Theodore Ruel

Tissue distribution and prevalence of Wolbachia infections in tsetse flies, Glossina spp.

Tsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ‐line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia‐specific PCR‐based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues.

Antiretroviral Agents and Prevention of Malaria in HIV-Infected Ugandan Children

BACKGROUND Human immunodeficiency virus (HIV) protease inhibitors show activity against Plasmodium falciparum in vitro. We hypothesized that the incidence of malaria in HIV-infected children would be lower among children receiving lopinavir-ritonavir-based antiretroviral therapy (ART) than among those receiving nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based ART. METHODS We conducted an open-label trial in which HIV-infected children 2 months to 5 years of age who were eligible for ART or were currently receiving NNRTI-based ART were randomly assigned to either lopinavir-ritonavir-based ART or NNRTI-based ART and were followed for 6 months to 2 years. Cases of uncomplicated malaria were treated with artemether-lumefantrine. The primary end point was the incidence of malaria. RESULTS We enrolled 176 children, of whom 170 received the study regimen: 86 received NNRTI-based ART, and 84 lopinavir-ritonavir-based ART. The incidence of malaria was lower among children receiving the lopinavir-ritonavir-based regimen than among those receiving the NNRTI-based regimen (1.32 vs. 2.25 episodes per person-year; incidence-rate ratio, 0.59; 95% confidence interval [CI], 0.36 to 0.97; P=0.04), as was the risk of a recurrence of malaria after treatment with artemether-lumefantrine (28.1% vs. 54.2%; hazard ratio, 0.41; 95% CI, 0.22 to 0.76; P=0.004). The median lumefantrine level on day 7 after treatment for malaria was significantly higher in the lopinavir-ritonavir group than in the NNRTI group. In the lopinavir-ritonavir group, lumefantrine levels exceeding 300 ng per milliliter on day 7 were associated with a reduction of more than 85% in the 63-day risk of recurrent malaria. A greater number of serious adverse events occurred in the lopinavir-ritonavir group than in the NNRTI group (5.6% vs. 2.3%, P=0.16). Pruritus occurred significantly more frequently in the lopinavir-ritonavir group, and elevated alanine aminotransferase levels significantly more frequently in the NNRTI group. CONCLUSIONS Lopinavir-ritonavir-based ART as compared with NNRTI-based ART reduced the incidence of malaria by 41%, with the lower incidence attributable largely to a significant reduction in the recurrence of malaria after treatment with artemether-lumefantrine. Lopinavir-ritonavir-based ART was accompanied by an increase in serious adverse events. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development; ClinicalTrials.gov number, NCT00978068.).

Neurocognitive and Motor Deficits in HIV-Infected Ugandan Children With High CD4 Cell Counts

BACKGROUND Human immunodeficiency virus (HIV) infection causes neurocognitive or motor function deficits in children with advanced disease, but it is unclear whether children with CD4 cell measures above the World Health Organization (WHO) thresholds for antiretroviral therapy (ART) initiation suffer significant impairment. METHODS The neurocognitive and motor functions of HIV-infected ART-naive Ugandan children aged 6-12 years with CD4 cell counts of >350 cells/μL and CD4 cell percentage of >15% were compared with those of HIV-uninfected children, using the Test of Variables of Attention (TOVA), the Kaufman Assessment Battery for Children, second edition (KABC-2), and the Bruininks-Oseretsky Test of Motor Proficiency, second edition (BOT-2). RESULTS Ninety-three HIV-infected children (median CD4 cell count, 655 cells/μL; plasma HIV RNA level, 4.7 log(10) copies/mL) were compared to 106 HIV-uninfected children. HIV-infected children performed worse on TOVA visual reaction times (multivariate analysis of covariance; P = .006); KABC-2 sequential processing (P = .005), simultaneous processing (P = .039), planning/reasoning (P = .023), and global performance (P = .024); and BOT-2 total motor proficiency (P = .003). High plasma HIV RNA level was associated with worse performance in 10 cognitive measures and 3 motor measures. In analysis of only WHO clinical stage 1 or 2 HIV-infected children (n = 68), significant differences between the HIV-infected and HIV-uninfected groups (P < .05) remained for KABC-2 sequential processing, KABC-2 planning/reasoning, and BOT-2 motor proficiency. CONCLUSIONS Significant motor and cognitive deficits were found in HIV-infected ART-naive Ugandan children with CD4 cell counts of ∼350 cells/μL and percentages of >15%. Study of whether early initiation of ART could prevent or reverse such deficits is needed.

