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Abstract Background Plasmodium falciparum (P. falciparum) remains a significant public health challenge globally, especially in sub-Saharan Africa (SSA), where it accounts for 99% of all malaria infections. The outcomes of P. falciparum infection vary, ranging from asymptomatic to severe, and are associated with factors such as host immunity, parasite genetic diversity, and multiplicity of infection (MOI). Using seven neutral microsatellite markers, the current study investigated P. falciparum genetic diversity and MOI in both asymptomatic and symptomatic malaria individuals in Uganda. Methods This cross-sectional study analyzed 225 P. falciparum isolates from both asymptomatic and symptomatic malaria patients, ranging in age from 6 months to ≥ 18 years. P. falciparum genetic diversity, MOI, and multi-locus linkage disequilibrium (LD) were assessed through genotyping of seven neutral microsatellite markers: Poly-α, TA1, TA109, PfPK2, 2490, C2M34–313, and C3M69–383. Genetic data analysis was performed using appropriate genetic analysis software. Results P. falciparum infections exhibited high genetic diversity in both asymptomatic and symptomatic individuals. The mean expected heterozygosity (He) ranged from 0.79 in symptomatic uncomplicated malaria cases to 0.81 in asymptomatic individuals. There was no significant difference (p = 0.33) in MOI between individuals with asymptomatic and symptomatic infections, with the mean MOI ranging from 1.92 in symptomatic complicated cases to 2.10 in asymptomatic individuals. Polyclonal infections were prevalent, varying from 58.5% in symptomatic complicated malaria to 63% in symptomatic uncomplicated malaria cases. A significant linkage disequilibrium (LD) was observed between asymptomatic and symptomatic uncomplicated/complicated infections (p

Abstract Background Reports on the impact of COVID-19 pandemic on the quality of malaria care and burden in sub Saharan Africa have provided a mixed picture to date. The impact of the 2nd (Delta) and 3rd (Omicron) COVID-19 waves on outpatient malaria indicators and case management practices was assessed at three public health facilities with varying malaria transmission intensities in Uganda. Methods Individual level data from all patients presenting to the out-patient departments (OPD) of the three facilities (Kasambya, Walukuba and Lumino) between January 2019 and February 2022 were included in the analysis. Outcomes of interest included total number of outpatient (OPD) visits, proportion of patients suspected to have malaria, proportion of suspected malaria cases tested with a malaria diagnostic test, test positivity rates (TPR) and proportion of malaria cases prescribed artemether-lumefantrine (AL). Using the pre-COVID-19 trends between January 2019 and February 2020, interrupted time series analysis was used to predict the expected trends for these study outcomes during the 2nd wave (May 2021–August 2021) and 3rd wave (November 2021–February 2022). The observed trends of the study outcomes were compared with the expected trends. Results There were no significant differences between the observed versus expected overall outpatient visits in the 2nd wave, however, a significant decline in OPD attendance was observed during the 3rd wave (15,101 vs 31,154; incidence rate ratio (IRR) = 0.48 [0.41–0.56]). No significant differences in the overall observed versus expected proportions of suspected malaria cases and test positivity rates in both COVID waves. However, a significant decrease in the overall proportion of suspected malaria cases tested with a malaria diagnostic test was observed during the 3rd wave (99.86% vs 99.99%; relative percent ratio [RPR] = 0.99 [0.99–0.99]). Finally, a significant decline in the overall proportion of malaria cases prescribed AL was observed during the 2nd wave (94.99% vs 99.85%; RPR = 0.95 [0.92–0.98]) but not the 3rd wave. Conclusion Significant declines in OPD attendance and suspected malaria cases tested with malaria diagnostic test were observed during the 3rd COVID-19 wave, while AL prescription significantly reduced during the 2nd COVID-19 wave. These findings add to the body of knowledge highlighting the adverse impact of COVID-19 pandemic on the malaria which could explain the increase in the malaria burden observed during this period.

Abstract Background Scale up of proven malaria control interventions has not been sufficient to control malaria in Uganda, emphasizing the need to explore innovative new approaches. Improved housing is one such promising strategy. This paper describes housing characteristics and their association with malaria burden in a moderate to high transmission setting in Uganda. Methods Between October and November 2021, a household survey was conducted in 1500 randomly selected households in Jinja and Luuka districts. Information on demographics, housing characteristics, use of malaria prevention measures, and proxy indicators of wealth were collected for each household. A finger-prick blood sample was obtained for thick blood smears for malaria from all children aged 6 months to 14 years in the surveyed households. Febrile children had a malaria rapid diagnostics test (RDT) done; positive cases were managed according to national treatment guidelines. Haemoglobin was assessed in children aged

Abstract Background Well-built housing limits mosquito entry and can reduce malaria transmission. The association between community-level housing and malaria burden in Uganda was assessed using data from randomly selected households near 64 health facilities in 32 districts. Methods Houses were classified as ‘improved’ (synthetic walls and roofs, eaves closed or absent) or ‘less-improved’ (all other construction). Associations between housing and parasitaemia were made using mixed effects logistic regression (individual-level) and multivariable fractional response logistic regression (community-level), and between housing and malaria incidence using multivariable Poisson regression. Results Between November 2021 and March 2022, 4.893 children aged 2–10 years were enrolled from 3.518 houses; of these, 1.389 (39.5%) were classified as improved. Children living in improved houses had 58% lower odds (adjusted odds ratio = 0.42, 95% CI 0.33–0.53, p 67% of houses improved had a 63% lower parasite prevalence (adjusted prevalence ratio 0.37, 95% CI 0.19–0.70, p

Abstract Background Disruptions in malaria control due to COVID-19 mitigation measures were predicted to increase malaria morbidity and mortality in Africa substantially. In Uganda, long-lasting insecticidal nets (LLINs) are distributed nationwide every 3–4 years, but the 2020–2021 campaign was altered because of COVID-19 restrictions so that the timing of delivery of new nets was different from the original plans made by the National Malaria Control Programme. Methods A transmission dynamics modelling exercise was conducted to explore how the altered delivery of LLINs in 2020–2021 impacted malaria burden in Uganda. Data were available on the planned LLIN distribution schedule for 2020–2021, and the actual delivery. The transmission model was used to simulate 100 health sub-districts, and parameterized to match understanding of local mosquito bionomics, net use estimates, and seasonal patterns based on data collected in 2017–2019 during a cluster-randomized trial (LLINEUP). Two scenarios were compared; simulated LLIN distributions matching the actual delivery schedule, and a comparable scenario simulating LLIN distributions as originally planned. Model parameters were otherwise matched between simulations. Results Approximately 70% of the study population received LLINs later than scheduled in 2020–2021, although some areas received LLINs earlier than planned. The model indicates that malaria incidence in 2020 was substantially higher in areas that received LLINs late. In some areas, early distribution of LLINs appeared less effective than the original distribution schedule, possibly due to attrition of LLINs prior to transmission peaks, and waning LLIN efficacy after distribution. On average, the model simulations predicted broadly similar overall mean malaria incidence in 2021 and 2022. After accounting for differences in cluster population size and LLIN distribution dates, no substantial increase in malaria burden was detected. Conclusions The model results suggest that the disruptions in the 2020–2021 LLIN distribution campaign in Uganda did not substantially increase malaria burden in the study areas.

