The East African Community mobile laboratory network prepares for monkeypox outbreaks.

In response to the largest recorded monkeypox virus outbreak outside of endemic Central and Western Africa, the East African Community (EAC), in cooperation with the Bernhard-Nocht- Institute for Tropical Medicine, coordinated an emergency monkeypox diagnostic training for the East African Region. As of June 2022, the Democratic Republic of Congo reported a steady increase of suspected monkeypox cases, increasing the risk of spill-over into the remaining six EAC Partner States. Within the existing EAC Mobile Laboratories project, laboratory experts of the National Public Health Laboratories of the remaining six EAC Partner States (Burundi, Rwanda, Tanzania, Kenya, Uganda, and South Sudan) participated in the workshop and were trained in the reception of suspect samples, DNA extraction and diagnosis using real-time polymerase chain reaction (RT-PCR). The EAC region is now equipped with the tools to prepare and rapidly respond to any emerging monkeypox outbreak.

Regional Evaluation of Two SARS-CoV-2 Antigen Rapid Diagnostic Tests in East Africa.

The clinical performance of two rapid antigen tests for the diagnosis of Severe Acute Respiratory Coronavirus (SARS-CoV-2) were regionally evaluated in East African populations. Swabs were collected from 1,432 individuals from five Partner States of the East African Community (Tanzania, Uganda, Burundi, Rwanda and South Sudan). The two rapid antigen tests (Bionote NowCheck COVID-19 Ag and SD Biosensor STANDARD Q COVID-19 Ag) were evaluated against the detection of SARS-CoV-2 RNA by the Reverse Transcription PCR (RT-PCR) gold standard. Of the concordant results with both RT-PCR and rapid antigen test data (862 for Bionote and 852 for SD Biosensor), overall clinical sensitivity was 60% and 50% for the Bionote NowCheck and the SD Biosensor STANDARD Q, respectively. Stratification by viral load, including samples with RT-PCR cycle thresholds (Ct) of <25, improved sensitivity to 90% for both rapid diagnostic tests (RDTs). Overall specificity was good at 99% for both antigen tests. Taken together, the clinical performance of both Ag-RDTs in real world settings within the East African target population was lower than has been reported elsewhere and below the acceptable levels for sensitivity of >80%, as defined by the WHO. Therefore, the rapid antigen test alone should not be used for diagnosis but could be used as part of an algorithm to identify potentially infectious individuals with high viral load. IMPORTANCE Accurate diagnostic tests are essential to both support the management and containment of outbreaks, as well as inform appropriate patient care. In the case of the SARS-CoV-2 pandemic, antigen Rapid Diagnostic Tests (Ag-RDTs) played a major role in this function, enabling widespread testing by untrained individuals, both at home and within health facilities. In East Africa, a number of SARS-CoV-2 Ag-RDTs are available; however, there remains little information on their true test performance within the region, in the hands of the health workers routinely carrying out SARS-CoV-2 diagnostics. This study contributes test performance data for two commonly used SARS-CoV-2 Ag-RDTs in East Africa, which will help inform the use of these RDTs within the region.

Molecular Characterization and Phylogenetic Analysis of Dengue Fever Viruses in Three Outbreaks in Tanzania Between 2017 and 2019.

