HIV viral load assays when used with whole blood perform well as a diagnostic assay for infants.

OBJECTIVE: Over the past several years, only approximately 50% of HIV-exposed infants received an early infant diagnosis test within the first two months of life. While high attrition and mortality account for some of the shortcomings in identifying HIV-infected infants early and putting them on life-saving treatment, fragmented and challenging laboratory systems are an added barrier. We sought to determine the accuracy of using HIV viral load assays for infant diagnosis of HIV. METHODS: We enrolled 866 Ugandan infants between March-April 2018 for this study after initial laboratory diagnosis. The median age was seven months, while 33% of infants were less than three months of age. Study testing was done using either the Roche or Abbott molecular technologies at the Central Public Health Laboratory. Dried blood spot samples were prepared according to manufacturer-recommended protocols for both the qualitative and quantitative assays. Viral load test samples for the Roche assay were processed using two different buffers: phosphate-buffered saline (PBS: free virus elution viral load protocol [FVE]) and Sample Pre-Extraction Reagent (SPEX: qualitative buffer). Dried blood spot samples were processed for both assays on the Abbott using the manufacturer's standard infant diagnosis protocol. All infants received a qualitative test for clinical management and additional paired quantitative tests. RESULTS: 858 infants were included in the analysis, of which 50% were female. Over 75% of mothers received antiretroviral therapy, while approximately 65% of infants received infant prophylaxis. The Roche SPEX and Abbott technologies had high sensitivity (>95%) and specificity (>98%). The Roche FVE had lower sensitivity (85%) and viral load values. CONCLUSIONS: To simplify and streamline laboratory practices, HIV viral load may be used to diagnose HIV infection in infants, particularly using the Roche SPEX and Abbott technologies.

The Impact of Control Interventions on Malaria Burden in Young Children in a Historically High-Transmission District of Uganda: A Pooled Analysis of Cohort Studies from 2007 to 2018.

There is limited evidence on whether malaria elimination is feasible in high-transmission areas of Africa. Between 2007 and 2018, we measured the impact of malaria control interventions in young children enrolled in three clinical trials and two observational studies in Tororo, Uganda, a historically high-transmission area. Data were pooled from children aged 0.5-2 years. Interventions included individually assigned chemoprevention and repeated rounds of indoor residual spraying (IRS) of insecticide. All children received long-lasting insecticidal nets (LLINs) and treatment for symptomatic malaria with artemisinin-based combination therapy. Malaria incidence was measured using passive surveillance and parasite prevalence by microscopy and molecular methods at regular intervals. Poisson's generalized linear mixed-effects models were used to estimate the impact of various control interventions. In total, 939 children were followed over 1,221.7 person years. In the absence of chemoprevention and IRS (reference group), malaria incidence was 4.94 episodes per person year and parasite prevalence 47.3%. Compared with the reference group, implementation of IRS was associated with a 97.6% decrease (95% CI: 93.3-99.1%, P = 0.001) in the incidence of malaria and a 96.0% decrease (95% CI: 91.3-98.2%, P < 0.001) in parasite prevalence (both measured after the fifth and sixth rounds of IRS). The addition of chemoprevention with monthly dihydroartemisinin-piperaquine to IRS was associated with a 99.5% decrease (95% CI: 98.6-99.9%, P < 0.001) in the incidence of malaria. In a historically high-malaria burden area of Uganda, a combination of LLINs, effective case management, IRS, and chemoprevention was associated with almost complete elimination of malaria in young children.

Results from a proactive follow-up intervention to improve linkage and retention among people living with HIV in Uganda: a pre-/post- study.