Abstract Background In sub-Saharan Africa (SSA), Plasmodium falciparum causes most of the malaria cases. Despite its crucial roles in disease severity and drug resistance, comprehensive data on Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are sparse in SSA. This study summarizes available information on genetic diversity and MOI, focusing on key markers (msp-1, msp-2, glurp, and microsatellites). The systematic review aimed to evaluate their influence on malaria transmission dynamics and offer insights for enhancing malaria control measures in SSA. Methods The review was conducted following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. Two reviewers conducted article screening, assessed the risk of bias (RoB), and performed data abstraction. Meta-analysis was performed using the random-effects model in STATA version 17. Results The review included 52 articles: 39 cross-sectional studies and 13 Randomized Controlled Trial (RCT)/cohort studies, involving 11,640 genotyped parasite isolates from 23 SSA countries. The overall pooled mean expected heterozygosity was 0.65 (95% CI: 0.51–0.78). Regionally, values varied: East (0.58), Central (0.84), Southern (0.74), and West Africa (0.69). Overall pooled allele frequencies of msp-1 alleles K1, MAD20, and RO33 were 61%, 44%, and 40%, respectively, while msp-2 I/C 3D7 and FC27 alleles were 61% and 55%. Central Africa reported higher frequencies (K1: 74%, MAD20: 51%, RO33: 48%) than East Africa (K1: 46%, MAD20: 42%, RO33: 31%). For msp-2, East Africa had 60% and 55% for I/C 3D7 and FC27 alleles, while West Africa had 62% and 50%, respectively. The pooled allele frequency for glurp was 66%. The overall pooled mean MOI was 2.09 (95% CI: 1.88–2.30), with regional variations: East (2.05), Central (2.37), Southern (2.16), and West Africa (1.96). The overall prevalence of polyclonal Plasmodium falciparum infections was 63% (95% CI: 56–70), with regional prevalences as follows: East (62%), West (61%), Central (65%), and South Africa (71%). Conclusion The study shows substantial regional variation in Plasmodium falciparum parasite genetic diversity and MOI in SSA. These findings suggest a need for malaria control strategies and surveillance efforts considering regional-specific factors underlying Plasmodium falciparum infection.

Abstract Background Provision of effective care to all women and newborns during the perinatal period is a viable strategy for achieving the Sustainable Development Goal 3 targets on reducing maternal and neonatal mortality. This study examined perinatal care (antenatal, intrapartum, postpartum) and its association with perinatal deaths at three district hospitals in Bunyoro region, Uganda. Methods A cross-sectional study was conducted in which a questionnaire was administered consecutively to 872 postpartum women before discharge who had attended antenatal care and given birth in the study hospitals. Data on care received during antenatal, labour, delivery, and postpartum period, and perinatal outcome were extracted from medical records of the enrolled postnatal women using a pre-tested structured tool. The care received from antenatal to 24 h postpartum period was assessed against the standard protocol of care established by World Health Organization (WHO). Poisson regression was used to assess the association between care received and perinatal death. Results The mean age of the women was 25 years (standard deviation [SD] 5.95). Few women had their blood tested for hemoglobin levels, HIV, and Syphilis (n = 53, 6.1%); had their urine tested for glucose and proteins (n = 27, 3.1%); undertook an ultrasound scan (n = 262, 30%); and had their maternal status assessed (n = 122, 14%) during antenatal care as well as had their uterus assessed for contraction and bleeding during postpartum care (n = 63, 7.2%). There were 19 perinatal deaths, giving a perinatal mortality rate of 22/1,000 births (95% Confidence interval [CI] 8.1–35.5). Of these 9 (47.4%) were stillbirths while the remaining 10 (52.6%) were early neonatal deaths. In the antenatal phase, only fetal examination was significantly associated with perinatal death (adjusted prevalence ratio [aPR] = 0.22, 95% CI 0.1–0.6). No significant association was found between perinatal deaths and care during labour, delivery, and the early postpartum period. Conclusion Women did not receive all the required perinatal care during the perinatal period. Perinatal mortality rate in Bunyoro region remains high, although it’s lower than the national average. The study shows a reduction in the proportion of perinatal deaths for pregnancies where the mother received fetal monitoring. Strategies focused on strengthened fetal status monitoring such as fetal movement counting methods and fetal heart rate monitoring devices during pregnancy need to be devised to reduce the incidence of perinatal deaths. Findings from the study provide valuable information that would support the strengthening of perinatal care services for improved perinatal outcomes.

Abstract Background Understanding the burden of dyslipidemia and its associated factors among adult people living with HIV on dolutegravir (DTG) based anti-retroviral therapy (ART) is critical to provide clinical guidance and risk reduction strategies in our setting. Methods We conducted a cross-sectional study on adult people living with HIV on DTG based ART between July and August 2022 at Mengo Hospital, a private not for profit missionary hospital owned by the Church of Uganda. Dyslipidemia was defined as: Total cholesterol (TC) ≥ 5.2 mmol/l, or high-density lipoprotein (HDL)

Abstract Background Uganda’s current guidelines recommend immediate initiation of Anti-Retroviral Therapy (ART) for persons living with HIV in order to reduce HIV/AIDS related morbidity and mortality. However, not all eligible PLHIV initiate ART within the recommended time following HIV diagnosis. We assessed the prevalence and factors associated with delayed ART initiation among PLHIV referred for ART initiation, five years since rolling out the test and treat guidelines. Methods In this cross-sectional study, we enrolled adult patients referred to Mulago Immune Suppressive Syndrome (Mulago ISS) clinic for ART initiation from January 2017 to May 2021. We collected data on socio-demographics, HIV diagnosis and referral circumstances, and time to ART initiation using a questionnaire. The outcome of interest was proportion of patients that delayed ART, defined as spending more than 30 days from HIV diagnosis to ART initiation. We performed multivariable logistic regression and identified significant factors. Results A total of 312 patients were enrolled of which 62.2% were female. The median (inter-quartile range [IQR]) age and baseline CD4 count of the patients were 35 (28–42) years and 315 (118.8–580.5) cells/μL respectively. Forty-eight (15.4%) patients delayed ART initiation and had a median (IQR) time to ART of 92 (49.0–273.5) days. The factors associated with delayed ART initiation were; 1) having had the HIV diagnosis made from a private health facility versus public, (adjusted odds ratio [aOR] = 2.4 (95% confidence interval [CI] 1.1–5.5); 2) initial denial of positive HIV test results, aOR = 5.4 (95% CI: 2.0–15.0); and, 3) having not received a follow up phone call from the place of HIV diagnosis, aOR = 2.8 (95% CI: 1.2–6.8). Conclusion There was significant delay of ART initiation among referred PLHIV within 5 years after the rollout of test and treat guidelines in Uganda. Health system challenges in the continuity of HIV care services negatively affects timely ART initiation among referred PLHIV in Uganda.

Abstract Background Malaria is one of the leading causes of morbidity and mortality among children under 5 years of age in Uganda. Although Karamoja sub-region has the highest prevalence of malaria, and one of the highest case fatality rates in children under 5 years, information on malaria case management for the sub-region is scarce. The study evaluated the malaria diagnostic and treatment practices, as well as the factors associated with inappropriate care for children under 5 years of age presenting with fever in two public hospitals within the sub-region. Methods A cross-sectional study was conducted amongst 857 children under 5 years of age who presented with fever at Abim and Kaabong general hospitals between February and March 2020. A questionnaire was administered to the primary caregiver during exit/bedside interviews to collect socio-demographic information. The participant clinical notes were reviewed to capture information on laboratory tests conducted, diagnosis given, and treatment prescribed. In addition, a health facility assessment was conducted and information on healthcare workers was collected. The healthcare worker and facility data was linked to the participant’s hospital visit. Main outcome measures were malaria diagnostic and treatment practices. Results Of the 857 children enrolled, 820 (95.7%) had a malaria diagnostic test done and 623 (76.0%) tested positive for malaria. All test positive children received anti-malarial treatment, however, only 424/623 (68.1%) received the recommended anti-malarial drug and 376/424 (88.7%) received the right dose of the treatment. Inappropriate diagnosis/treatment was in 321 (37.5%) of the enrolled participants. Factors associated with inappropriate diagnosis/treatment included: lack of recommended anti-malarials on the day of the visit (Prevalence Ratio [PR] = 2.1, 95% confidence interval [CI] 1.8–2.4), hospital where care was sought (PR = 0.4, 95% CI 0.3–0.5), being managed by a recently supervised health worker (PR = 0.5, 95% CI 0.2–0.9), and health worker cadre (PR = 0.8, 95% CI 0.7–0.9). Conclusion The prevalence of inappropriate malaria diagnosis and treatment in the Karamoja sub-region was high with approximately one in every three children receiving inappropriate care. This was majorly influenced by health system factors, which if improved upon may reduce malaria-related mortalities in the sub-region a vital step in meeting the country’s target of zero deaths from malaria by 2030.