BACKGROUND: Dengue is a disease of public health interest, and Tanzania experienced major outbreaks in 2014 and 2019. Here, we report our findings on the molecular characterization of dengue viruses (DENV) that circulated during two smaller outbreaks (2017 and 2018) and one major epidemic (2019) in Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: We tested archived serum samples from 1,381 suspected dengue fever patients, with a median age of 29 (IQR:22-40) years, referred to the National Public Health Laboratory for confirmation of DENV infection. DENV serotypes were identified by reverse transcription polymerase chain reaction (RT-PCR), and specific genotypes were identified by sequencing the envelope glycoprotein gene and phylogenetic inference methods. DENV was confirmed in 823 (59.6%) cases. More than half (54.7%) of patients with dengue fever infection were males, and nearly three-quarters (73%) of the infected individuals were living in Kinondoni district, Dar es Salaam. DENV-3 Genotype III caused the two smaller outbreaks in 2017 and 2018, while DENV-1 Genotype V caused the 2019 epidemic. DENV-1 Genotype I was also detected in one patient in 2019. CONCLUSION/SIGNIFICANCE: This study has demonstrated the molecular diversity of dengue viruses circulating in Tanzania. We found that contemporary circulating serotypes did not cause the major epidemic of 2019 but rather due to a serotype shift from DENV-3 (2017/2018) to DENV-1 in 2019. Such a change increases the risk for patients previously infected with a particular serotype to develop severe symptoms upon potential re-infection with a heterologous serotype due to antibody-dependent enhancement of infection. Therefore, the circulation of serotypes emphasizes the need to strengthen the country's dengue surveillance system for better management of patients, early detection of outbreaks, and vaccine development.

Asymptomatic Plasmodium falciparum carriage and clinical disease: a 5-year community-based longitudinal study in The Gambia.

BACKGROUND: Carriers of persistent asymptomatic Plasmodium falciparum infections constitute an infectious reservoir that maintains malaria transmission. Understanding the extent of carriage and characteristics of carriers specific to endemic areas could guide use of interventions to reduce infectious reservoir. METHODS: In eastern Gambia, an all-age cohort from four villages was followed up from 2012 to 2016. Each year, cross-sectional surveys were conducted at the end of the malaria transmission season (January) and just before the start of the next one (June) to determine asymptomatic P. falciparum carriage. Passive case detection was conducted during each transmission season (August to January) to determine incidence of clinical malaria. Association between carriage at the end of the season and at start of the next one and the risk factors for this were assessed. Effect of carriage before start of the season on risk of clinical malaria during the season was also examined. RESULTS: A total of 1403 individuals-1154 from a semi-urban village and 249 from three rural villages were enrolled; median age was 12 years (interquartile range [IQR] 6, 30) and 12 years (IQR 7, 27) respectively. In adjusted analysis, asymptomatic P. falciparum carriage at the end of a transmission season and carriage just before start of the next one were strongly associated (adjusted odds ratio [aOR] = 19.99; 95% CI 12.57-31.77, p < 0.001). The odds of persistent carriage (i.e. infected both in January and in June) were higher in rural villages (aOR = 13.0; 95% CI 6.33-26.88, p < 0.001) and in children aged 5-15 years (aOR = 5.03; 95% CI 2.47-10.23, p = < 0.001). In the rural villages, carriage before start of the season was associated with a lower risk of clinical malaria during the season (incidence risk ratio [IRR] 0.48, 95% CI 0.27-0.81, p = 0.007). CONCLUSIONS: Asymptomatic P. falciparum carriage at the end of a transmission season strongly predicted carriage just before start of the next one. Interventions that clear persistent asymptomatic infections when targeted at the subpopulation with high risk of carriage may reduce the infectious reservoir responsible for launching seasonal transmission.

Lessons Learned From a Large Cross-Border Field Simulation Exercise to Strengthen Emergency Preparedness in East Africa, 2019.

Field simulation exercises (FSXs) require substantial time, resources, and organizational experience to plan and implement and are less commonly undertaken than drills or tabletop exercises. Despite this, FSXs provide an opportunity to test the full scope of operational capacities, including coordination across sectors. From June 11 to 14, 2019, the East African Community Secretariat conducted a cross-border FSX at the Namanga One Stop Border Post between the Republic of Kenya and the United Republic of Tanzania. The World Health Organization Department of Health Security Preparedness was the technical lead responsible for developing and coordinating the exercise. The purpose of the FSX was to assess and further enhance multisectoral outbreak preparedness and response in the East Africa Region, using a One Health approach. Participants included staff from the transport, police and customs, public health, animal health, and food inspection sectors. This was the first FSX of this scale, magnitude, and complexity to be conducted in East Africa for the purpose of strengthening emergency preparedness capacities. The FSX provided an opportunity for individual learning and national capacity strengthening in emergency management and response coordination. In this article, we describe lessons learned and propose recommendations relevant to FSX design, management, and organization to inform future field exercises.

