Neutralization of HIV subtypes A and D by breast milk IgG from women with HIV infection in Uganda.

OBJECTIVES: Among HIV-exposed infants in resource-limited countries, 8-12% are infected postnatally by breastfeeding. However, most of those uninfected at birth remain uninfected over time despite daily exposure to HIV in breast milk. Thus, we assessed the HIV-inhibitory activity of breast milk. METHODS: We measured cross-clade neutralization in activated PBMC of Ugandan subtype A (92UG031) and D (92UG005) primary HIV by breast milk or purified milk IgG and IgA from 25 HIV-infected Ugandan women. Isotype-specific antigen recognition was resolved by immunoblot. We determined HIV subtype from envelope population sequences in cells from 13 milk samples by PCR. RESULTS: Milk inhibited p24 production by ≥50% (dose-dependent) by subtype A (21/25; 84%) and subtype D (11/25; 44%). IgG consistently reacted with multiple HIV antigens, including gp120/gp41, but IgA primarily recognized p24 alone. Depletion of IgG (n = 5), not IgA, diminished neutralization (mean 78 ± 33%) that was largely restored by IgG repletion. Mothers infected with subtype A more effectively neutralized subtype A than D. CONCLUSIONS: Breast milk from HIV-infected women showed homotypic and cross-subtype neutralization of HIV by IgG-dependent and -independent mechanisms. These data direct further investigations into mechanisms of resistance against postnatal transmission of HIV to infants from their mothers.

Long-term follow-up of children in the HIVNET 012 perinatal HIV prevention trial: five-year growth and survival.

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

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).

Human papillomavirus prevalence and cytopathology correlation in young Ugandan women using a low-cost liquid-based Pap preparation.

Screening for HPV-driven cervical dysplasia and neoplasia is a significant public health concern in the developing world. The purpose of this study was to use a manual, low-cost liquid-based Pap preparation to determine HPV prevalence in HIV-positive and HIV-negative young women in Kampala, Uganda and to correlate cervical cytopathology with HPV-DNA genotype. About 196 post-partum women aged 18-30 years underwent rapid HIV testing and pelvic examination. Liquid-based cervical cytology samples were processed using a low-cost manual technique. A DNA collection device was used to collect specimens for HPV genotyping. HIV and HPV prevalence was 18 and 64%, respectively. Overall, 49% of women were infected with a high-risk HPV genotype. The most common high-risk HPV genotypes were 16 (8.2%), 33 (7.7%), 35 (6.6%), 45 (5.1%), and 58 (5.1%). The prevalence of HPV 18 was 3.6%. HIV-positive women had an HPV prevalence of 86% compared to 59% in HIV-negative women (P = 0.003). The prevalence of HPV 16/18 did not differ by HIV status. HIV-positive women were infected with a significantly greater number of HPV genotypes compared to HIV-negative women. By multivariate analysis, the main risk factor for HPV infection was coinfection with HIV. HIV-positive women were four times more likely to have abnormal cytology than HIV-negative women (43% vs. 11.6%, P < 0.001). These data highlight that HIV infection is a strong risk factor for HPV infection and resultant abnormal cervical cytology. Notably, the manual low-cost liquid-based Pap preparation is practical in this setting and offers an alternate method for local studies of HPV vaccine efficacy.

Early weaning of HIV-exposed uninfected infants and risk of serious gastroenteritis: Findings from two perinatal HIV prevention trials in Kampala, Uganda.

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

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.

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 < or =56 days). Incidence of MTCT after 56 days was low [0.7% per month (95% confidence interval, CI: 0.4 to 1.0)], but continued through 18 months. In multivariate analyses, early MTCT "factors" included NVP versus zidovudine (hazard ratio (HR) = 0.57, 95% CI: 0.38 to 0.86), pre-entry maternal viral load (VL, HR = 1.76, 95% CI: 1.28 to 2.41), and CD4 cell count (HR = 1.16, 95% CI: 1.05 to 1.28). Maternal VL (6-8 weeks) was associated with late MTCT (HR = 3.66, 95% CI: 1.78 to 7.50), whereas maternal NVP resistance (6-8 weeks) was not. CONCLUSIONS: Maternal VL was the best predictor of both early and late transmission. Maternal NVP resistance at 6-8 weeks did not predict late transmission.