Abstract Background In 2020–2021, long-lasting insecticidal nets (LLINs) were distributed nationwide in Uganda during the COVID-19 pandemic. A cross-sectional survey was conducted in 12 districts to evaluate the impact of the campaign 1–5 months after LLIN distribution. Methods During April–May 2021, households were randomly selected from target areas (1–7 villages) surrounding 12 government-run health facilities established as Malaria Reference Centres; at least 50 households were enrolled per cluster. Outcomes included household ownership of LLINs distributed through the universal coverage campaign (UCC) (at least one UCC LLIN), adequate coverage of UCC LLINs (at least one UCC LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Multivariate logistic regression models were used to identify household- and individual-level factors associated with outcomes, controlling for clustering around health facilities. Results In total, 634 households, with 3342 residents and 1631 bed-nets, were included. Most households (93.4%) owned at least 1 UCC LLIN, but only 56.8% were adequately covered by UCC LLINs. In an adjusted analysis, the factor most strongly associated with adequate coverage by UCC LLINs was fewer household residents (1–4 vs 7–14; adjusted odds ratio [aOR] 12.96, 95% CI 4.76–35.26, p

Abstract Studies have estimated the impact of the environment on malaria incidence although few have explored the differential impact due to malaria control interventions. Therefore, the objective of the study was to evaluate the effect of indoor residual spraying (IRS) on the relationship between malaria and environment (i.e. rainfall, temperatures, humidity, and vegetation) using data from a dynamic cohort of children from three sub-counties in Uganda. Environmental variables were extracted from remote sensing sources and averaged over different time periods. General linear mixed models were constructed for each sub-counties based on a log-binomial distribution. The influence of IRS was analysed by comparing marginal effects of environment in models adjusted and unadjusted for IRS. Great regional variability in the shape (linear and non-linear), direction, and magnitude of environmental associations with malaria risk were observed between sub-counties. IRS was significantly associated with malaria risk reduction (risk ratios vary from RR = 0.03, CI 95% [0.03–0.08] to RR = 0.35, CI95% [0.28–0.42]). Model adjustment for this intervention changed the magnitude and/or direction of environment-malaria associations, suggesting an interaction effect. This study evaluated the potential influence of IRS in the malaria-environment association and highlighted the necessity to control for interventions when they are performed to properly estimate the environmental influence on malaria. Local models are more informative to guide intervention program compared to national models.

Abstract Background Routine malaria surveillance data in Africa primarily come from public health facilities reporting to national health management information systems. Although information on gender is routinely collected from patients presenting to these health facilities, stratification of malaria surveillance data by gender is rarely done. This study evaluated gender difference among patients diagnosed with parasitological confirmed malaria at public health facilities in Uganda. Methods This study utilized individual level patient data collected from January 2020 through April 2021 at 12 public health facilities in Uganda and cross-sectional surveys conducted in target areas around these facilities in April 2021. Associations between gender and the incidence of malaria and non-malarial visits captured at the health facilities from patients residing within the target areas were estimated using poisson regression models controlling for seasonality. Associations between gender and data on health-seeking behaviour from the cross-sectional surveys were estimated using poisson regression models controlling for seasonality. Results Overall, incidence of malaria diagnosed per 1000 person years was 735 among females and 449 among males (IRR = 1.72, 95% CI 1.68–1.77, p

Abstract Background In March 2020, the government of Uganda implemented a strict lockdown policy in response to the COVID-19 pandemic. Interrupted time series analysis (ITSA) was performed to assess whether major changes in outpatient attendance, malaria burden, and case management occurred after the onset of the COVID-19 epidemic in rural Uganda. Methods Individual level data from all outpatient visits collected from April 2017 to March 2021 at 17 facilities were analysed. Outcomes included total outpatient visits, malaria cases, non-malarial visits, proportion of patients with suspected malaria, proportion of patients tested using rapid diagnostic tests (RDTs), and proportion of malaria cases prescribed artemether-lumefantrine (AL). Poisson regression with generalized estimating equations and fractional regression was used to model count and proportion outcomes, respectively. Pre-COVID trends (April 2017-March 2020) were used to predict the’expected’ trend in the absence of COVID-19 introduction. Effects of COVID-19 were estimated over two six-month COVID-19 time periods (April 2020-September 2020 and October 2020–March 2021) by dividing observed values by expected values, and expressed as ratios. Results A total of 1,442,737 outpatient visits were recorded. Malaria was suspected in 55.3% of visits and 98.8% of these had a malaria diagnostic test performed. ITSA showed no differences between observed and expected total outpatient visits, malaria cases, non-malarial visits, or proportion of visits with suspected malaria after COVID-19 onset. However, in the second six months of the COVID-19 time period, there was a smaller mean proportion of patients tested with RDTs compared to expected (relative prevalence ratio (RPR) = 0.87, CI (0.78–0.97)) and a smaller mean proportion of malaria cases prescribed AL (RPR = 0.94, CI (0.90–0.99)). Conclusions In the first year after the COVID-19 pandemic arrived in Uganda, there were no major effects on malaria disease burden and indicators of case management at these 17 rural health facilities, except for a modest decrease in the proportion of RDTs used for malaria diagnosis and the mean proportion of malaria cases prescribed AL in the second half of the COVID-19 pandemic year. Continued surveillance will be essential to monitor for changes in trends in malaria indicators so that Uganda can quickly and flexibly respond to challenges imposed by COVID-19.

Abstract Background Intensive malaria control may have additional benefits beyond reducing the incidence of symptomatic malaria. We compared antibiotic treatment of children before and after the implementation of highly effective malaria control interventions in Tororo, a historically high transmission area of Uganda. Methods Two successive cohorts of children, aged 0.5 to 10 years, were followed from September 2011 to October 2019 in a dedicated study clinic. Universal distribution of long-lasting insecticidal nets was conducted in 2013 and 2017. Sustained indoor residual spraying of insecticide (IRS) was initiated in December 2014. Generalized linear mixed-effects models were used to compare the incidence of antimalarial and antibiotic treatments before and after vector control measures were implemented. Results Comparing the period prior to the implementation of IRS to the period after IRS had been sustained for 4–5 years, the adjusted incidence of malaria treatments decreased from 2.68 to 0.05 per person-year (incidence rate ratio [IRR] = 0.02, 95% CI 0.01–0.03, p < 0.001), and the adjusted incidence of antibiotic treatments decreased from 4.14 to 1.26 per person-year (IRR = 0.30, 95% CI 0.27–0.34, p < 0.001). The reduction in antibiotic usage was primarily associated with fewer episodes of symptomatic malaria and fewer episodes of fever with sub-microscopic parasitemia, both of which were frequently treated with antibiotics. Conclusions In a historically high transmission setting, the implementation of highly effective vector control interventions was followed by a marked reduction in antibiotic treatment of children. This added benefit of malaria control could have important implications for antibiotic prescribing practices, efforts to curtail antimicrobial resistance, and health system costs.

