Improved adherence to test, treat, and track (T3) malaria strategy among Over-the-Counter Medicine Sellers (OTCMS) through interventions implemented in selected rural communities of Fanteakwa North district, Ghana.

BACKGROUND: Prompt diagnosis and treatment of malaria prevents a mild case from developing into severe disease and death. Unfortunately, parasitological testing of febrile children is greater in the public and formal private sector than in the informal private sector where many patients with malaria-like symptoms first seek treatment. This study was aimed at improving implementation of the T3 policy among OTCMS using some interventions that could be scaled-up easily at the national level. METHODS: Interventions were evaluated using a two-arm, cluster randomized trial across 8 rural communities (4 clusters per arm), in two adjacent districts of Ghana. A total of 7 OTCMS in the intervention arm and 5 OTCMS in the control arm in the selected communities participated in the study. Five interventions were implemented in the intervention arm only. These were acquisition of subsidized malaria rapid diagnostic test (RDT) kits, training of OTCMS, supportive visits to OTCMS, community sensitization on malaria, and introduction of malaria surveillance tool. The primary outcome was the proportion of children under 10 years with fever or suspected to have malaria visiting OTCMS and getting tested (using RDT) before treatment. Secondary outcomes included OTCMS adherence to national malaria treatment guidelines and the recommended RDT retail price. Outcomes were measured using mystery client (an adult who pretends to be a real patient) surveys supplemented by a household survey. Proportions were compared using chi-square test or Fisher exact test. RESULTS: Following deployment of interventions, mystery client survey showed that OTCMS' adherence to malaria protocol in the intervention arm increased significantly (p < 0.05) compared to the control arm. Household surveys in the intervention arm showed that caregivers self-treating their children or visiting drug vendors significantly decreased in favour of visits to OTCMS shops for treatment (p < 0.001). End-line malaria testing rate was higher compared with the baseline rate, though not statistically significant (30.8% vs 10.5%; p = 0.1238). OTCMS in the intervention arm also adhered to the subsidized RDT retail price of GHc2.40. CONCLUSION: Interventions targeting OTCMS in rural communities have the potential of improving adherence to the T3 malaria policy and subsequently improving management of uncomplicated malaria in Ghana. TRIAL REGISTRATION: ISRCTN registry ISRCTN77836926. Registered on 4 November 2019.

Guiding placement of health facilities using multiple malaria criteria and an interactive tool.

BACKGROUND: Access to healthcare is important in controlling malaria burden and, as a result, distance or travel time to health facilities is often a significant predictor in modelling malaria prevalence. Adding new health facilities may reduce overall travel time to health facilities and may decrease malaria transmission. To help guide local decision-makers as they scale up community-based accessibility, the influence of the spatial allocation of new health facilities on malaria prevalence is evaluated in Bunkpurugu-Yunyoo district in northern Ghana. A location-allocation analysis is performed to find optimal locations of new health facilities by separately minimizing three district-wide objectives: malaria prevalence, malaria incidence, and average travel time to health facilities. METHODS: Generalized additive models was used to estimate the relationship between malaria prevalence and travel time to the nearest health facility and other geospatial covariates. The model predictions are then used to calculate the optimisation criteria for the location-allocation analysis. This analysis was performed for two scenarios: adding new health facilities to the existing ones, and a hypothetical scenario in which the community-based healthcare facilities would be allocated anew. An interactive web application was created to facilitate efficient presentation of this analysis and allow users to experiment with their choice of health facility location and optimisation criteria. RESULTS: Using malaria prevalence and travel time as optimisation criteria, two locations that would benefit from new health facilities were identified, regardless of scenarios. Due to the non-linear relationship between malaria incidence and prevalence, the optimal locations chosen based on the incidence criterion tended to be inequitable and was different from those based on the other optimisation criteria. CONCLUSIONS: This study findings underscore the importance of using multiple optimisation criteria in the decision-making process. This analysis and the interactive application can be repurposed for other regions and criteria, bridging the gap between science, models and decisions.

Trends and predictive factors for treatment failure following artemisinin-based combination therapy among children with uncomplicated malaria in Ghana: 2005-2018.