BACKGROUND: Despite gains in HIV testing and treatment access in sub-Saharan Africa, patient attrition from care remains a problem. Evidence is needed of real-world implementation of low-cost, scalable, and sustainable solutions to reduce attrition. We hypothesized that more proactive patient follow-up and enhanced counseling by health facilities would improve patient linkage and retention. METHODS: At 20 health facilities in Central Uganda, we implemented a quality of care improvement intervention package that included training lay health workers in best practices for patient follow-up and counseling, including improved appointment recordkeeping, phone calls and home visits to lost patients, and enhanced adherence counseling strategies; and strengthening oversight of these processes. We compared patient linkage to and retention in HIV care in the 9 months before implementation of the intervention to the 9 months after implementation. Data were obtained from facility-based registers and files and analysed using multivariable logistic regression. RESULTS: Among 1900 patients testing HIV-positive during the study period, there was not a statistically significant increase in linkage to care after implementing the intervention (52.9% versus 54.9%, p = 0.63). However, among 1356 patients initiating antiretroviral therapy during the follow-up period, there were statistically significant increases in patient adherence to appointment schedules (44.5% versus 55.2%, p = 0.01) after the intervention. There was a small increase in Ministry of Health-defined retention in care (71.7% versus 75.7%, p = 0.12); when data from the period of intervention ramp-up was dropped, this increase became statistically significant (71.7% versus 77.6%, p = 0.01). The increase in retention was more dramatic for patients under age 19 years (N = 84; 64.0% versus 83.9%, p = 0.01). The cost per additional patient retained in care was $47. CONCLUSIONS: Improving patient tracking and counseling practices was relatively low cost and enhanced patient retention in care, particularly for pediatric and adolescent patients. This approach should be considered for scale-up in Uganda and elsewhere. However, no impact was seen in improved patient linkage to care with this proactive follow-up intervention. TRIAL REGISTRATION: Pan African Clinical Trial Registry #PACTR201611001756166 . Registered August 31, 2016.

Assessing linkage to and retention in care among HIV patients in Uganda and identifying opportunities for health systems strengthening: a descriptive study.

BACKGROUND: While antiretroviral therapy (ART) availability for HIV patients has increased dramatically in Uganda, patient linkage to and retention in care remains a challenge. We assessed patterns of engagement in care in 20 Ugandan health facilities with low retention based on national reporting. METHODS: We assessed patient linkage to care (defined as registering for pre-ART or ART care at the facility within 1 month of HIV diagnosis) and 6-month retention in care (having a visit 3-6 months after ART initiation) and associations with patient-/facility-level factors using multivariate logistic regression. RESULTS: Among 928 newly HIV-diagnosed patients, only 53.0% linked to care within 1 month. Of these, 83.7% linked within 1 week. Among 678 newly initiated ART patients, 14.5% never returned for a follow-up visit at the facility. Retention was 71.7% according to our primary definition but much lower if stricter definitions were used. Most patients were already falling behind appointment schedules at their first ART follow-up (median: 28 days post-initiation vs. recommended 14 days). 27.3% of newly-initiated patients had follow-up appointments scheduled 45+ days apart rather than monthly per national guidelines. Linkage and retention were not strongly correlated with each other within facilities (rs = 0.06; p = 0.82). Females, adolescents, and patients in rural settings tended to have lower linkage and retention in multivariable-adjusted models. CONCLUSIONS: Linkage support may be most critical immediately after testing positive, as patients are less likely to link over time. More information is needed on reasons for appointment schedules by clinicians and implications on retention. TRIAL REGISTRATION: This study was registered in the Pan African Clinical Trial Registry database (#PACTR201611001756166).

Where have all the children gone? High HIV prevalence in infants attending nutrition and inpatient entry points.

INTRODUCTION: Despite notable progress towards PMTCT, only 50% of HIV-exposed infants in sub-Saharan Africa were tested within the first 2 months of life and only 30% of HIV-infected infants are on antiretroviral treatment. This study assessed HIV prevalence in infants and children receiving care at various service entry points in primary healthcare facilities in Uganda. METHODS: A total of 3600 infants up to 24 months of age were systematically enrolled and tested at four regional hospitals across Uganda. Six hundred infants were included and tested from six facility entry points: PMTCT, immunization, inpatient, nutrition, outpatient and community outreach services. FINDINGS: The traditional EID entry point, PMTCT, had a prevalence of 3.8%, representing 19.6% of the total HIV-positive infants identified in the study. Fifty percent of the 117 identified HIV-positive infants were found in the nutrition wards, which had a prevalence of 9.8% (p < 0.001 compared to PMTCT). Inpatient wards had a prevalence of 3.5% and yielded 17.9% of the HIV-positive infants identified. Infants tested at immunization wards and through outreach services identified 0.8% and 1.7% of the HIV-positive infants respectively, and had a prevalence of less than 0.3%. CONCLUSIONS: Expanding routine early infant diagnosis screening beyond the traditional PMTCT setting to nutrition and inpatient entry points will increase the identification of HIV-infected infants. Careful reflection for appropriate testing strategies, such as maternal re-testing to identify new HIV infections and HIV-exposed infants in need of follow-up testing and care, at immunization and outreach services should be considered given the expectedly low prevalence rates. These findings may help HIV care programmes significantly expand testing to improve access to early infant diagnosis and paediatric treatment.