Abstract Background Environmental factors such as temperature, rainfall, and vegetation cover play a critical role in malaria transmission. However, quantifying the relationships between environmental factors and measures of disease burden relevant for public health can be complex as effects are often non-linear and subject to temporal lags between when changes in environmental factors lead to changes in malaria incidence. The study investigated the effect of environmental covariates on malaria incidence in high transmission settings of Uganda. Methods This study leveraged data from seven malaria reference centres (MRCs) located in high transmission settings of Uganda over a 24-month period. Estimates of monthly malaria incidence (MI) were derived from MRCs’ catchment areas. Environmental data including monthly temperature, rainfall, and normalized difference vegetation index (NDVI) were obtained from remote sensing sources. A distributed lag nonlinear model was used to investigate the effect of environmental covariates on malaria incidence. Results Overall, the median (range) monthly temperature was 30 °C (26–47), rainfall 133.0 mm (3.0–247), NDVI 0.66 (0.24–0.80) and MI was 790 per 1000 person-years (73–3973). Temperature of 35 °C was significantly associated with malaria incidence compared to the median observed temperature (30 °C) at month lag 2 (IRR: 2.00, 95% CI: 1.42–2.83) and the increased cumulative IRR of malaria at month lags 1–4, with the highest cumulative IRR of 8.16 (95% CI: 3.41–20.26) at lag-month 4. Rainfall of 200 mm significantly increased IRR of malaria compared to the median observed rainfall (133 mm) at lag-month 0 (IRR: 1.24, 95% CI: 1.01–1.52) and the increased cumulative IRR of malaria at month lags 1–4, with the highest cumulative IRR of 1.99(95% CI: 1.22–2.27) at lag-month 4. Average NVDI of 0.72 significantly increased the cumulative IRR of malaria compared to the median observed NDVI (0.66) at month lags 2–4, with the highest cumulative IRR of 1.57(95% CI: 1.09–2.25) at lag-month 4. Conclusions In high-malaria transmission settings, high values of environmental covariates were associated with increased cumulative IRR of malaria, with IRR peaks at variable lag times. The complex associations identified are valuable for designing strategies for early warning, prevention, and control of seasonal malaria surges and epidemics.

Abstract Background Although microscopy remains the gold standard for malaria diagnosis, little is known about its accuracy in the private health facilities in Uganda. This study evaluated the accuracy of malaria microscopy, and factors associated with inaccurate smear results at private health facilities in Entebbe Municipality, Uganda. Methods Between April and May 2018, all patients referred for a malaria smear in 16 private health facilities in Entebbe municipality were screened, and 321 patients were enrolled. A questionnaire was administered to collect demographic and clinical information, facility-based smear results were recorded from the participant’s consultation notes, and a research slide was obtained for expert microscopy during exit interview. A health facility assessment was conducted, and information on experience in performing malaria microscopy was collected from all facility personnel reading smears and the data was linked to the participant’s clinic visit. Results The test positivity rate of malaria parasitaemia was 15.0% by expert microscopy. The sensitivity, specificity and negative predictive value of the facility-based microscopy were high (95.8%, 90.1 and 99.2%, respectively). However; the positive predictive value (PPV) was low with 27/73 (63%) patients diagnosed with malaria not having the disease. Majority of the inaccurate results were from 2 of the 23 laboratory personnel reading the smears. The factors associated with inaccurate smear readings included being read by a technician; (1) who had less than 5 years’ experience in reading malaria smears (adjusted Odds Ratio [aOR] = 9.74, 95% confidence interval [CI] (1.06–89.5), p-value = 0.04), and (2) who was examining less than 5 smears a day (aOR = 38.8, 95% CI 9.65–156, p-value

Abstract Background Travel is a well-recognized risk factor for malaria. Within sub-Saharan Africa, travellers from areas of lower to higher transmission intensity are potentially at high risk of malaria. Long-lasting insecticidal nets (LLINs) are the primary tool for prevention of malaria, and their widespread use has contributed to substantial reductions in malaria burden. However, travellers often fail to use LLINs. To further explore the challenges and opportunities of using LLINs, travellers were interviewed in Uganda. Methods In August and September 2019, 20 participants attending outpatient clinics at Naguru General Hospital in Kampala with a history of travel out of Kampala within the previous 60 days were purposively selected. Data were collected through in-depth interviews and analysed thematically using NVivo 12. Results Of the 20 participants, 13 were male. Thirteen of the 20 participants tested positive for malaria by microscopy, and 5 reported using of LLINs during travel. The main reasons for travel were to attend social events (weddings, funerals, overnight prayers) and for work. travellers who attended social events reported using LLINs less commonly than those who travelled for work. Challenges to using LLINs during travel included: (1) limited access to LLINs; (2) challenges in planning ahead of travel; (3) lack of space or ability to hang LLINs while travelling; (4) impression that LLINs in lodging places were unhygienic; (5) cultural beliefs discouraging use of LLINs during social events; (6) participation in overnight ceremonies; and (7) doubts about efficacy of LLINs. Positive factors influencing use of LLINs during travel included knowledge regarding malaria prevention and good affordability and availability of LLINs. Conclusions Despite good traveller knowledge regarding malaria control measures, use of LLINs was limited. Use of LLINs in the prevention of malaria among travellers from low to high transmission settings needs to be prioritized. This calls for increased behaviour change oriented communication to improve traveller preparedness and consideration of use of repellents in situations where LLINs may not be feasible. The Uganda Ministry of Health and Malaria Control Division should use educational messages to increase awareness about the risks of getting malaria during overnight travel through the media. Truck drivers should be sensitized through their companies to use the available space at the back of the trucks for hanging nets and consider using pop-up nets.

Abstract Background Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. Methods A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2–10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher’s exact test as appropriate at P ≤ 0.05. Results Overall, 75.5% (95% CI 61.1–85.8) and 24.5% (95% CI14.2–38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8–89.1), P

Indoor residual spraying (IRS) of insecticide is one of the primary malaria vector control initiatives, but implementation is limited. Here, the authors show that discontinuation of IRS in Uganda was associated with increased malaria incidence, and introduction of IRS was associated with decreased incidence.

Abstract Background Uganda's clinical management guidelines recommend a malaria laboratory test in all patients presenting with fever (history of fever or an axillary temperature ≥ 37.5 °C), and only those with a positive test receive anti-malarial treatment. However, the current practice in areas with declining malaria transmission remains unknown. This study assessed the clinicians’ diagnostic practices, the factors associated with recommending a test, and the risk of missing a malaria case when a test is not recommended in patients presenting with fever in Kampala, an area of declining malaria transmission in Uganda. Methods Between January and March 2020, 383 participants aged ≥ 12 years and presenting to Kisenyi Health Centre IV in Kampala district with fever were enrolled in the study. A questionnaire was administered during exit interviews, routine diagnostic practices were recorded from participant clinical notes, and a research blood slide was obtained for later reading. Results Of the enrolled participants, 356 (93%) had a malaria diagnostic test recommended by the clinician. Factors associated with increasing prevalence of having a test recommended included; history of overnight travel (adjusted prevalence ratio [aPR] 1.07, 95% confidence interval [CI] 1.02–1.13, p = 0.011), being married (aPR = 1.07, 95% CI 1.01–1.13, p = 0.022), and having tertiary education (aPR = 1.09 95% CI 1.01–1.17, p = 0.031). Among the 27 participants where a malaria diagnostic test was not recommended, 4 (14.8%) had a positive study smear. Conclusion Despite having significant declines in malaria transmission in Kampala in the last decade, clinicians at the study health facility highly adhered to the clinical management guidelines, recommending a malaria test in almost all patients presenting with fever. However, a significant proportion of malaria cases was missed when a test was not recommended. These results highlight the importance of laboratory testing for malaria in all patients who present with fevers and live in endemic settings even when the transmission has significantly declined.