Effects of trimethoprim-sulfamethoxazole and insecticide-treated bednets on malaria among HIV-infected Ugandan children

Background:Trimethoprim–sulfamethoxazole (TMP/SMX) prophylaxis and insecticide-treated bednets reduce malaria risk among HIV-infected adults. The efficacy of TMP/SMX may be diminished where antifolate resistance to malaria is high. We evaluated the efficacy of these interventions for malaria prevention among Ugandan children. Methods:We concurrently followed 300 HIV-infected children aged 1–10 years and a community-based cohort of 561 healthy children aged 1–11 years over 11 months in Kampala, Uganda. The HIV-infected children received TMP/SMX prophylaxis and insecticide treated bednets. In the community cohort, insecticide-treated bednets were introduced during the observation period. Children from both cohorts were followed using a standardized protocol to measure the incidence of malaria. Results:Only nine episodes of malaria were diagnosed among HIV-infected children (incidence = 0.07/person-year) in comparison with 440 episodes among children from the community (incidence = 0.90/person-year; P < 0.0001). The use of insecticide-treated bednets was associated with a 43% reduction in malaria incidence (P < 0.001), and a combination of TMP/SMX and use of insecticide-treated bednets with a 97% reduction in malaria incidence (P < 0.001). The prevalence of five mutations associated with antifolate resistance was high among malaria cases detected in both the HIV (100%) and community cohorts (75%). Malaria accounted for only 4% of febrile episodes in the HIV cohort in comparison with 33% in the community-based cohort (P < 0.0001). Conclusion:In a malaria endemic area with a high level of molecular markers of antifolate resistance, the combined use of TMP/SMX prophylaxis and insecticide-treated bednets was associated with a dramatic reduction in malaria incidence among HIV-infected children.

High Risk of Neutropenia in HIV-Infected Children following Treatment with Artesunate plus Amodiaquine for Uncomplicated Malaria in Uganda

BACKGROUND Artemisinin-based combination therapies are rapidly being adopted for the treatment of malaria in Africa; however, there are limited data on their safety and efficacy among human immunodeficiency virus (HIV)-infected populations. METHODS We compared malaria treatment outcomes between cohorts of HIV-infected and HIV-uninfected children in Uganda who were observed for 18 and 29 months, respectively. Malaria was treated with artesunate plus amodiaquine, and outcomes were assessed using standardized guidelines. HIV-infected children received trimethoprim-sulfamethoxazole prophylaxis and antiretroviral therapy in accordance with current guidelines. RESULTS Twenty-six HIV-infected participants experiencing 35 episodes of malaria and 134 HIV-uninfected children experiencing 258 episodes of malaria were included in the study. Twelve HIV-infected children were receiving antiretroviral therapy, 11 of whom were receiving zidovudine. Malaria treatment was highly efficacious in both the HIV-infected and HIV-uninfected cohorts (28-day risk of recrudescence, 0% and 3.6%, respectively); however, there was a trend towards increased risk of recurrent malaria among the HIV-uninfected children (2.9% vs. 13.2%; p = .08). Importantly, the risk of neutropenia 14 days after initiation of treatment with artesunate plus amodiaquine was higher among HIV-infected children than among HIV-uninfected children (45% vs. 6%; p < .001). The severity of all episodes of neutropenia in HIV-uninfected children was mild to moderate, and 16% of episodes of neutropenia in the HIV-infected cohort were severe or life-threatening (neutrophil count, <750 cells/mm(3)). In the HIV-infected cohort, the risk of neutropenia was significantly higher among children who received antiretroviral therapy than among those who did not receive antiretroviral therapy (75% vs. 26%; p < .001). CONCLUSIONS Artesunate plus amodiaquine was highly efficacious for malaria treatment in HIV-infected children but was associated with a high risk of neutropenia, especially in the context of concurrent antiretroviral use. Our findings highlight an urgent need for evaluation of alternative antimalarial therapies for HIV-infected individuals.

