HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

OBJECTIVES: The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. DESIGN: Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. METHODS: DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. RESULTS: HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. CONCLUSION: HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

Longitudinal analysis of the humoral response to Kaposi's sarcoma-associated herpesvirus after primary infection in children.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS)-one of the most common pediatric cancers in sub-Saharan Africa-however, the factors that lead to disease progression are not fully understood. HIV infection, immunosuppression, and high KSHV viral load increase the risk of developing KS, suggesting that the loss of an effective anti-KSHV immune response may be an important risk factor. However, very little is known about the KSHV-specific immune response prior to KS and less is known about the anti-KSHV immune response during the very early stages of infection. We therefore prospectively followed a cohort of 86 Zambian children for 2 years after primary KSHV seroconversion to characterize the humoral immune response during the early stages of KSHV infection. Plasma, peripheral blood mononuclear cells, and oral swabs were collected from patients every 3 months and analyzed for KSHV-specific antibodies and presence of viral DNA. We observed an approximately 40% KSHV seropositive rate among infected children at time points after primary seroconversion, indicating that seroreversion is common after primary KSHV infection. At the time of primary KSHV seroconversion HIV-infected ART-naïve children had a more robust KSHV antibody response compared to HIV-infected children taking ART and HIV-uninfected children. Conversely, the longitudinal anti-KSHV antibody response was highly variable and did not correlate with available clinical information, HIV/ART status, or presence of KSHV DNA. Collectively, our data suggest that there is limited impact by the variations in the humoral immune response in young children after infection. J. Med. Virol. 88:1973-1981, 2016. © 2016 Wiley Periodicals, Inc.

Effects of Antiretroviral Therapy on Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Transmission Among HIV-Infected Zambian Children.

BACKGROUND: The risk of Kaposi's sarcoma-associated herpesvirus (KSHV) acquisition among children is increased by HIV infection. Antiretroviral therapy (ART) was recently made widely available to HIV-infected children in Zambia. However, the impact of early ART on KSHV transmission to HIV-infected children is unknown. METHODS: We enrolled and followed a cohort of 287 HIV-exposed, KSHV-negative children under 12 months of age from Lusaka, Zambia, to identify KSHV seroconversion events. Potential factors associated with KSHV infection-with an emphasis on HIV, ART, and immunological measures-were assessed through structured questionnaires and blood analyses. Incidence rate, Kaplan-Meier, and multivariable Cox regression models were used to assess differences in time to event (KSHV seroconversion) between groups. All statistical tests were two-sided. RESULTS: During follow-up, 151 (52.6%) children underwent KSHV seroconversion. Based on 3552 months of follow-up, we observed similar KSHV incidence rates between HIV-infected and uninfected children. Among HIV-infected children, ART-naïve children had statistically significantly increased risk of KSHV acquisition (adjusted hazard ratio [AHR] = 5.04, 95% confidence interval [CI] = 2.36 to 10.80, P < .001). Time-updated CD4(+) T-cell percentage was also statistically significantly associated with risk of KSHV acquisition (AHR = 0.82, 95% CI = 0.74 to 0.92, P < .001), such that each 5% increase of CD4(+) T-cells represented an 18% decrease in risk of acquiring KSHV. CONCLUSIONS: Our data suggest that early ART and prevention of immune suppression reduce the risk of KSHV acquisition among HIV-infected children in an area where both viruses are highly endemic. This study highlights the importance of programs in Africa to provide children with ART immediately after HIV infection is diagnosed.

Low-abundance resistant mutations in HIV-1 subtype C antiretroviral therapy-naive individuals as revealed by pyrosequencing.

Given the recent scale-up of antiretroviral therapy (ART) in sub-Saharan Africa, we sought to determine how often and at what levels do drug-resistant mutant variants exist in ART-naive HIV subtype C infected individuals. Samples from 10 ART-naive Zambian individuals were subjected to ultra-deep pyrosequencing (UDPS) to characterize the frequency of low-abundance drug resistance mutations in the pol gene. Low-abundance clinically relevant variants were detected for nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in eight of the ten subjects. Intermediate to high-level resistance was predicted for the majority of NRTIs. Mutations conferring resistance to most first-line and some second-line therapy drugs were also observed. UDPS detected a number of additional major resistant mutations suggesting that these individuals may have an increased risk of virological failure after initiating ART. Moreover, the effectiveness of first-line and even some secondline ART may be compromised in this setting.

Early childhood infection of Kaposi's sarcoma-associated herpesvirus in Zambian households: a molecular analysis.

Sub-Saharan Africa is endemic for Kaposi's sarcoma-associated herpesvirus (KSHV) and there is a high rate of early childhood infection; however, the transmission sources are not well characterized. We examined household members as potential KSHV transmission sources to young children in the KSHV-endemic country of Zambia. To this end, we enrolled and followed Zambian households with at least one KSHV-seropositive child and collected longitudinal buccal swab samples. KSHV burden was evaluated and K1 sequences from the children were determined and analyzed for differences to K1 sequences from household members. The K1 sequences were also analyzed for evolution over time. We generated K1 sequences from 31 individuals across 16 households. Nine households contained multiple KSHV-positive members, including at least one child. In six out of the nine households, the child had 100% sequence identity to all household members. However, in two households the child and mother had distinct K1 sequences. In the remaining household, the children were the only KSHV-infected individuals. Furthermore, we report that 1 out of 18 individuals had K1 sequence variation within the timespan analyzed. In our study, we provide evidence that (i) early childhood KSHV transmission occurs from both within and outside the household, (ii) intrahousehold transmission can occur via nonmaternal sources, (iii) viral shedding in the buccal cavity is highly variable and (iv) the dominant K1 sequence within an individual did not rapidly evolve over time. These results are important for developing KSHV intervention strategies.

Short communication: antiretroviral therapy resistance mutations present in the HIV type 1 subtype C pol and env regions from therapy-naive patients in Zambia.

The prevalence of antiretroviral therapy (ART) resistance mutations present in HIV-1 subtype C pol and env regions of the proviral DNA was analyzed and compared from therapy-naive individuals before (Cohort A) and after (Cohort B) the availability of free ART in Zambia. Mutations present in sequences published in a previous study from Zambian ART-naive individuals infected with subtype C were analyzed using current parameters for the classification of ART drug resistance and compared with Cohorts A and B. No statistically significant differences were observed when comparing mutations present in the pol and env of these cohorts. However, an increase in the number of minor, borderline, or partial resistance mutations as well as the presence of major resistance mutations were observed in Cohort B. These results suggest there is an increasing trend of drug resistance-associated mutations that could be a result of the availability of free ART in Zambia. Moreover, the high prevalence of resistance mutations observed for maraviroc and vicriviroc in both cohorts may suggest a limited efficacy of entry inhibitors on HIV-1 subtype C.

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia.

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.