Abstract Background Evaluation of genetic relatedness of malaria parasites is a useful tool for understanding transmission patterns, but patterns are not easily detectable in areas with moderate to high malaria transmission. To evaluate the feasibility of detecting genetic relatedness in a moderate malaria transmission setting, relatedness of Plasmodium falciparum infections was measured in cohort participants from randomly selected households in the Kihihi sub-county of Uganda (annual entomological inoculation rate of 27 infectious bites per person). Methods All infections detected via microscopy or Plasmodium-specific loop mediated isothermal amplification from passive and active case detection during August 2011-March 2012 were genotyped at 26 microsatellite loci, providing data for 349 samples from 230 participants living in 80 households. Pairwise genetic relatedness was calculated using identity by state (IBS). Results As expected, genetic diversity was high (mean heterozygosity [He] = 0.73), and the majority (76.5 %) of samples were polyclonal. Despite the high genetic diversity, fine-scale population structure was detectable, with significant spatiotemporal clustering of highly related infections. Although the difference in malaria incidence between households at higher (mean 1127 metres) versus lower elevation (mean 1015 metres) was modest (1.4 malaria cases per person-year vs. 1.9 per person-year, respectively), there was a significant difference in multiplicity of infection (2.2 vs. 2.6, p = 0.008) and, more strikingly, a higher proportion of highly related infections within households (6.3 % vs. 0.9 %, p = 0.0005) at higher elevation compared to lower elevation. Conclusions Genetic data from a relatively small number of diverse, multiallelic loci reflected fine scale patterns of malaria transmission. Given the increasing interest in applying genetic data to augment malaria surveillance, this study provides evidence that genetic data can be used to inform transmission patterns at local spatial scales even in moderate transmission areas.

Abstract Background Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. Methods High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results The prevalence of KIR3DS1, KIR2DL5, KIR2DS5, and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6 vs 13.2%: p = 0.006, 57.2 vs 66.4%: p = 0.005, 33.2 vs 46.6%: p

Abstract Background Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. Methods Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. Results Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

Abstract Background Malaria surveillance is critical for monitoring changes in malaria morbidity over time. National Malaria Control Programmes often rely on surrogate measures of malaria incidence, including the test positivity rate (TPR) and total laboratory confirmed cases of malaria (TCM), to monitor trends in malaria morbidity. However, there are limited data on the accuracy of TPR and TCM for predicting temporal changes in malaria incidence, especially in high burden settings. Methods This study leveraged data from 5 malaria reference centres (MRCs) located in high burden settings over a 15-month period from November 2018 through January 2020 as part of an enhanced health facility-based surveillance system established in Uganda. Individual level data were collected from all outpatients including demographics, laboratory test results, and village of residence. Estimates of malaria incidence were derived from catchment areas around the MRCs. Temporal relationships between monthly aggregate measures of TPR and TCM relative to estimates of malaria incidence were examined using linear and exponential regression models. Results A total of 149,739 outpatient visits to the 5 MRCs were recorded. Overall, malaria was suspected in 73.4% of visits, 99.1% of patients with suspected malaria received a diagnostic test, and 69.7% of those tested for malaria were positive. Temporal correlations between monthly measures of TPR and malaria incidence using linear and exponential regression models were relatively poor, with small changes in TPR frequently associated with large changes in malaria incidence. Linear regression models of temporal changes in TCM provided the most parsimonious and accurate predictor of changes in malaria incidence, with adjusted R2 values ranging from 0.81 to 0.98 across the 5 MRCs. However, the slope of the regression lines indicating the change in malaria incidence per unit change in TCM varied from 0.57 to 2.13 across the 5 MRCs, and when combining data across all 5 sites, the R2 value reduced to 0.38. Conclusions In high malaria burden areas of Uganda, site-specific temporal changes in TCM had a strong linear relationship with malaria incidence and were a more useful metric than TPR. However, caution should be taken when comparing changes in TCM across sites.

Abstract Background Accurate measures of malaria incidence are essential to track progress and target high-risk populations. While health management information system (HMIS) data provide counts of malaria cases, quantifying the denominator for incidence using these data is challenging because catchment areas and care-seeking behaviours are not well defined. This study’s aim was to estimate malaria incidence using HMIS data by adjusting the population denominator accounting for travel time to the health facility. Methods Outpatient data from two public health facilities in Uganda (Kihihi and Nagongera) over a 3-year period (2011–2014) were used to model the relationship between travel time from patient village of residence (available for each individual) to the facility and the relative probability of attendance using Poisson generalized additive models. Outputs from the model were used to generate a weighted population denominator for each health facility and estimate malaria incidence. Among children aged 6 months to 11 years, monthly HMIS-derived incidence estimates, with and without population denominators weighted by probability of attendance, were compared with gold standard measures of malaria incidence measured in prospective cohorts. Results A total of 48,898 outpatient visits were recorded across the two sites over the study period. HMIS incidence correlated with cohort incidence over time at both study sites (correlation in Kihihi = 0.64, p

Abstract Background The burden of malaria in Uganda remains high, but has become increasingly heterogenous following intensified malaria control. Travel within Uganda is recognized as a risk factor for malaria, but behaviours associated with travel are not well-understood. To address this knowledge gap, malaria-relevant behaviours of cohort participants were assessed during travel and at home in Uganda. Methods Residents from 80 randomly selected households in Nagongera sub-county, Tororo district were enrolled into a cohort to study malaria in rural Uganda. All participants were given long-lasting insecticidal nets (LLINs) at enrolment and were evaluated every 4 weeks at the study clinic. Participants were asked if they had travelled overnight from their home, and if so, a questionnaire was administered to capture information on travel details and behaviours. Behaviour while travelling was assessed within 4 weeks following travel during the study clinic visit. Behaviour while at home was assessed using a similar questionnaire during two-weekly home visits. Behaviours while travelling vs at home were compared using log binomial regression models with generalized estimating equations adjusting for repeated measures in the same individual. Analysis of factors associated with LLIN adherence, such as destination and duration of travel, time to bed during travel, gender and age at time of travel, were assessed using log binomial regression models with generalized estimating equations adjusting for repeated measures in the same individual. Results Between October 2017 and October 2019, 527 participants were enrolled and assessed for travel. Of these, 123 (23.2%) reported taking 211 overnight trips; 149 (70.6%) trips were within Tororo. Participants were less likely to use LLINs when travelling than when at home (41.0% vs. 56.2%, relative risk [RR] 0.73, 95% CI 0.60–0.89, p = 0.002); this difference was noted for women (38.8% vs 59.2%, RR 0.66, 95% CI 0.52–0.83, p = 0.001) but not men (48.3% vs 46.6%, RR 0.96, 95% CI 0.67–1.40, p = 0.85). In an adjusted analysis, factors associated with LLIN use when travelling included destination (travelling to districts not receiving indoor residual spraying [IRS] 65.8% vs Tororo district 32.2%, RR 1.80, 95% CI 1.31–2.46, p 7 nights 60.3% vs one night 24.4%, RR 1.97, 95% CI 1.07–3.64, p = 0.03). Conclusions Travellers, particularly women, were less likely to use LLINs when travelling than when at home. LLIN adherence was higher among those who travelled to non-IRS districts and for more than 1 week, suggesting that perceived malaria risk influences LLIN use. Strategies are needed to raise awareness of the importance of using LLINs while travelling.