BACKGROUND: Since the introduction of artemisinin-based combination therapy (ACT) in Ghana in 2005 there has been a surveillance system by the National Malaria Control Programme (NMCP) and the University of Ghana Noguchi Memorial Institute for Medical Research (UG-NMIMR) to monitor the therapeutic efficacy of ACTs for the treatment of uncomplicated malaria in the country. We report trends and determinants of failure following treatment of Ghanaian children with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) combinations. METHODS: Per protocol analyses as well as cumulative incidence of day 28 treatment failure from Kaplan Meier survival analyses were used to describe trends of failure over the surveillance period of 2005-2018. Univariable and multivariable cox regression analyses were used to assess the determinants of treatment failure over the period. RESULTS: Day 28 PCR-corrected failure, following treatment with ASAQ, significantly increased from 0.0% in 2005 to 2.0% (95% CI: 1.1-3.6) in 2015 (p = 0.013) but significantly decreased to 0.4% (95% CI: 0.1-1.6) in 2018 (p = 0.039). Failure, following treatment with AL, decreased from 4.5% (95% CI: 2.0-9.4) in 2010 to 2.7% (95% CI: 1.4-5.1) in 2018, though not statistically significant (p = 0.426). Risk of treatment failure, from multivariable cox regression analyses, was significantly lower among children receiving ASAQ compared with those receiving AL (HR = 0.24; 95% CI: 0.11-0.53; p < 0.001); lower among children with no parasitaemia on day 3 compared with those with parasitaemia on day 3 (HR = 0.02; 95% CI: 0.01-0.13; p < 0.001); and higher among children who received ASAQ and had axillary temperature ≥ 37.5 °C on day 1 compared with those with axillary temperature < 37.5 °C (HR = 3.96; 95% CI: 1.61-9.75; p = 0.003). CONCLUSIONS: Treatment failures for both ASAQ and AL have remained less than 5% (below WHO's threshold of 10%) in Ghana since 2005. Predictors of treatment failure that need to be considered in the management of uncomplicated malaria in the country should include type of ACT, day 3 parasitaemia, and day 1 axillary temperature of patients being treated.

Factors impacting test-based management of suspected malaria among caregivers of febrile children and private medicine retailers within rural communities of Fanteakwa North District, Ghana.

BACKGROUND: Prompt diagnosis and treatment prevents a mild case of malaria from developing into severe disease and death. Unfortunately, parasitological testing of febrile children is greater in the public and formal private sector than in the informal private sector in sub-Saharan Africa. METHODS: A mixed method study was carried out to determine factors limiting test-based management of suspected malaria cases among caregivers of febrile children and Over-the-Counter medicine sellers (OTCMS) in eight rural communities in Ghana. Structured questionnaires were used to interview 254 adult caregivers. Fourteen in-depth interviews were conducted with OTCMS. The interviews were audio-recorded, transcribed verbatim, and analysed thematically. RESULTS: The most frequently sought health providers by caregivers of febrile children in descending order were Community Health-Based Planning Services (CHPS) compounds; drug vendors; and OTCMS. Malaria parasitological testing rate of febrile children was highest (94.9%) at the CHPS compound and lowest (10.5%) at the OTCMS shops. Proportion of febrile children not subjected to malaria blood test is 28.3%. Among caregivers who did not ask for malaria blood test, 15.2% reported that healthcare provider did not offer a malaria blood test; 21.7% were financially handicapped to visit the Health Centre; and 63% lacked knowledge of malaria blood test and where to get it. From OTCMS point of view, clients' inability to pay for malaria blood test, community perception that OTCMS are unqualified to perform malaria blood test, financial loss when unused RDT kits expires, clients' demand for half dose of ACT, and activities of drug peddlers are factors limiting adherence to WHO recommended policy on testing before treating uncomplicated malaria cases. CONCLUSION: The study results suggest the need to implement community friendly interventions aimed at improving test-based management of suspected malaria in febrile children. These may include educating caregivers and community members on the need to test and confirm malaria in febrile children before treating them, and supply of subsidized RDT kits to OTCMS and re-training them to provide testing services to their clients. Further studies pertaining to influence of gender roles on healthcare seeking attitude for febrile children is also suggested.

Malaria parasitaemia and mRDT diagnostic performances among symptomatic individuals in selected health care facilities across Ghana.