The East African Community (EAC) mobile laboratory networks in Kenya, Burundi, Tanzania, Rwanda, Uganda, and South Sudan-from project implementation to outbreak response against Dengue, Ebola, COVID-19, and epidemic-prone diseases.

BACKGROUND: East Africa is home to 170 million people and prone to frequent outbreaks of viral haemorrhagic fevers and various bacterial diseases. A major challenge is that epidemics mostly happen in remote areas, where infrastructure for Biosecurity Level (BSL) 3/4 laboratory capacity is not available. As samples have to be transported from the outbreak area to the National Public Health Laboratories (NPHL) in the capitals or even flown to international reference centres, diagnosis is significantly delayed and epidemics emerge. MAIN TEXT: The East African Community (EAC), an intergovernmental body of Burundi, Rwanda, Tanzania, Kenya, Uganda, and South Sudan, received 10 million € funding from the German Development Bank (KfW) to establish BSL3/4 capacity in the region. Between 2017 and 2020, the EAC in collaboration with the Bernhard-Nocht-Institute for Tropical Medicine (Germany) and the Partner Countries' Ministries of Health and their respective NPHLs, established a regional network of nine mobile BSL3/4 laboratories. These rapidly deployable laboratories allowed the region to reduce sample turn-around-time (from days to an average of 8h) at the centre of the outbreak and rapidly respond to epidemics. In the present article, the approach for implementing such a regional project is outlined and five major aspects (including recommendations) are described: (i) the overall project coordination activities through the EAC Secretariat and the Partner States, (ii) procurement of equipment, (iii) the established laboratory setup and diagnostic panels, (iv) regional training activities and capacity building of various stakeholders and (v) completed and ongoing field missions. The latter includes an EAC/WHO field simulation exercise that was conducted on the border between Tanzania and Kenya in June 2019, the support in molecular diagnosis during the Tanzanian Dengue outbreak in 2019, the participation in the Ugandan National Ebola response activities in Kisoro district along the Uganda/DRC border in Oct/Nov 2019 and the deployments of the laboratories to assist in SARS-CoV-2 diagnostics throughout the region since early 2020. CONCLUSIONS: The established EAC mobile laboratory network allows accurate and timely diagnosis of BSL3/4 pathogens in all East African countries, important for individual patient management and to effectively contain the spread of epidemic-prone diseases.

G6PD Polymorphisms and Hemolysis After Antimalarial Treatment With Low Single-Dose Primaquine: A Pooled Analysis of Six African Clinical Trials.

Primaquine (PQ) is an antimalarial drug with the potential to reduce malaria transmission due to its capacity to clear mature Plasmodium falciparum gametocytes in the human host. However, the large-scale roll-out of PQ has to be counterbalanced by the additional risk of drug-induced hemolysis in individuals suffering from Glucose-6-phospate dehydrogenase (G6PD) deficiency, a genetic condition determined by polymorphisms on the X-linked G6PD gene. Most studies on G6PD deficiency and PQ-associated hemolysis focused on the G6PD A- variant, a combination of the two single nucleotide changes G202A (rs1050828) and A376G (rs1050829), although other polymorphisms may play a role. In this study, we tested the association of 20 G6PD single nucleotide polymorphisms (SNPs) with hemolysis measured seven days after low single dose of PQ given at the dose of 0.1 mg/kg to 0.75 mg/kg in 957 individuals from 6 previously published clinical trials investigating the safety and efficacy of this drug spanning five African countries. After adjusting for inter-study effects, age, gender, baseline hemoglobin level, PQ dose, and parasitemia at screening, our analysis showed putative association signals from the common G6PD mutation, A376G [-log10(p-value) = 2.44] and two less-known SNPs, rs2230037 [-log10(p-value] = 2.60), and rs28470352 [-log10(p-value) = 2.15]; A376G and rs2230037 were in very strong linkage disequilibrium with each other (R 2 = 0.978). However, when the effects of these SNPs were included in the same regression model, the subsequent associations were in the borderline of statistical significance. In conclusion, whilst a role for the A- variant is well established, we did not observe an important additional role for other G6PD polymorphisms in determining post-treatment hemolysis in individuals treated with low single-dose PQ.