Hiv-subtype A is associated with poorer neuropsychological performance compared with subtype D in antiretroviral therapy-naive Ugandan children

Background:HIV-subtype D is associated with more rapid disease progression and higher rates of dementia in Ugandan adults compared with HIV-subtype A. There are no data comparing neuropsychological function by HIV subtype in Ugandan children. Design:One hundred and two HIV-infected antiretroviral therapy (ART) naive Ugandan children 6–12 years old (mean 8.9) completed the Kaufman Assessment Battery for Children, second edition (KABC-2), the Test of Variables of Attention (TOVA), and the Bruininks–Oseretsky Test for Motor Proficiency, second edition (BOT-2). Using a PCR-based multiregion assay with probe hybridization in five different regions (gag, pol, vpu, env, gp-41), HIV subtype was defined by hybridization in env and by total using two or more regions. Analysis of covariance was used for multivariate comparison. Results:The env subtype was determined in 54 (37 A, 16 D, 1 C) children. Subtype A and D groups were comparable by demographics, CD4 status, and WHO stage. Subtype A infections had higher log viral loads (median 5.0 vs. 4.6, P = 0.02). Children with A performed more poorly than those with D on all measures, especially on KABC-2 Sequential Processing (memory) (P = 0.01), Simultaneous Processing (visual–spatial analysis) (P = 0.005), Learning (P = 0.02), and TOVA visual attention (P = 0.04). When adjusted for viral load, Sequential and Simultaneous Processing remained significantly different. Results were similar comparing by total HIV subtype. Conclusion:HIV subtype A children demonstrated poorer neurocognitive performance than those with HIV subtype D. Subtype-specific neurocognitive deficits may reflect age-related differences in the neuropathogenesis of HIV. This may have important implications for when to initiate ART and the selection of drugs with greater central nervous system penetration.

Effect of trimethoprim-sulphamethoxazole on the risk of malaria in HIV-infected Ugandan children living in an area of widespread antifolate resistance

BackgroundDaily trimethoprim-sulfamethoxazole (TS) protects against malaria, but efficacy may be diminished as anti-folate resistance increases. This study assessed the incidence of falciparum malaria and the prevalence of resistance-conferring Plasmodium falciparum mutations in HIV-infected children receiving daily TS and HIV-uninfected children not taking TS.Materials and methodsSubjects were 292 HIV-infected and 517 uninfected children from two cohort studies in Kampala, Uganda observed from August 2006 to December 2008. Daily TS was given to HIV-infected, but not HIV-uninfected children and all participants were provided an insecticide-treated bed net. Standardized protocols were used to measure the incidence of malaria and identify markers of antifolate resistance.ResultsSixty-five episodes of falciparum malaria occurred in HIV-infected and 491 episodes in uninfected children during the observation period. TS was associated with a protective efficacy of 80% (0.10 vs. 0.45 episodes per person year, p < 0.001), and efficacy did not vary over three consecutive 9.5 month periods (81%, 74%, 80% respectively, p = 0.506). The prevalences of dhfr 51I, 108N, and 59R and dhps 437G and 540E mutations were each over 90% among parasites infecting both HIV-infected and uninfected children. Prevalence of the dhfr 164L mutation, which is associated with high-level resistance, was significantly higher in parasites from HIV-infected compared to uninfected children (8% vs. 1%, p = 0.001). Sequencing of the dhfr and dhps genes identified only one additional polymorphism, dhps 581G, in 2 of 30 samples from HIV-infected and 0 of 54 samples from uninfected children.ConclusionDespite high prevalence of known anti-folate resistance-mediating mutations, TS prophylaxis was highly effective against malaria, but was associated with presence of dhfr 164L mutation.

The potential role of rodents in the enzootic cycle of Rift Valley fever virus in Senegal.

