randomized pregnant mothers to HIVNET 012 nevirapine versus short-course antenatal zidovudine, we compared breast milk HIV-1 RNA viral shedding and administration of single-dose nevirapine between mothers who transmitted HIV-1 to their infants at 6 weeks postpartum and those who did not. RESULTS: In multivariate analyses, maximum breast milk HIV-1 RNA levels (hazard ratio [HR] = 2.50, 95% confidence interval [CI]: 1.25 to 4.99; P = 0.01) and nevirapine use (HR = 0.12, 95% CI: 0.02 to 0.97; P = 0.05) were each independently associated with perinatal transmission at 6 weeks postpartum. Mothers who transmitted HIV-1 to their infants had significantly higher HIV-1 RNA levels in their breast milk between the second day and sixth week postpartum. Among mothers with maximum breast milk virus levels less than a median of 3.5 log(10) copies/mL, the administration of nevirapine further decreased HIV-1 transmission risk from 22.2% to 0.0% (P = 0.04). CONCLUSIONS: Peripartum administration of single-dose nevirapine to mother and infant decreases early perinatal HIV-1 transmission by means of breast milk HIV-1 RNA suppression and, independently, by providing the infant with exposure prophylaxis.     

Long-term mitochondrial toxicity in HIV-uninfected infants born to HIV-infected mothers

Although children born to HIV-infected (HIV+) women receiving antiretroviral therapy during pregnancy show virtually no adverse clinical effects at birth, the antiretroviral nucleoside analog drugs are known to damage nuclear and mitochondrial DNA. In this study, biomarkers of mitochondrial toxicity and genotoxicity have been examined in a well-characterized sample set consisting of infants born to HIV-uninfected (HIV-) mothers (n = 30), and HIV- infants (n = 20) born to HIV-infected (HIV+) mothers who received either no antiretroviral therapy (n = 10) or zidovudine (3'-azido-3'-deoxythymidine [AZT]) during pregnancy (n = 10). DNA from cord blood leukocytes and peripheral blood leukocytes taken at 1 and 2 years of age was examined for loss of mitochondrial DNA (mtDNA) and telomere integrity. Telomere length, a measure of nuclear DNA damage, was the same in all infants at birth and at age 1 year. The quantity of mtDNA was assessed relative to nuclear DNA using a polymerase chain reaction-based chemiluminescence detection (PCR-CID) method that determined mitochondrial D Loop gene copies relative to nuclear 18S RNA gene copies by comparison with a standard curve. MtDNA quantity was expressed as a ratio of gene copy numbers. In infants of uninfected mothers (AZT-/HIV-) at the three time points, the ratios were 442 to 515, whereas in infants of untreated AZT-/HIV+ mothers the ratios were 261 to 297, and in infants of AZT-treated (AZT+/HIV+) mothers the ratios were 146 to 203. At all three time points, differences between the AZT-/HIV- group and the two HIV+ groups were statistically significant (p <.05), and differences between the AZT-/HIV+ and AZT+/HIV+ groups were also statistically significant (p <.05), demonstrating that AZT exposure causes a persistent depletion of mtDNA. The study shows that children of HIV+ mothers are at risk for mitochondrial damage that is further increased in infants of mothers receiving AZT during pregnancy.     

Sensitive oligonucleotide ligation assay for low-level detection of nevirapine resistance mutations in human immunodeficiency virus type 1 quasispecies

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.     

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 subtype B DNA in dried blood samples for diagnosis in infants in the field

The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 microl of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45 degrees C for 20 h. The primers used for nested PCR of the HIV-1 pol gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.     

艾滋病临床用药分析

目的 探讨艾滋病临床用药方案与CD4细胞的关系,以期为艾滋病后续治疗起到积极的理论支持。方法 选取某临床医院2012年12月~2017年10月统计的732例艾滋病患者基线和随访数据,分析所有患者基线及随访治疗方案,将CD4个数作为评判标准,主要研究基线治疗以及随访治疗后患者CD4细胞个数的变化情况。结果 临床数据中患者的所有治疗方案中,有五组治疗方案:3TC+AZT+EFV、3TC+AZT+NVP、3TC+D4T+EFV、3TC+D4T+NVP和3TC+TDF+EFV占总方案的93.03%,且其中我国抗HIV治疗方案主要应用3TC+AZT+EFV和3TC+D4T+EFV,全部数据中未换药组占比79.97%;在治疗过程中,CD4提高的患者占64.30%。结论 艾滋病临床治疗中,治疗方案3TC+AZT+EFV、3TC+AZT+NVP、3TC+D4T+EFV、3TC+D4T+NVP和3TC+TDF+EFV被广泛应用,我国临床治疗以方案3TC+AZT+EFV和3TC+D4T+EFV为主,且各种治疗方案对于HIV病情有相对较好的稳定作用。     