Commentary: mobile laboratories for SARS-CoV-2 diagnostics: what Europe could learn from the East African Community to assure trade in times of border closures.

BACKGROUND: The emergence of SARS-CoV-2 mutants might lead to European border closures, which impact on trade and result in serious economic losses. In April 2020, similar border closures were observed during the first SARS-CoV-2 wave in East Africa. MAIN BODY: Since 2017 the East African Community EAC together with the Bernhard-Nocht-Institute for Tropical Medicine BNITM established a mobile laboratory network integrated into the National Public Health Laboratories of the six Partner States for molecular diagnosis of viral haemorrhagic fevers and SARS-CoV-2. Since May 2020, the National Public Health Laboratories of Kenya, Rwanda, Burundi, Uganda and South Sudan deployed these mobile laboratories to their respective borders, issuing a newly developed "Electronic EAC COVID-19 Digital Certificate" to SARS-CoV-2 PCR-negative truck drivers, thus assuring regional trade. CONCLUSION: Considering the large financial damages of border closures, such a mobile laboratory network as demonstrated in East Africa is cost-effective, easy to implement and feasible. The East African Community mobile laboratory network could serve as a blueprint for Europe and other countries around the globe.

Improved housing versus usual practice for additional protection against clinical malaria in The Gambia (RooPfs): a household-randomised controlled trial.

BACKGROUND: In malaria-endemic areas, residents of modern houses have less malaria than those living in traditional houses. We aimed to assess whether children in The Gambia received an incremental benefit from improved housing, where current best practice of insecticide-treated nets, indoor residual spraying, seasonal malaria chemoprevention in children younger than 5 years, and prompt treatment against clinical malaria was in place. METHODS: In this randomised controlled study, 800 households with traditional thatched-roofed houses were randomly selected from 91 villages in the Upper River Region of The Gambia. Within each village, equal numbers of houses were randomly allocated to the control and intervention groups using a sampling frame. Houses in the intervention group were modified with metal roofs and screened doors and windows, whereas houses in the control group received no modifications. In each group, clinical malaria in children aged 6 months to 13 years was monitored by active case detection over 2 years (2016-17). We did monthly collections from indoor light traps to estimate vector densities. Primary endpoints were the incidence of clinical malaria in study children with more than 50% of observations each year and household vector density. The trial is registered at ISRCTN02622179. FINDINGS: In June, 2016, 785 houses had one child each recruited into the study (398 in unmodified houses and 402 in modified houses). 26 children in unmodified houses and 28 children in modified houses did not have at least 50% of visits in a year and so were excluded from analysis. 38 children in unmodified houses were recruited after study commencement, as were 21 children in modified houses, meaning 410 children in unmodified houses and 395 in modified houses were included in the parasitological analyses. At the end of the study, 659 (94%) of 702 children were reported to have slept under an insecticide-treated net; 662 (88%) of 755 children lived in houses that received indoor residual spraying; and 151 (90%) of 168 children younger than 5 years had seasonal malaria chemoprevention. Incidence of clinical malaria was 0·12 episodes per child-year in children in the unmodified houses and 0·20 episodes per child-year in the modified houses (unadjusted incidence rate ratio [RR] 1·68 [95% CI 1·11-2·55], p=0·014). Household vector density was 3·30 Anopheles gambiae per house per night in the unmodified houses compared with 3·60 in modified houses (unadjusted RR 1·28 [0·87-1·89], p=0·21). INTERPRETATION: Improved housing did not provide protection against clinical malaria in this area of low seasonal transmission with high coverage of insecticide-treated nets, indoor residual spraying, and seasonal malaria chemoprevention. FUNDING: Global Health Trials funded by Medical Research Council, UK Department for International Development, and Wellcome Trust.