Abstract Background Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. Methods Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. Results Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6–13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4–5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7–20.0%) compared to the western region 3.1% (95% CI 0.8–7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02–23.55), p = 0.003. Conclusions This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

Abstract Background Over the last two decades, there has been remarkable progress in malaria control in sub-Saharan Africa, due mainly to the massive deployment of long-lasting insecticidal nets and indoor residual spraying. Despite these gains, it is clear that in many situations, additional interventions are needed to further reduce malaria transmission. The World Health Organization (WHO) has promoted the Integrated Vector Management (IVM) approach through its Global Vector Control Response 2017–2030. However, prior roll-out of larval source management (LSM) as part of IVM, knowledge on ecology of larval aquatic habitats is required. Methods Aquatic habitats colonized by immature Anopheles and culicines vectors were characterized at three sites of low, medium and high malaria transmission in Uganda from October 2011 to June 2015. Larval surveys were conducted along transects in each site and aquatic habitats described according to type and size. Immature Anopheles, culicines and pupae from the described habitats were sampled using standard dipping methods to determine larval and pupae densities. Larvae were identified as anopheline or culicine, and counted. Pupae were not identified further. Binary logistic regression analysis was used to identify factors associated with the presence of immature Anopheles and culicines in each site. Results A total of 1205 larval aquatic habitats were surveyed and yielded a total of 17,028 anopheline larvae, 26,958 culicine larvae and 1189 pupae. Peaks in larval abundance occurred in all sites in March–May and August-October coinciding with the rainy seasons. Anopheles larvae were found in 52.4% (n = 251) of aquatic habitats in Tororo, a site of high transmission, 41.9% (n = 536) of habitats in Kanungu, a site with moderate malaria transmission, and 15.8% (n = 418) in Jinja, a site with low malaria transmission. The odds of finding larvae was highest in rice fields compared to pools in both Tororo (odds ratio, OR = 4.21, 95% CI 1.22–14.56, p = 0.02) and Kanungu (OR = 2.14, 95% CI 1.12–4.07, p = 0.02), while in Jinja the odd were highest in containers (OR = 4.55, 95% CI = 1.09–19.14, p = 0.03). In Kanungu, larvae were less likely to be found in containers compared to pools (OR = 0.26, 95% CI 0.09–0.66, p = 0.008) and river fringe (OR = 0.19, 95% CI 0.07–0.52, p = 0.001). Medium sized habitats were associated with high odds of finding larvae compared to small habitats (OR = 3.59, 95% CI 1.18–14.19, p = 0.039). Conclusions These findings show that immature Anopheles and culicines were common in areas of high and moderate transmission but were rare in areas of low transmission. Although immature Anopheles and culicines were found in all types of water bodies, they were most common in rice fields and less common in open drains and in river fringes. Methods are needed to reduce the aquatic stages of anopheline mosquitoes in human-made habitats, particularly rice fields.

Abstract Background Malaria control using long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) has been associated with reduced transmission throughout Africa. However, the impact of transmission reduction on the age distribution of malaria cases remains unclear. Methods Over a 10-year period (January 2009 to July 2018), outpatient surveillance data from four health facilities in Uganda were used to estimate the impact of control interventions on temporal changes in the age distribution of malaria cases using multinomial regression. Interventions included mass distribution of LLINs at all sites and IRS at two sites. Results Overall, 896,550 patient visits were included in the study; 211,632 aged 15 years. Over time, the age distribution of patients not suspected of malaria and those malaria negative either declined or remained the same across all sites. In contrast, the age distribution of suspected and confirmed malaria cases increased across all four sites. In the two LLINs-only sites, the proportion of malaria cases in 15 years increased from 40 to 61% and 29 to 39%, respectively. In the sites receiving LLINs plus IRS, these proportions increased from 19 to 44% and 18 to 31%, respectively. Conclusions These findings demonstrate a shift in the burden of malaria from younger to older individuals following implementation of successful control interventions, which has important implications for malaria prevention, surveillance, case management and control strategies.

Abstract Background Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear. Methods Data from a longitudinal cohort study conducted in 3 sub-counties in Uganda was used to quantify associations between three red blood cell variants Hb [AA, AS, S (rs334)], alpha thalassaemia 3.7 kb deletion, and glucose-6-phosphate dehydrogenase deficiency A—(G6PD 202A genotype) and malaria incidence, parasite prevalence, parasite density (a measure of anti-parasite immunity) and body temperature adjusted for parasite density (a measure of anti-disease immunity). All analyses were adjusted for age, average household entomological inoculation rate, and study site. Results for all variants were compared to those for wild type genotypes. Results In children, HbAS was associated, compared to wild type, with a lower incidence of malaria (IRR = 0.78, 95% CI 0.66–0.92, p = 0.003), lower parasite density upon infection (PR = 0.66, 95% CI 0.51–0.85, p = 0.001), and lower body temperature for any given parasite density (− 0.13 ℃, 95% CI − 0.21, − 0.05, p = 0.002). In children, HbSS was associated with a lower incidence of malaria (IRR = 0.17, 95% CI 0.04–0.71, p = 0.02) and lower parasite density upon infection (PR = 0.31, 95% CI 0.18–0.54, p

Five years of sustained indoor residual spraying (IRS) of insecticide from 2014 to 2019, first using a carbamate followed by an organophosphate, was associated with a marked reduction in the incidence of malaria in five districts of Uganda. We assessed changes in malaria incidence over an additional 21 months, corresponding to a change in IRS formulations using clothianidin with and without deltamethrin. Using enhanced health facility surveillance data, our objectives were to 1) estimate the impact of IRS on monthly malaria case counts at five surveillance sites over a 6.75 year period, and 2) compare monthly case counts at five facilities receiving IRS to ten facilities in neighboring districts not receiving IRS. For both objectives, we specified mixed effects negative binomial regression models with random intercepts for surveillance site adjusting for rainfall, season, care-seeking, and malaria diagnostic. Following the implementation of IRS, cases were 84% lower in years 4–5 (adjusted incidence rate ratio [aIRR] = 0.16, 95% CI 0.12–0.22), 43% lower in year 6 (aIRR = 0.57, 95% CI 0.44–0.74), and 39% higher in the first 9 months of year 7 (aIRR = 1.39, 95% CI 0.97–1.97) compared to pre-IRS levels. Cases were 67% lower in IRS sites than non-IRS sites in year 6 (aIRR = 0.33, 95% CI 0.17–0.63) but 38% higher in the first 9 months of year 7 (aIRR = 1.38, 95% CI 0.90–2.11). We observed a resurgence in malaria to pre-IRS levels despite sustained IRS. The timing of this resurgence corresponded to a change of active ingredient. Further research is needed to determine causality.