Rapid Serological Tests Ineffectively Screen for HIV Exposure in HIV-Positive Infants.

BACKGROUND: Data on the performance and utility of rapid serological tests in infants to determine HIV exposure are unclear and in some instances contradictory. This study sought to understand the performance of rapid serological tests in high HIV burden, high Option B+ coverage settings to be used as an HIV exposure screening tool. METHODS: A total of 3600 infants up to 24 months of age at 4 regional hospitals in Uganda were systematically enrolled and tested simultaneously using both HIV rapid serological and nucleic acid-based tests. RESULTS: Only 58 of the 94 HIV-positive infants who received both rapid serological and nucleic acid-based tests were positive with the rapid serological test (sensitivity: 61.7%; 95% confidence interval: 51.1 to 71.5). Using rapid serological tests to screen infants for exposure to HIV and follow-up nucleic acid-based testing would have missed 38.3% (36 of 94) of HIV-positive infants. Finally, several HIV-positive infants who were negative by rapid serological test presented to well-child entry points and were considered healthy. All 3 HIV-positive infants presenting to outreach and immunization were negative by rapid serological testing and 73% (8 of 11) presenting to outpatient. CONCLUSIONS: These data suggest that the use of rapid serological tests may have inadequate performance as an indicator of exposure and potential HIV infection among infants presenting at both well-child (immunization and community outreach) and sick-infant (nutrition and inpatient) entry points. To improve the identification of HIV-positive infants, nucleic acid-based testing should instead be considered in infants aged younger than 18 months.

Population Pharmacokinetics and Pharmacodynamics of Lumefantrine in Young Ugandan Children Treated With Artemether-Lumefantrine for Uncomplicated Malaria.

BACKGROUND: The pharmacokinetics and pharmacodynamics of lumefantrine, a component of the most widely used treatment for malaria, artemether-lumefantrine, has not been adequately characterized in young children. METHODS: Capillary whole-blood lumefantrine concentration and treatment outcomes were determined in 105 Ugandan children, ages 6 months to 2 years, who were treated for 249 episodes of Plasmodium falciparum malaria with artemether-lumefantrine. RESULTS: Population pharmacokinetics for lumefantrine used a 2-compartment open model with first-order absorption. Age had a significant positive correlation with bioavailability in a model that included allometric scaling. Children not receiving trimethoprim-sulfamethoxazole with capillary whole blood concentrations <200 ng/mL had a 3-fold higher hazard of 28-day recurrent parasitemia, compared with those with concentrations >200 ng/mL (P = .0007). However, for children receiving trimethoprim-sulfamethoxazole, the risk of recurrent parasitemia did not differ significantly on the basis of this threshold. Day 3 concentrations were a stronger predictor of 28-day recurrence than day 7 concentrations. CONCLUSIONS: We demonstrate that age, in addition to weight, is a determinant of lumefantrine exposure, and in the absence of trimethoprim-sulfamethoxazole, lumefantrine exposure is a determinant of recurrent parasitemia. Exposure levels in children aged 6 months to 2 years was generally lower than levels published for older children and adults. Further refinement of artemether-lumefantrine dosing to improve exposure in infants and very young children may be warranted.

Malaria illness mediated by anaemia lessens cognitive development in younger Ugandan children.

BACKGROUND: Asymptomatic falciparum malaria is associated with poorer cognitive performance in African schoolchildren and intermittent preventive treatment of malaria improves cognitive outcomes. However, the developmental benefits of chemoprevention in early childhood are unknown. Early child development was evaluated as a major outcome in an open-label, randomized, clinical trial of anti-malarial chemoprevention in an area of intense, year-round transmission in Uganda. METHODS: Infants were randomized to one of four treatment arms: no chemoprevention, daily trimethoprim-sulfamethoxazole, monthly sulfadoxine-pyrimethamine, or monthly dihydroartemisinin-piperaquine (DP), to be given between enrollment (4-6 mos) and 24 months of age. Number of malaria episodes, anaemia (Hb < 10) and neurodevelopment [Mullen Scales of Early Learning (MSEL)] were assessed at 2 years (N = 469) and at 3 years of age (N = 453); at enrollment 70 % were HIV-unexposed uninfected (HUU) and 30 % were HIV-exposed uninfected (HEU). RESULTS: DP was highly protective against malaria and anaemia, although trial arm was not associated with MSEL outcomes. Across all treatment arms, episodes of malarial illness were negatively predictive of MSEL cognitive performance both at 2 and 3 years of age (P = 0.02). This relationship was mediated by episodes of anaemia. This regression model was stronger for the HEU than for the HUU cohort. Compared to HUU, HEU was significantly poorer on MSEL receptive language development irrespective of malaria and anaemia (P = 0.01). CONCLUSIONS: Malaria with anaemia and HIV exposure are significant risk factors for poor early childhood neurodevelopment in malaria-endemic areas in rural Africa. Because of this, comprehensive and cost/effective intervention is needed for malaria prevention in very young children in these settings.