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.

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.

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.

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 and 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 HIV Network for Prevention Trials 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 per microliter (CD4 cell %) was 4150 (41%) at birth, 4900 (24%) at 12 months, 4300 (19%) at 24 months, 4150 (19%) at 36 months, 4100 (18%) at 48 months, and 3800 (20%) at 60 months. The highest risk of mortality within 12 months was 34% - 37% at birth and declined to 13%-15% at 24 months regardless of TLC measurement. The correlation between CD4 cell % and TLC was extremely low overall (r = 0.01). CONCLUSIONS: The TLC did not predict a risk of progression to death within 12 months in HIV-infected Ugandan children. Therefore, TLC alone may not be a useful surrogate marker for determining those children at highest risk of death, who require antiretroviral therapy most urgently.

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.

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.

Detection of K103N in Ugandan women after repeated exposure to single dose nevirapine.

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

The costs associated with adverse event procedures for an international HIV clinical trial determined by activity-based costing.

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

HIV type 1 variants with nevirapine resistance mutations are rarely detected in antiretroviral drug-naive African women with subtypes A, C, and D.

K103N is frequently detected in HIV-infected women after single dose (SD) nevirapine (NVP). K103N-containing variants were detected more frequently by the ViroSeq HIV-1 Genotyping System in women with subtype C (69.2%) than subtypes A (19.4%, p < 0.0001) or D (36.1%, p < 0.0001). K103N-containing variants were also detected more frequently and at higher levels in women with subtype C by the LigAmp assay. In this report, we analyzed samples collected prior to or within hours after SD NVP administration from antiretroviral drug-naive African women with subtypes A, C, and D. Only 1/254 samples had an NVP resistance mutation detected with the ViroSeq system, and only 4/236 samples had K103N detected at < 0.5% with the LigAmp assay [2/110 (1.8%) with subtype A, 1/46 (2.2%) with subtype C, and 1/80 (1.3%) with subtype D] (p = 0.92). We did not detect significant differences in the pre-NVP frequency of NVP resistance mutations or the pre-NVP levels of K103N-containing variants in women with subtypes A, C, and D that explain the dramatic subtype-based differences in emergence of HIV-1 variants with these mutations after SD NVP exposure.

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 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.

Persistence of K103N-containing HIV-1 variants after single-dose nevirapine for prevention of HIV-1 mother-to-child transmission.

K103N-containing human immunodeficiency virus (HIV)-1 variants are selected in some women who receive single-dose (SD) nevirapine (NVP) for prevention of HIV-1 mother-infant transmission. We examined the persistence of K103N in women who received SD NVP prophylaxis. K103N was detected using the LigAmp assay (assay cutoff, 0.5% K103N). K103N was detected at 6-8 weeks in 60 (41.7%) of 144 women. Fading (lack of detection) of K103N was documented in 16 women by 2 years, 43 women by 3 years, and 55 women by 4 and 5 years. Slower fading was independently associated with HIV-1 subtype (D>A) and higher pre-NVP viral load.

Sensitivity of the ViroSeq HIV-1 genotyping system for detection of the K103N resistance mutation in HIV-1 subtypes A, C, and D.

The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping methods detect antiretroviral drug resistance mutations present in the major viral population of a test sample. These assays also detect some mutations in viral variants that are present as mixtures. We compared detection of the K103N nevirapine resistance mutation using ViroSeq and a sensitive, quantitative point mutation assay, LigAmp. The LigAmp assay measured the percentage of K103N-containing variants in the viral population (percentage of K103N). We analyzed 305 samples with HIV-1 subtypes A, C, and D collected from African women after nevirapine administration. ViroSeq detected K103N in 100% of samples with >20% K103N, 77.8% of samples with 10 to 20% K103N, 71.4% of samples with 5 to 10% K103N, and 16.9% of samples with 1 to 5% K103N. The sensitivity of ViroSeq for detection of K103N was similar for subtypes A, C, and D. These data indicate that the ViroSeq system reliably detects the K103N mutation at levels above 20% and frequently detects the mutation at lower levels. Further studies are needed to compare the sensitivity of different assays for detection of HIV-1 drug resistance mutations and to determine the clinical relevance of HIV-1 minority variants.