Sero-epidemiological evaluation of malaria transmission in The Gambia before and after mass drug administration.

BACKGROUND: As The Gambia aims to achieve malaria elimination by 2030, serological assays are a useful surveillance tool to monitor trends in malaria incidence and evaluate community-based interventions. METHODS: Within a mass drug administration (MDA) study in The Gambia, where reduced malaria infection and clinical disease were observed after the intervention, a serological sub-study was conducted in four study villages. Spatio-temporal variation in transmission was measured with a panel of recombinant Pf antigens on a multiplexed bead-based assay. Village-level antibody levels were quantified as under-15 sero-prevalence, sero-conversion rates, and age-adjusted antibody acquisition rates. Antibody levels prior to MDA were assessed for association with persistent malaria infection after community chemoprophylaxis. RESULTS: Seasonal changes in antibodies to Etramp5.Ag1 were observed in children under 15 years in two transmission settings-the West Coast and Upper River Regions (4.32% and 31.30% Pf prevalence, respectively). At the end of the malaria season, short-lived antibody responses to Etramp5.Ag1, GEXP18, HSP40.Ag1, EBA175 RIII-V, and Rh2.2030 were lower amongst 1-15 year olds in the West Coast compared to the Upper River, reflecting known differences in transmission. Prior to MDA, individuals in the top 50th percentile of antibody levels had two-fold higher odds of clinical malaria during the transmission season, consistent with previous findings from the Malaria Transmission Dynamics Study, where individuals infected before the implementation of MDA had two-fold higher odds of re-infection post-MDA. CONCLUSIONS: Serological markers can serve dual functions as indicators of malaria exposure and incidence. By monitoring age-specific sero-prevalence, the magnitude of age-stratified antibody levels, or identifying groups of individuals with above-average antibody responses, these antigens have the potential to complement conventional malaria surveillance tools. Further studies, particularly cluster randomised trials, can help establish standardised serological protocols to reliably measure transmission across endemic settings.

Antibody responses to a suite of novel serological markers for malaria surveillance demonstrate strong correlation with clinical and parasitological infection across seasons and transmission settings in The Gambia.

BACKGROUND: As malaria transmission declines, sensitive diagnostics are needed to evaluate interventions and monitor transmission. Serological assays measuring malaria antibody responses offer a cost-effective detection method to supplement existing surveillance tools. METHODS: A prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions in The Gambia. Serological analysis included samples from the West Coast Region at the start and end of the season (July and December 2013) and from the Upper River Region in July and December 2013 and April and December 2014. Antigen-specific antibody responses to eight Plasmodium falciparum (P. falciparum) antigens-Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175 RIII-V, PfMSP119, PfAMA1, and PfGLURP.R2-were quantified using a multiplexed bead-based assay. The association between antibody responses and clinical and parasitological endpoints was estimated at the individual, household, and population level. RESULTS: Strong associations were observed between clinical malaria and concurrent sero-positivity to Etramp5.Ag1 (aOR 4.60 95% CI 2.98-7.12), PfMSP119 (aOR 4.09 95% CI 2.60-6.44), PfAMA1 (aOR 2.32 95% CI 1.40-3.85), and PfGLURP.R2 (aOR 3.12, 95% CI 2.92-4.95), while asymptomatic infection was associated with sero-positivity to all antigens. Village-level sero-prevalence amongst children 2-10 years against Etramp5.Ag1, HSP40.Ag1, and PfMSP119 showed the highest correlations with clinical and P. falciparum infection incidence rates. For all antigens, there were increased odds of asymptomatic P. falciparum infection in subjects residing in a compound with greater than 50% sero-prevalence, with a 2- to 3-fold increase in odds of infection associated with Etramp5.Ag1, GEXP18, Rh2.2030, PfMSP119, and PfAMA1. For individuals residing in sero-positive compounds, the odds of clinical malaria were reduced, suggesting a protective effect. CONCLUSIONS: At low transmission, long-lived antibody responses could indicate foci of malaria transmission that have been ongoing for several seasons or years. In settings where sub-patent infections are prevalent and fluctuate below the detection limit of polymerase chain reaction (PCR), the presence of short-lived antibodies may indicate recent infectivity, particularly in the dry season when clinical cases are rare. Serological responses may reflect a persistent reservoir of infection, warranting community-targeted interventions if individuals are not clinically apparent but have the potential to transmit. Therefore, serological surveillance at the individual and household level may be used to target interventions where there are foci of asymptomatically infected individuals, such as by measuring the magnitude of age-stratified antibody levels or identifying areas with clustering of above-average antibody responses across a diverse range of serological markers.