Effective Antimalarial Chemoprevention in Childhood Enhances the Quality of CD4+ T Cells and Limits Their Production of Immunoregulatory Interleukin 10.

BACKGROUND: Experimental inoculation of viable Plasmodium falciparum sporozoites administered with chemoprevention targeting blood-stage parasites results in protective immunity. It is unclear whether chemoprevention similarly enhances immunity following natural exposure to malaria. METHODS: We assessed P. falciparum-specific T-cell responses among Ugandan children who were randomly assigned to receive monthly dihydroartemisinin-piperaquine (DP; n = 87) or no chemoprevention (n = 90) from 6 to 24 months of age, with pharmacologic assessments for adherence, and then clinically followed for an additional year. RESULTS: During the intervention, monthly DP reduced malaria episodes by 55% overall (P < .001) and by 97% among children who were highly adherent to DP (P < .001). In the year after the cessation of chemoprevention, children who were highly adherent to DP had a 55% reduction in malaria incidence as compared to children given no chemoprevention (P = .004). Children randomly assigned to receive DP had higher frequencies of blood-stage specific CD4(+) T cells coproducing interleukin-2 and tumor necrosis factor α (P = .003), which were associated with protection from subsequent clinical malaria and parasitemia, and fewer blood-stage specific CD4(+) T cells coproducing interleukin-10 and interferon γ (P = .001), which were associated with increased risk of malaria. CONCLUSIONS: In this setting, effective antimalarial chemoprevention fostered the development of CD4(+) T cells that coproduced interleukin 2 and tumor necrosis factor α and were associated with prospective protection, while limiting CD4(+) T-cell production of the immunoregulatory cytokine IL-10.

Frequent Malaria Drives Progressive Vδ2 T-Cell Loss, Dysfunction, and CD16 Up-regulation During Early Childhood.

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

Variable piperaquine exposure significantly impacts protective efficacy of monthly dihydroartemisinin-piperaquine for the prevention of malaria in Ugandan children.

BACKGROUND: Anti-malarial chemoprevention with dihydroartemisinin-piperaquine (DHA/PQ) is a promising tool for malaria control, but its efficacy in children may be limited by inadequate drug exposure. METHODS: Children were enrolled in a non directly-observed trial of DHA/PQ chemoprevention in a high transmission setting in Uganda. Children were randomized at 6 months of age to no chemoprevention (n = 89) or monthly DHA/PQ (n = 87) and followed through 24 months of age, with pharmacokinetic sampling performed at variable times following monthly dosing of DHA/PQ. A previously published pharmacokinetic model was used to estimate piperaquine (PQ) exposure in each child, and associations between PQ exposure and the protective efficacy (PE) of DHA/PQ were explored. RESULTS: The incidence of malaria was 6.83 and 3.09 episodes per person year at risk in the no chemoprevention and DHA/PQ arms, respectively (PE 54 %, 95 % CI 39-66 %, P < 0.001). Among children randomized to DHA/PQ, 493 pharmacokinetic samples were collected. Despite nearly 100 % reported adherence to study drug administration at home, there was wide variability in PQ exposure, and children were stratified into three groups based on average PQ exposure during the intervention that was determined by model generated percentiles (low, n = 40; medium, n = 37, and high, n = 10). Gender and socioeconomic factors were not significantly associated with PQ exposure. In multivariate models, the PE of DHA/PQ was 31 % in the low PQ exposure group (95 % CI 6-49 %, P = 0.02), 67 % in the medium PQ exposure group (95 % CI 54-76 %, P < 0.001), and 97 % in the high PQ exposure group (95 % CI 89-99 %, P < 0.001). CONCLUSIONS: The protective efficacy of DHA/PQ chemoprevention in young children was strongly associated with higher drug exposure; in children with the highest PQ exposure, monthly DHA/PQ chemoprevention was nearly 100 % protective against malaria. Strategies to ensure good adherence to monthly dosing and optimize drug exposure are critical to maximize the efficacy of this promising malaria control strategy. TRIAL REGISTRATION: Current Controlled Trials Identifier NCT00948896.