House construction is rapidly modernizing across Africa but the potential benefits for human health are poorly understood. We hypothesised that improvements to housing would be associated with reductions in malaria, acute respiratory infection (ARI) and gastrointestinal illness in an area of low malaria endemicity in Uganda. Data were analysed from a cohort study of male and female child and adult residents (n = 531) of 80 randomly-selected households in Nagongera sub-county, followed for 24 months (October 4, 2017 to October 31, 2019). Houses were classified as modern (brick walls, metal roof and closed eaves) or traditional (all other homes). Light trap collections of mosquitoes were done every two weeks in all sleeping rooms. Every four weeks, we measured malaria infection (using microscopy and qPCR to detect malaria parasites), incidence of malaria, ARI and gastrointestinal illness. We collected 15,780 adult female Anopheles over 7,631 nights. We collected 13,277 blood samples of which 10.2% (1,347) were positive for malaria parasites. Over 958 person years we diagnosed 38 episodes of uncomplicated malaria (incidence 0.04 episodes per person-year at risk), 2,553 episodes of ARI (incidence 2.7 episodes per person-year) and 387 episodes of gastrointestinal illness (incidence 0.4 episodes per person-year). Modern houses were associated with a 53% lower human biting rate compared to traditional houses (adjusted incidence rate ratio [aIRR] 0.47, 95% confidence interval [CI] 0.32–0.67, p

Background Perinatal mortality remains high globally and remains an important indicator of the quality of a health care system. To reduce this mortality, it is important to provide the recommended care during the perinatal period. We assessed the prevalence and factors associated with appropriate perinatal care (antenatal, intrapartum, and postpartum) in Bunyoro region, Uganda. Results from this study provide valuable information on the perinatal care services and highlight areas of improvement for better perinatal outcomes. Methods A cross sectional survey was conducted among postpartum mothers attending care at three district hospitals in Bunyoro. Following consent, a questionnaire was administered to capture the participants’ demographics and data on care received was extracted from their antenatal, labour, delivery, and postpartum records using a pre-tested structured tool. The care received by women was assessed against the standard protocol established by World Health Organization (WHO). Poisson regression with robust standard errors was used to assess factors associated with appropriate postpartum care. Results A total of 872 mothers receiving care at the participating hospitals between March and June 2020 were enrolled in the study. The mean age of the mothers was 25 years (SD = 5.95). None of the mothers received appropriate antenatal or intrapartum care, and only 3.8% of the participants received appropriate postpartum care. Factors significantly associated with appropriate postpartum care included mothers being >35 years of age (adjusted prevalence ratio [aPR] = 11.9, 95% confidence interval [CI] 2.8–51.4) and parity, with low parity (2–3) and multiparous (>3) mothers less likely to receive appropriate care than prime gravidas (aPR = 0.3, 95% CI 0.1–0.9 and aPR = 0.3, 95% CI 0.1–0.8 respectively). Conclusions Antenatal, intrapartum, and postpartum care received by mothers in this region remains below the standard recommended by WHO, and innovative strategies across the continuum of perinatal care need to be devised to prevent mortality among the mothers. The quality of care also needs to be balanced for all mothers irrespective of the age and parity.

Abstract Background Long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are widely recommended for the prevention of malaria in endemic regions. Data from human landing catches provide information on the impact of vector control on vector populations. Here, malaria transmission indoors and outdoors, before and after mass deployment of LLINs and IRS in Uganda was compared. Methods The study took place in Tororo district, a historically high transmission area where universal LLIN distribution was conducted in November 2013 and May 2017 and 6 rounds of IRS implemented from December 2014 to July 2018. Human landing catches were performed in 8 houses monthly from October 2011 to September 2012 (pre-intervention period) and every 4 weeks from November 2017 to October 2018 (post-intervention period). Mosquitoes were collected outdoors from 18:00 to 22:00 h and indoors from 18:00 to 06:00 h. Female Anopheles were tested for the presence of Plasmodium falciparum sporozoites and species identification performed using gross dissection and polymerase chain reaction (PCR). Results The interventions were associated with a decline in human biting rate from 19.6 to 2.3 female Anopheles mosquitoes per house per night (p

Abstract Background Loss to follow-up (LTFU) from care among people living with HIV (PLHIV) is thought to be more common in the public setting compared to the private health care. It is anticipated that the problem may become worse with the current “test and treat” policy in Uganda due to the likely increases in patient loads and its attendant pressure on health care providers to support patient counseling. This study determined the incidence and factors associated with LTFU from HIV care among adult PLHIV in public health facilities in Wakiso district, Uganda. Methods This was a retrospective cohort study that involved the review of 646 records of patients initiated on antiretroviral therapy (ART) between January 1st, 2015 and December 31st, 2017 at 13 randomly selected public health facilities in Wakiso district. The cox proportional hazards regression was used to determine the factors associated with LTFU. The results were supported by sequential in-depth and key informant interviews to explore reasons for LTFU. Results Of the 646 patients enrolled, 391 were female (60.5%), 282 were below 30 years (43.6%) and 207 were married (50.1%). A total of 216 patients (33.4%) had no documented outcomes and were considered LTFU. The incidence of LTFU was 21 per 1000 person months (95% confidence interval (CI): 18–25 per 1000 person months). Factors associated with LTFU included having normal weight compared to underweight (adjusted hazard ratio (aHR) 0.64, 95% CI: 0.45–0.90, p = 0.011), receiving HIV care from hospitals compared to lower level facilities (aHR 0.22, 95% CI: 0.12–0.41, p

Abstract Background The human infectious reservoir for malaria consists of individuals capable of infecting mosquitoes. Oocyst prevalence and density are typical indicators of human infectivity to mosquitoes. However, identification of oocysts is challenging, particularly in areas of low malaria transmission intensity where few individuals may infect mosquitoes, and infected mosquitoes tend to have few oocysts. Here, features that differentiate oocysts from other oocyst-like in mosquito midguts are explained and illustrated. In addition, the establishment and maintenance of infrastructure to perform malaria transmission experiments is described. This work may support other initiatives to set up membrane feeding infrastructure and guide oocyst detection in low transmission settings. Methods In 2014, an insectary was developed and equipped in Tororo district, Uganda. A colony of Anopheles gambiae s.s. mosquitoes (Kisumu strain) was initiated to support infectivity experiments from participants enrolled in a large cohort study. Venous blood drawn from participants who were naturally infected with malaria parasites was used for membrane feeding assays, using 60–80 mosquitoes per experiment. Approximately 9–10 days after feeding, mosquitoes were dissected, and midguts were stained in mercurochrome and examined by light microscopy for Plasmodium falciparum oocysts and similar structures. In supportive experiments, different staining procedures were compared using in vitro cultured parasites. Results A stable colony of the Kisumu strain of An. gambiae s.s. was achieved, producing 5000–10,000 adult mosquitoes on a weekly basis. Challenges due to temperature fluctuations, mosquito pathogens and pests were successfully overcome. Oocysts were characterized by: presence of malaria pigment, clearly defined edge, round shape within the mosquito midgut or on the peripheral tissue and always attached to the epithelium. The main distinguishing feature between artifacts and mature oocysts was the presence of defined pigment within the oocysts. Conclusions Oocysts may be mistaken for other structures in mosquito midguts. Distinguishing real oocysts from oocyst-like structures may be challenging for inexperienced microscopists due to overlapping features. The characteristics and guidelines outlined here support identification of oocysts and reliable detection at low oocyst densities. Practical advice on sustaining a healthy mosquito colony for feeding experiments is provided. Following the reported optimization, the established infrastructure in Tororo allows assessments of infectivity of naturally infected parasite carriers.

Understanding how immunity to malaria is affected by declining transmission is important to aid vaccine design and understand disease resurgence. Both IgG subclasses and avidity of antigen-specific responses are important components of an effective immune response. Using a multiplex bead array assay, we measured the total IgG, IgG subclasses, and avidity profiles of responses to 18 P. falciparum blood stage antigens in samples from 160 Ugandans collected at two time points during high malaria transmission and two time points following a dramatic reduction in transmission. Results demonstrated that, for the antigens tested, (i) the rate of decay of total IgG following infection declined with age and was driven consistently by the decrease in IgG3 and occasionally the decrease in IgG1; (ii) the proportion of IgG3 relative to IgG1 in the absence of infection increased with age; (iii) the increase in avidity index (the strength of association between the antibody and antigen) following infection was largely due to a rapid loss of non-avid compared to avid total IgG; and (iv) both avid and non-avid total IgG in the absence of infection increased with age. Further studies are required to understand the functional differences between IgG1 and IgG3 in order to determine their contribution to the longevity of protective immunity to malaria. Measuring changes in antibody avidity may be a better approach of detecting affinity maturation compared to avidity index due to the differential expansion and contraction of high and low avidity total IgG.

