Analysis of HIV tropism in Ugandan infants.

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).

Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay.

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.

Analysis of drug resistance in children receiving antiretroviral therapy for treatment of HIV-1 infection in Uganda.

We analyzed drug resistance in HIV-infected Ugandan children who received antiretroviral therapy in a prospective, observational study (2004-2006); some children had prior single-dose nevirapine (sdNVP) exposure. Children received stavudine (d4T), lamivudine (3TC), and nevirapine (NVP); treatment was continued if they were clinically and immunologically stable. Samples with >1,000 copies/ml HIV RNA were analyzed by using the ViroSeq HIV Genotyping System (ViroSeq). Subtype A and D pretreatment samples also were analyzed with the LigAmp assay (for K103N, Y181C, and G190A). ViroSeq results were obtained for 74 pretreatment samples (35 from sdNVP-exposed children (median age, 19 months) and 39 from sdNVP-unexposed children (median age, 84 months). This included 39 subtype A, 22 subtype D, 1 subtype C, and 12 inter-subtype recombinant samples. One sample had nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance, one had nucleoside reverse transcriptase inhibitor (NRTI) resistance, and three had protease inhibitor (PI) resistance. Y181C was detected by using LigAmp in five pretreatment samples [four (14.8%) of 37 samples from sdNVP-exposed children, one (4.2%) of 24 samples from children without prior sdNVP exposure; p = 0.35]. Among children who were not virally suppressed at 48 weeks of treatment, all 12 tested had NNRTI resistance, as well as resistance to 3TC and emtricitibine (FTC); three had resistance to other NRTIs. Seven of those children had a ViroSeq result at 96 weeks of treatment; four of the seven acquired resistance to additional NRTIs by 96 weeks. In Uganda, clinically and immunologically stable children receiving nonsuppressive antiretroviral treatment regimens are at risk for development of drug resistance.

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.

Analysis of HIV type 1 gp41 and enfuvirtide susceptibility among men in the United States who were HIV infected prior to availability of HIV entry inhibitors.

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.

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.

Analysis of nevirapine resistance mutations in cloned HIV type 1 variants from HIV-infected Ugandan infants using a single-step amplification-sequencing method (AmpliSeq).

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.

Analysis of nevirapine (NVP) resistance in Ugandan infants who were HIV infected despite receiving single-Dose (SD) NVP versus SD NVP plus daily NVP up to 6 weeks of age to prevent HIV vertical transmission.

BACKGROUND: Single-dose nevirapine (SD NVP) at birth plus NVP prophylaxis for the infant up to 6 weeks of age is superior to SD NVP alone for prevention of vertical transmission of human immunodeficiency virus (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: When infants were 6 weeks old, ViroSeq detected NVP resistance in a higher proportion of infants in the 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 prophylaxis was also associated with detection of NVP resistance by ViroSeq at 6 months (7 of 7 [100%] infants in the extended NVP arm had resistance detected, compared with 1 of 6 [16.7%] infants in the SD NVP arm; P = .005). CONCLUSIONS: The use of extended NVP prophylaxis was associated with increased selection for and persistence of NVP resistance in HIV-infected Ugandan infants.

Nevirapine resistance in women and infants after first versus repeated use of single-dose nevirapine for prevention of HIV-1 vertical transmission.

Single-dose (SD) nevirapine (NVP) significantly reduces mother-to-child transmission of human immunodeficiency virus (HIV). We analyzed NVP resistance after receipt of SD NVP in 57 previously SD NVP-naive women, in 34 SD NVP-experienced women, and in 17 HIV-infected infants. The proportion of women infected with variants with resistance mutations, the types of mutations detected, and the frequency and level of K103N were similar in the two groups of women at 6 weeks and 6 months post partum. NVP resistance was detected in a similar proportion of infants born to SD NVP-naive versus SD NVP-experienced women. Repeated use of SD NVP to prevent HIV transmission does not appear to influence NVP resistance.

HIV-1 tropism and survival in vertically infected Ugandan infants.

BACKGROUND: 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. METHODS: Plasma or serum was analyzed using a commercial coreceptor tropism assay. HIV env subtype was determined by phylogenetic methods. RESULTS: 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. CONCLUSIONS: 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.