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.

Poor housing construction associated with increased malaria incidence in a cohort of young Ugandan children.

Despite the use of accepted interventions to combat malaria, such as insecticide-treated bed nets and artemisinin-based combination therapy, malaria remains a leading cause of morbidity and mortality in Uganda. We investigated associations between household factors and malaria incidence in a cohort of children living in a highly endemic region of Uganda. Living in a modern house, defined as the use of non-earth floors, non-thatched roofs, and non-mud walls, was associated with approximately half malaria incidence compared with living in a traditional home (incidence rate ratio [IRR] = 0.54, P = 0.001). Other factors found to be associated with a lower incidence of malaria included living in town versus rural setting; sleeping in a room with openings to the outside (windows, eaves, and airbricks); and having an older and more educated primary caregiver. This study adds to the growing body of evidence that improved house construction may be associated with a lower risk of malaria.

Impact of antimalarial treatment and chemoprevention on the drug sensitivity of malaria parasites isolated from ugandan children.

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated malaria in the setting of three different chemopreventive regimens.

BACKGROUND: The burden of malaria remains high for children in parts of Africa despite the use of insecticide-treated bed nets (ITNs). Chemoprevention has the potential of reducing the malaria burden; however, limited data exist on the efficacy and safety of anti-malarial therapy in the setting of chemoprevention. METHODS: 600 children 4-5 months of age were enrolled in Tororo, Uganda, an area of high transmission intensity. Participants were given ITNs, and caregivers instructed to bring their child to a study clinic whenever they were ill. Starting at six months of age, 579 were randomized to no chemoprevention, monthly sulphadoxine-pyrimethamine (SP), daily trimethoprim-sulphamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP). Study drugs were administered unsupervised at home until 24 months of age. Episodes of uncomplicated malaria were treated with artemether-lumefantrine (AL) with active follow-up for 28 days. The cumulative risk of recurrent malaria within 84 days and the risk of adverse events within 28 days were compared across study arms using a Cox proportional hazards model and generalized estimating equations, respectively. RESULTS: A total of 1007, 919, 736, and 451 episodes of malaria were treated in the no chemoprevention, SP, TS, and DP arms, respectively. Only 19 (0.6%) treatments were for severe malaria. Early response to therapy with AL was excellent with 96.5% fever clearance and 99.4% parasite clearance by day 3. However, over 50% of AL treatments were followed by recurrent parasitaemia within 28 days. Compared to the no chemoprevention arm, the cumulative risk of recurrent malaria within 84 days following treatment of uncomplicated malaria with AL was significantly lower in the DP arm (HR = 0.77, 95% CI 0.63-0.95, p = 0.01) but not the SP or TS arms. Compared to the no chemoprevention arm, none of the chemopreventive regimens were associated with an increased risk of adverse events following treatment of malaria with AL. CONCLUSIONS: The risk of severe malaria was very low in this cohort of young children living in a high transmission setting. In the setting of chemoprevention, treatment of uncomplicated malaria with AL was safe and efficacious, with moderate protection against recurrent malaria among children assigned monthly DP. TRIAL REGISTRATION: ClinicalTrials.gov NCT00948896 .

IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

BACKGROUND: The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. METHODS: We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. RESULTS: IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). CONCLUSIONS: In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.

Protective efficacy of prolonged co-trimoxazole prophylaxis in HIV-exposed children up to age 4 years for the prevention of malaria in Uganda: a randomised controlled open-label trial.