A survey of Anopheles species composition and insecticide resistance on the island of Bubaque, Bijagos Archipelago, Guinea-Bissau.

BACKGROUND: Bubaque is the most populous island of the Bijagos archipelago, a group of malaria-endemic islands situated off the coast of Guinea-Bissau, West Africa. Malaria vector control on Bubaque relies almost exclusively on the use of long-lasting insecticidal nets (LLINs). However, there is little information on local vector bionomics and insecticide resistance. METHODS: A survey of mosquito species composition was performed at the onset of the wet season (June/July) and the beginning of the dry season (November/December). Sampling was performed using indoor adult light-traps and larval dipping. Anopheles mosquitoes were identified to species level and assessed for kdr allele frequency by TaqMan PCR. Females were analysed for sporozoite positivity by CSP-ELISA. Resistance to permethrin and α-cypermethrin was measured using the CDC-bottle bioassay incorporating the synergist piperonyl-butoxide. RESULTS: Several Anopheles species were found on the island, all belonging to the Anopheles gambiae sensu lato (s.l.) complex, including An. gambiae sensu stricto, Anopheles coluzzii, Anopheles melas, and An. gambiae/An. coluzzii hybrids. Endophagic Anopheles species composition and abundance showed strong seasonal variation, with a majority of An. gambiae (50% of adults collected) caught in June/July, while An. melas was dominant in November/December (83.9% of adults collected). Anopheles gambiae had the highest sporozoite rate in both seasons, with infection rates of 13.9% and 20% in June/July and November/December, respectively. Moderate frequencies of the West African kdr allele were found in An. gambiae (36%), An. coluzzii (35%), An. gambiae/An. coluzzii hybrids (42%). Bioassays suggest moderate resistance to α-cypermethrin, but full susceptibility to permethrin. CONCLUSIONS: The island of Bubaque maintained an An. gambiae s.l. population in both June/July and November/December. Anopheles gambiae was the primary vector at the onset of the wet season, while An. melas is likely to be responsible for most dry season transmission. There was moderate kdr allele frequency and synergist assays suggest likely metabolic resistance, which could reduce the efficacy of LLINs. Future control of malaria on the islands should consider the seasonal shift in mosquito species, and should employ continuous monitoring for insecticide resistance.

Clustering of asymptomatic Plasmodium falciparum infection and the effectiveness of targeted malaria control measures.