Wild rodents (214) of fourteen species were trapped at seven sites in Senegal. Arvicanthis niloticus and Mastomys erythroleucus were among the most frequently collected species (77.2% of total capture). All rodents were examined for the presence of anti-Rift Valley fever virus (RVFV) antibody; the prevalence over all sampled species was 3.8%, varying widely with respect to species and location. Four of 14 species of rodents were found to have anti-RVFV antibodies: Rattus rattus (one positive of two tested) Mastomys huberti (13.5%), A. niloticus (4.3%), and M. erthroleucus (2.4%). The highest prevalence of anti-RVFV antibody was recorded within the enzootic area of the Senegal River delta, at Richard Toll (9.6%). A. niloticus and M. erythroleucus and a strain of laboratory-bred mice were experimentally inoculated with two strains of RVFV and examined for viremia, illness, seroconversion and mortality. A. niloticus and M. erythroleucus demonstrated a limited resistance to infection, thus potentially allowing for the replication of virus in these animals and making these species possible candidates as hosts in the maintenance cycle of RVFV in nature.

Hair and plasma data show that lopinavir, ritonavir, and efavirenz all transfer from mother to infant in utero, but only efavirenz transfers via breastfeeding.

Background:As efforts intensify to eliminate perinatal HIV transmission, understanding kinetics of maternal-to-child transfer of antiretrovirals during pregnancy and breastfeeding is critical. Antiretroviral levels in plasma, cord blood, and breastmilk reflect exposure over short intervals. Hair concentrations reflect cumulative exposure and can uniquely quantify in utero transfer of maternal medications to infants. We measured plasma and hair antiretroviral levels in HIV-infected Ugandan mothers and their infants at delivery and during breastfeeding to assess transfer. Methods:HIV-infected pregnant women were randomized to lopinavir/ritonavir- or efavirenz-based therapy in a larger trial (the Prevention of Malaria and HIV disease in Tororo, PROMOTE). At 0, 8, and 12 weeks postpartum, plasma antiretroviral levels were measured in 117 mother–infant pairs; hair levels were assayed at 12 weeks. Ratios and correlations of infant:maternal concentrations were calculated. Results:By 12 weeks, 90.4% of mothers reported exclusive breastfeeding. Hair and plasma levels over time suggest moderate (47%) to extensive (87%) in utero transfer of lopinavir and ritonavir, respectively, but negligible transfer of either via breastfeeding. Moderate transfer of efavirenz occurs during pregnancy and breastfeeding (40% cumulative; 15% during breastfeeding). Despite differences in exposure, no infant seroconversions or correlations between infant hair/plasma antiretroviral levels and adverse effects were observed. Conclusions:Using a unique approach combining hair and plasma data, we found that different antiretrovirals have distinct kinetics of mother-to-infant transfer. Efavirenz transfers during both pregnancy and breastfeeding, whereas lopinavir and ritonavir transfer only in utero. Further study of the degree and timing of maternal-to-child transfer by antiretroviral will help optimize strategies that protect infants and minimize toxicities during periods of risk.

Early virologic failure and the development of antiretroviral drug resistance mutations in HIV-infected Ugandan children.

Background:Without virologic testing, HIV-infected African children starting antiretroviral (ARV) therapy are at risk for undetected virologic failure and the development of ARV resistance. We sought to determine the prevalence of early virologic failure (EVF), to characterize the evolution of ARV-resistance mutations and to predict the impact on second-line therapy. Methods:The prevalence of EVF (HIV RNA >400 copies/mL on sequential visits after 6 months of therapy) was identified among 120 HIV-infected Ugandan children starting ARV therapy. ARV mutations were identified by population sequencing of HIV-1 pol in sequential archived specimens. Composite discrete genotypic susceptibility scores were determined for second-line ARV regimens. Results:EVF occurred in 16 children (13%) and persisted throughout a median (interquartile ratio) 938 (760-1066) days of follow-up. M184V and nonnucleoside reverse transcriptase inhibitor-associated mutations emerged within 6 months of EVF; thymidine-analog-mutations arose after 12 months. Worse discrete genotypic susceptibility scores correlated with increasing duration of failure (Spearman R = −0.47; P = 0.001). Only 1 child met World Health Organization CD4 criteria for ARV failure at the time of EVF or during the follow-up period. Conclusions:A significant portion of HIV-infected African children experience EVF that would be undetected using CD4/clinical monitoring and resulted in the accumulation of ARV mutations that could compromise second-line therapy options.