BACKGROUND: WHO recommends daily co-trimoxazole for children born to HIV-infected mothers from 6 weeks of age until breastfeeding cessation and exclusion of HIV infection. We have previously reported on the effectiveness of continuation of co-trimoxazole prophylaxis up to age 2 years in these children. We assessed the protective efficacy and safety of prolonging co-trimoxazole prophylaxis until age 4 years in HIV-exposed children. METHODS: We undertook an open-label randomised controlled trial alongside two observational cohorts in eastern Uganda, an area with high HIV prevalence, malaria transmission intensity, and antifolate resistance. We enrolled HIV-exposed infants between 6 weeks and 9 months of age and prescribed them daily co-trimoxazole until breastfeeding cessation and HIV-status confirmation. At the end of breastfeeding, children who remained HIV-uninfected were randomly assigned (1:1) to discontinue co-trimoxazole or to continue taking it up to age 2 years. At age 2 years, children who continued co-trimoxazole prophylaxis were randomly assigned (1:1) to discontinue or continue prophylaxis from age 2 years to age 4 years. The primary outcome was incidence of malaria (defined as the number of treatments for new episodes of malaria diagnosed with positive thick smear) at age 4 years. For additional comparisons, we observed 48 HIV-infected children who took continuous co-trimoxazole prophylaxis and 100 HIV-unexposed uninfected children who never received prophylaxis. We measured grade 3 and 4 serious adverse events and hospital admissions. All children were followed up to age 5 years and all analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00527800. FINDINGS: 203 HIV-exposed infants were enrolled between Aug 10, 2007, and March 28, 2008. After breastfeeding ended, 185 children were not infected with HIV and were randomly assigned to stop (n=87) or continue (n=98) co-trimoxazole up to age 2 years. At age 2 years, 91 HIV-exposed children who had remained on co-trimoxazole prophylaxis were randomly assigned to discontinue (n=46) or continue (n=45) co-trimoxazole from age 2 years to age 4 years. We recorded 243 malaria episodes (2·91 per person-years) in the 45 HIV-exposed children assigned to continue co-trimoxazole until age 4 years compared with 503 episodes (5·60 per person-years) in the 46 children assigned to stop co-trimoxazole at age 2 years (incidence rate ratio 0·53, 95% CI 0·39-0·71; p< 0·0001). There was no evidence of malaria incidence rebound in the year after discontinuation of co-trimoxazole in the HIV-exposed children who stopped co-trimoxazole at age 2 years, but incidence increased significantly in HIV-exposed children who stopped co-trimoxazole at age 4 years (odds ratio 1·78, 95% CI 1·19-2·66; p= 0·005). Incidence of grade 3 or 4 serious adverse events, hospital admissions, or deaths did not significantly differ between HIV-exposed, HIV-unexposed, and HIV-infected children.

Efficacy and safety of three regimens for the prevention of malaria in young HIV-exposed Ugandan children: a randomized controlled trial.

OBJECTIVE: Trimethoprim-sulfamethoxazole prophylaxis is recommended for HIV-exposed infants until breastfeeding ends and HIV infection has been excluded. Extending prophylaxis with a focus on preventing malaria may be beneficial in high transmission areas. We investigated three regimens for the prevention of malaria in young HIV-exposed children. DESIGN: An open-label, randomized controlled trial. SETTING: Tororo, Uganda, a rural area with intense, year-round, malaria transmission. PARTICIPANTS: Two hundred infants aged 4-5 months enrolled and 186 randomized after cessation of breastfeeding and confirmed to be HIV uninfected (median 10 months of age). INTERVENTION: No chemoprevention, monthly sulfadoxine-pyrimethamine, daily trimethoprim-sulfamethoxazole or monthly dihydroartemisinin-piperaquine given from randomization to 24 months of age. MAIN OUTCOME MEASURES: The primary outcome was the incidence of malaria during the intervention period. Secondary outcomes included the incidence of hospitalization, diarrhoeal illness, or respiratory tract infection; prevalence of anaemia and asymptomatic parasitemia; measures of safety; and incidence of malaria over 1 year after the intervention was stopped. RESULTS: During the intervention, the incidence of malaria in the no chemoprevention group was 6.28 episodes per person-year at risk. Protective efficacy was 69% [95% confidence interval (95% CI) 53-80, P < 0.001] for dihydroartemisinin-piperaquine, 49% (95% CI 23-66, P = 0.001) for trimethoprim-sulfamethoxazole and 9% for sulfadoxine-pyrimethamine (95% CI -35 to 38, P = 0.65). There were no significant differences in any secondary outcomes, with the exception of a lower prevalence of asymptomatic parasitemia in the dihydroartemisinin-piperaquine arm. CONCLUSION: Monthly chemoprevention with dihydroartemisinin-piperaquine was well tolerated and associated with a significant reduction in malaria in young HIV-exposed children.

Polymorphisms in K13 and falcipain-2 associated with artemisinin resistance are not prevalent in Plasmodium falciparum isolated from Ugandan children.

The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006-7 (n = 49) and from 2010-12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.