BACKGROUND: Because clustering of Plasmodium falciparum infection had been noted previously, the clustering of infection was examined at four field sites in West Africa: Dangassa and Dioro in Mali, Gambissara in The Gambia and Madina Fall in Senegal. METHODS: Clustering of infection was defined by the percent of persons with positive slides for asexual P. falciparum sleeping in a house which had been geopositioned. Data from each site were then tested for spatial, temporal and spatio-temporal clustering in relation to the prevalence of infection from smear surveys. RESULTS: These studies suggest that clustering of P. falciparum infection also affects the effectiveness of control interventions. For example, the clustering of infection in Madina Fall disappeared in 2014-2016 after vector control eliminated the only breeding site in 2013. In contrast, the temporal clustering of infection in Dioro (rainy season of 2014, dry season of 2015) was consistent with the loss of funding for Dioro in the second quarter of 2014 and disappeared when funds again became available in late 2015. The clustering of infection in rural (western) areas of Gambissara was consistent with known rural-urban differences in the prevalence of infection and with the thatched roofs, open eaves and mud walls of houses in rural Gambissara. In contrast, the most intense transmission was in Dangassa, where the only encouraging observation was a lower prevalence of infection in the dry season. Taken together, these results suggest: (a) the transmission of infection was stopped in Madina Fall by eliminating the only known breeding site, (b) the prevalence of infection was reduced in Dioro after financial support became available again for malaria control in the second half of 2015, (c) improvements in housing should improve malaria control by reducing the number of vectors in rural communities such as western Gambissara, and (d) beginning malaria control during the dry season may reduce transmission in hyperendemic areas such as Dangassa. CONCLUSIONS: From a conceptual perspective, testing for spatial, temporal and spatio-temporal clustering based on epidemiologic data permits the generation of hypotheses for the clustering observed and the testing of candidate interventions to confirm or refute those hypotheses.

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest. METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods. RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype. CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.

Mass Drug Administration With Dihydroartemisinin-piperaquine and Malaria Transmission Dynamics in The Gambia: A Prospective Cohort Study.

BACKGROUND: Mass drug administration (MDA) may further reduce malaria transmission in low-transmission areas. The impact of MDA on the dynamics of malaria transmission was determined in a prospective cohort study. METHODS: Annual rounds of MDA with dihydroartemisinin-piperaquine (DP) were implemented were implemented in 2014 and 2015 in six village pairs before the malaria transmission season. Blood samples were collected from residents between July and December for microscopy and nested PCR. Incidence and prevalence of infection, clinical disease, and risk of malaria reinfection post-MDA were determined. RESULTS: Coverage of three DP doses was 68.2% (2014) and 65.6% (2015), compliance was greater than 80%. Incidence of infection was significantly lower in 2014 (incidence rate [IR] = 0.2 per person year [PPY]) than in 2013 (IR = 1.1 PPY; P < .01); monthly infection prevalence declined in the first three months post-MDA. Clinical malaria incidence was lower in 2014 (IR = 0.1 PPY) and 2015 (IR = 0.2 PPY) than in 2013 (IR = 0.4 PPY; P < .01), but remained higher in eastern Gambia. Individuals infected before MDA had a 2-fold higher odds of reinfection post-MDA (adjusted odds ratio = 2.5, 95% confidence interval 1.5-4.3; P < .01). CONCLUSIONS: MDA reduced malaria infection and clinical disease during the first months. The reduction was maintained in low-transmission areas, but not in eastern Gambia. Annual MDA could be followed by focal MDA targeting individuals infected during the dry season. Repeated MDA rounds, some during the dry season over larger geographical areas, may result in a more marked and sustained decrease of malaria transmission.

Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing.  Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22°C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Development of a data collection and management system in West Africa: challenges and sustainability.