Introduction Tuberculosis (TB) mortality estimates derived only from cohorts of patients initiated on TB treatment do not consider outcomes of patients with pretreatment loss to follow-up (LFU). We aimed to assess the effect of pretreatment LFU on TB-associated mortality in the six months following TB diagnosis at public health facilities in Uganda. Methods At ten public health facilities, we retrospectively reviewed treatment data for all patients with a positive Xpert®MTB/RIF test result from January to June 2018. Pretreatment LFU was defined as not initiating TB treatment within two weeks of a positive test. We traced patients with pretreatment LFU to ascertain their vital status. We performed Kaplan Meier survival analysis to compare the cumulative incidence of mortality, six months after diagnosis among patients who did and did not experience pretreatment LFU. We also determined the health facility level estimates of TB associated mortality before and after incorporating deaths prior to treatment initiation among patients who experienced pretreatment LFU. Results Of 510 patients with positive test, 100 (19.6%) experienced pretreatment LFU. Of these, we ascertained the vital status of 49 patients. In the six months following TB diagnosis, mortality was higher among patients who experienced pretreatment LFU 48.1/1000py vs 22.9/1000py. Hazard ratio [HR] 3.18, 95% confidence interval [CI] (1.61–6.30). After incorporating deaths prior to treatment initation among patients who experienced pretreatment LFU, health facility level estimates of TB associated mortality increased from 8.4% (95% CI 6.1%-11.6%) to 10.2% (95% CI 7.7%-13.4%). Conclusion Patients with confirmed TB who experience pretreatment LFU have high mortality within the first six months. Efforts should be made to prioritise linkage to treatment for this group of patients. Deaths that occur prior to treatment initation should be included when reporting TB mortality in order to more accurately reflect the health impact of TB.

Abstract Background Submicroscopic malaria parasitaemia is common in both high- and low-endemicity settings, but its clinical consequences are unclear. Methods A cohort of 364 children (0.5–10 years of age) and 106 adults was followed from 2011 to 2016 in Tororo District, Uganda using passive surveillance for malaria episodes and active surveillance for parasitaemia. Participants presented every 90 days for routine visits (n = 9075); a subset was followed every 30 days. Participants who presented with fever and a positive blood smear were treated for malaria. At all routine visits microscopy was performed and samples from subjects with a negative blood smear underwent loop-mediated isothermal amplification for detection of plasmodial DNA. Results Submicroscopic parasitaemia was common; the proportion of visits with submicroscopic parasitemia was 25.8% in children and 39.2% in adults. For children 0.5–10 years of age, but not adults, having microscopic and submicroscopic parasitaemia at routine visits was significantly associated with both fever (adjusted risk ratios [95% CI], 2.64 [2.16–3.22], 1.67 [1.37–2.03]) and non-febrile illness (aRR [CI], 1.52 [1.30–1.78], 1.26 [1.09–1.47]), compared to not having parasitaemia. After stratifying by age, significant associations were seen between submicroscopic parasitaemia and fever in children aged 2–

Abstract Background Chronic kidney disease (CKD) is a major cause of morbidity and mortality in Sub-Saharan Africa (SSA). The majority of studies on CKD in SSA have been conducted among HIV-infected populations and mainly from large health facilities. We determined the prevalence of CKD and its predictors among populations in communities in central Uganda. Methods A cross-sectional study was conducted in Wakiso district using multi-stage sampling. Data was collected on age, sex, socio-economic status, history of alcohol intake, diabetes mellitus, hypertension and smoking. Measurement of blood pressure, weight and height to determine body mass index (BMI) and investigations including HIV testing, fasting blood sugar, creatinine and urinalysis were conducted. Logistic regression was used to estimate the strength of the association between variables and the presence of CKD estimated using the Cockcroft Gault formula. Results A total of 955 participants aged 18–87 years were enrolled into the study. The median age was 31 years (Interquartile range 24–42) and majority (67%) were female. Up to 21.4% (204/955) had abnormal renal function with CKD stage 1 in 6.2% (59/955), stage 2 in 12.7% (121/955), stage 3 in 2.4% (23/955), CKD stage 4 in 0% and CKD stage 5 in 0.1% (1/995). Female gender OR 1.8 (95% Confidence Interval [CI] 1.2–2.8), age >30 years OR 2.2(95% CI 1.2–3.8) and high social economic status OR 2.1 (95% CI 1.3–3.6) were associated with increased risk of CKD while BMI > 25Kg/m2 was protective against CKD OR 0.1 (95% CI 0.04–0.2). Traditional risk factors such as HIV-infection, diabetes mellitus, smoking and alcohol intake were not found to be significantly associated with CKD. Conclusion We found a high prevalence of kidney disease in central Uganda. Interestingly the traditional risk factors associated with CKD previously documented, were not associated with CKD.

Abstract Background The World Health Organization (WHO) recommends injectable artesunate given either intravenously or by the intramuscular route for definitive treatment for severe malaria and recommends a single intramuscular dose of intramuscular artesunate or intramuscular artemether or intramuscular quinine, in that order of preference as pre-referral treatment when definitive treatment is not possible. Where intramuscular injections are not available, children under 6 years may be administered a single dose of rectal artesunate. Although the current malaria treatment guidelines in Ethiopia recommend intra-rectal artesunate or alternatively intramuscular artemether or intramuscular quinine as pre-referral treatment for severe malaria at the health posts, there are currently no WHO prequalified suppliers of intra-rectal artesunate and when available, its use is limited to children under 6 years of age leaving a gap for the older age groups. Intramuscular artesunate is not part of the drugs recommended for pre-referral treatment in Ethiopia. This study assessed the perspectives of health workers, and policy-makers on the use of intramuscular artesunate as a pre-referral and definitive treatment for severe malaria at the health post level. Methods In-depth interviews were held with 101 individuals including health workers, malaria focal persons, and Regional Health Bureaus from Oromia and southern nations, nationalities, and peoples’ region, as well as participants from the Federal Ministry of Health and development partners. An interview guide was used in the data collection and thematic content analysis was employed for analysis. Results Key findings from this study are: (1) provision of intramuscular artesunate as pre-referral and definitive treatment for severe malaria at health posts could be lifesaving; (2) with adequate training, and provision of facilities including beds, health posts can provide definitive treatment for severe malaria using intramuscular artesunate where referral is delayed or not possible; (3) health workers at health centres and hospitals frequently use the intravenous route because it allows for co-administration of other drugs, but they find the intramuscular route easier to use at the health post level; (4) the reasons commonly cited against the management of severe malaria using intramuscular artesunate at health post level were: lack of capacity to manage complications and fear of irrational drug use; (5) use of intramuscular artesunate at health post level will require evidence on safety and feasibility before policy shift. Conclusion From the perspective of health workers, use of intramuscular artesunate as pre-referral treatment of severe malaria cases at the health post is possible but dependent on training and availability of skilled workers. Use of intramuscular artesunate as definitive treatment at health posts was not supported, however, operational research to establish its feasibility, safety and efficacy was recommended to guide any implementation of such an intervention.