Prevention of mother-to-child transmission of HIV-1 through breast-feeding by treating infants prophylactically with lamivudine in Dar es Salaam, Tanzania: the Mitra Study

OBJECTIVE: To investigate the possibility of reducing mother-to-child transmission (MTCT) of HIV-1 through breast-feeding by prophylactic antiretroviral (ARV) treatment of the infant during the breast-feeding period. DESIGN: An open-label, nonrandomized, prospective cohort study in Tanzania (Mitra). METHODS: HIV-1-infected pregnant women were treated according to regimen A of the Petra trial with zidovudine (ZDV) and lamivudine (3TC) from week 36 to 1 week postpartum. Infants were treated with ZDV and 3TC from birth to 1 week of age (Petra arm A) and then with 3TC alone during breast-feeding (maximum of 6 months). Counseling emphasized exclusive breast-feeding. HIV transmission was analyzed using the Kaplan-Meier survival technique. Cox regression was used for comparison with the breast-feeding population in arm A of the Petra trial, taking CD4 cell count and other possible confounders into consideration. RESULTS: There were 398 infants included in the transmission analysis in the Mitra study. The estimated cumulative proportion of HIV-1-infected infants was 3.8% (95% confidence interval [CI]: 2.0 to 5.6) at week 6 after delivery and 4.9% (95% CI: 2.7 to 7.1) at month 6. The median time of breast-feeding was 18 weeks. High viral load and a low CD4 T-cell count at enrollment were associated with transmission. The Kaplan-Meier estimated risk of HIV-1 infection at 6 months in infants who were HIV-negative at 6 weeks was 1.2% (95% CI: 0.0 to 2.4). The cumulative HIV-1 infection or death rate at 6 months was 8.5% (95% CI: 5.7 to 11.4). No serious adverse events related to the ARV treatment of infants occurred. The HIV-1 transmission rate during breast-feeding in the Mitra study up to 6 months after delivery was more than 50% lower than in the breast-feeding population of Petra arm A (relative hazard=2.61; P=0.001; adjusted values). The difference in transmission up to 6 months was significant also in the subpopulation of mothers with CD4 counts>or=200 cells/microL. CONCLUSIONS: The rates of MTCT of HIV-1 in the Mitra study at 6 weeks and 6 months after delivery are among the lowest reported in a breast-feeding population in sub-Saharan Africa. Prophylactic 3TC treatment of infants to prevent MTCT of HIV during breast-feeding was well tolerated by the infants and could be a useful strategy to prevent breast milk transmission of HIV when mothers do not need ARV treatment for their own health.     

Relation between chemokine receptor use, disease stage, and HIV-1 subtypes A and D: results from a rural Ugandan cohort

OBJECTIVES: To determine whether there are differences in coreceptor use in subjects infected with HIV-1 envelope subtypes A and D that could explain the differences in progression rates between these subtypes in a rural Ugandan cohort. METHODS: HIV-1 was subtyped in env by V3 sequencing or heteroduplex mobility assay. Coreceptor use was determined by the ability of the isolates to replicate in U87 CD4 cells expressing different coreceptors. The Fisher exact test was used to examine the relation between coreceptor use and subtype, clinical stage, and V3 charge. The Kruskall-Wallis nonparametric test was used to examine the association between median CD4 cell counts, coreceptor use, and subtype. Logistic regression was used to examine predicted coreceptor use at different CD4 groupings. RESULTS: Isolates from 66 participants were analyzed. Thirty-one were infected with subtype A, and 35 were infected with subtype D. Although this work was based on a small sample size, we found statistically significant differences. The probability of having an X4 virus was higher in subtype D infections than in subtype A infections among those with a non-AIDS clinical status (Fisher exact test, P = 0.040). Logistic regression analysis, in which we predicted X4 use by subtype and stratified by CD4 group, confirmed these findings among those with a CD4 count >200 cells/microL (likelihood ratio test, P = 0.003). R5 viruses were associated with higher median CD4 cell counts than X4 or X4/R5 (Kruskall-Wallis test, P = 0.0045). A V3 charge of +5 and greater was highly associated with X4 virus (Fisher exact test, P = 0.006). CONCLUSIONS: These subtype differences in coreceptor use may partially explain the faster progression rates we have previously reported in individuals infected with subtype D compared with subtype A. Our observations may have implications for the future use of coreceptor inhibitors in this population.