BACKGROUND: Developing and sustaining a data collection and management system (DCMS) is difficult in malaria-endemic countries because of limitations in internet bandwidth, computer resources and numbers of trained personnel. The premise of this paper is that development of a DCMS in West Africa was a critically important outcome of the West African International Centers of Excellence for Malaria Research. The purposes of this paper are to make that information available to other investigators and to encourage the linkage of DCMSs to international research and Ministry of Health data systems and repositories. METHODS: We designed and implemented a DCMS to link study sites in Mali, Senegal and The Gambia. This system was based on case report forms for epidemiologic, entomologic, clinical and laboratory aspects of plasmodial infection and malarial disease for a longitudinal cohort study and included on-site training for Principal Investigators and Data Managers. Based on this experience, we propose guidelines for the design and sustainability of DCMSs in environments with limited resources and personnel. RESULTS: From 2012 to 2017, we performed biannual thick smear surveys for plasmodial infection, mosquito collections for anopheline biting rates and sporozoite rates and year-round passive case detection for malarial disease in four longitudinal cohorts with 7708 individuals and 918 households in Senegal, The Gambia and Mali. Major challenges included the development of uniform definitions and reporting, assessment of data entry error rates, unstable and limited internet access and software and technology maintenance. Strengths included entomologic collections linked to longitudinal cohort studies, on-site data centres and a cloud-based data repository. CONCLUSIONS: At a time when research on diseases of poverty in low and middle-income countries is a global priority, the resources available to ensure accurate data collection and the electronic availability of those data remain severely limited. Based on our experience, we suggest the development of a regional DCMS. This approach is more economical than separate data centres and has the potential to improve data quality by encouraging shared case definitions, data validation strategies and analytic approaches including the molecular analysis of treatment successes and failures.

Single low-dose primaquine for blocking transmission of Plasmodium falciparum malaria - a proposed model-derived age-based regimen for sub-Saharan Africa.

BACKGROUND: In 2012, the World Health Organization recommended blocking the transmission of Plasmodium falciparum with single low-dose primaquine (SLDPQ, target dose 0.25 mg base/kg body weight), without testing for glucose-6-phosphate dehydrogenase deficiency (G6PDd), when treating patients with uncomplicated falciparum malaria. We sought to develop an age-based SLDPQ regimen that would be suitable for sub-Saharan Africa. METHODS: Using data on the anti-infectivity efficacy and tolerability of primaquine (PQ), the epidemiology of anaemia, and the risks of PQ-induced acute haemolytic anaemia (AHA) and clinically significant anaemia (CSA), we prospectively defined therapeutic-dose ranges of 0.15-0.4 mg PQ base/kg for children aged 1-5 years and 0.15-0.5 mg PQ base/kg for individuals aged ≥6 years (therapeutic indices 2.7 and 3.3, respectively). We chose 1.25 mg PQ base for infants aged 6-11 months because they have the highest rate of baseline anaemia and the highest risks of AHA and CSA. We modelled an anthropometric database of 661,979 African individuals aged ≥6 months (549,127 healthy individuals, 28,466 malaria patients and 84,386 individuals with other infections/illnesses) by the Box-Cox transformation power exponential and tested PQ doses of 1-15 mg base, selecting dosing groups based on calculated mg/kg PQ doses. RESULTS: From the Box-Cox transformation power exponential model, five age categories were selected: (i) 6-11 months (n = 39,886, 6.03%), (ii) 1-5 years (n = 261,036, 45.46%), (iii) 6-9 years (n = 20,770, 3.14%), (iv) 10-14 years (n = 12,155, 1.84%) and (v) ≥15 years (n = 328,132, 49.57%) to receive 1.25, 2.5, 5, 7.5 and 15 mg PQ base for corresponding median (1st and 99th centiles) mg/kg PQ base of: (i) 0.16 (0.12-0.25), (ii) 0.21 (0.13-0.37), (iii) 0.25 (0.16-0.38), (iv) 0.26 (0.15-0.38) and (v) 0.27 (0.17-0.40). The proportions of individuals predicted to receive optimal therapeutic PQ doses were: 73.2 (29,180/39,886), 93.7 (244,537/261,036), 99.6 (20,690/20,770), 99.4 (12,086/12,155) and 99.8% (327,620/328,132), respectively. CONCLUSIONS: We plan to test the safety of this age-based dosing regimen in a large randomised placebo-controlled trial (ISRCTN11594437) of uncomplicated falciparum malaria in G6PDd African children aged 0.5 - 11 years. If the regimen is safe and demonstrates adequate pharmacokinetics, it should be used to support malaria elimination.