BACKGROUND: Parasitological diagnosis generates data to assist malaria-endemic countries determine their status within the malaria elimination continuum and also inform the deployment of proven interventions to yield maximum impact. This study determined prevalence of malaria parasitaemia and mRDT performances among febrile patients in selected health care facilities across Ghana. METHODS: This study was a cross-sectional survey conducted in the previously 10 regions of Ghana from May to August 2018. Each patient suspected to have uncomplicated malaria was tested using microscopy and two malaria rapid diagnostic tests (mRDTs): routinely used CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH). Main outcome variables were malaria slide and CareStart™ Malaria HRP2 (Pf) positivity rates; and diagnostic accuracy of CareStart™ Malaria HRP2 (Pf) and SD Bioline Malaria Ag Pf (HRP2/pLDH) using microscopy as "gold standard". RESULTS: Overall parasite positivity rates were 32.3% (6266/19402) by mRDT and 16.0% (2984/18616) by microscopy, with Plasmodium falciparum mono-infection accounting for 98.0% of all infections. The odds of parasitaemia by microscopy was significantly lower among female patients compared with males (OR = 0.78; 95% CI: 0.66-0.91), and among patients with history of previous antimalarial intake compared with those with no such history (OR = 0.72; 95% CI: 0.54-0.95). Overall sensitivity of CareStart™ Malaria HRP2 (Pf) was statistically similar to that of the HRP2 band of SD Bioline Malaria Ag Pf (HRP2/pLDH) combo kit (95.4%; 95% CI: 94.6-96.1 vs 94.3%; 95% CI: 93.4-95.1; p = 0.065) but significantly higher than the pLDH band (89.3%; 95% CI: 88.1-90.4; p < 0.001). The same pattern was observed for negative predictive value. CONCLUSIONS: Malaria control interventions should be targeted at the general population, and history of antimalarial intake considered a key predictor of malaria slide negativity. Furthermore, HRP2-based mRDTs remain effective diagnostic tool in the management of suspected uncomplicated malaria in the country.

Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in Ghana Using Molecular Inversion Probes and Next-Generation Sequencing.

A key drawback to monitoring the emergence and spread of antimalarial drug resistance in sub-Saharan Africa is early detection and containment. Next-generation sequencing methods offer the resolution, sensitivity, and scale required to fill this gap by surveilling for molecular markers of drug resistance. We performed targeted sequencing using molecular inversion probes to interrogate five Plasmodium falciparum genes (pfcrt, pfmdr1, pfdhps, pfdhfr, and pfk13) implicated in chloroquine, sulfadoxine-pyrimethamine (SP), and artemisinin resistance in two sites in Ghana. A total of 803 dried blood spots from children aged between 6 months and 14 years presenting with uncomplicated P. falciparum malaria at the Begoro District Hospital in Begoro and the Ewim Polyclinic in Cape Coast, Ghana, from 2014 to 2017 were prepared on filter paper. Thirteen years after the removal of drug pressure, chloroquine-sensitive parasite strains with pfcrt K76 have increased nearly to fixation in Begoro, in the forest area (prevalence = 95%), but at a lower rate in Cape Coast, in the coastal region (prevalence = 71%, Z = -3.5, P < 0.001). In addition, pfmdr1 184F-bearing parasites are under strong selection. The pfdhfr/pfdhps quadruple genotype ( IRNG K), associated with SP resistance, is near saturation. Our study identified at a 2 to 10% prevalence pfdhps 581G, which is a sulfadoxine resistance marker that correlates with the failure of SP prophylaxis in pregnancy and which has not been observed in Ghana. The differences in the reexpansion of chloroquine-sensitive strains observed at the two study sites, the stronger SP resistance, and the high prevalence of pfmdr1 184F should be further monitored to inform malaria control strategies in Ghana.

Plasmodium falciparum genetic factors rather than host factors are likely to drive resistance to ACT in Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) partner drugs, currently used in Ghana are lumefantrine, amodiaquine and piperaquine. Plasmodium falciparum isolates with reduced susceptibility to these partner drugs may affect treatment outcome. Mutations in pfmdr1 gene is linked to reduced parasite susceptibility to amodiaquine and lumefantrine. In addition, the potency of the partner drugs in vivo depends on the metabolism by the cytochrome P450 (CYP) enzyme in the host. Mutations in the CYP2C8 and CYP3A4 genes are linked to reduced metabolism of amodiaquine and lumefantrine in vitro, respectively. This study investigated the host and parasite genetic factors affecting the susceptibility of the malaria parasite to ACT partner drugs. METHODS: Archived samples from 240 patients age ≤ 9 years participating in anti-malarial drug resistance survey in Ghana, and given artemether with lumefantrine (AL) or artesunate with amodiaquine (AA), were selected and analysed. Polymerase chain reaction (PCR) followed by Sanger sequencing was used to determine the polymorphisms in CYP2C8, CYP3A4 and pfmdr1 genes. RESULTS: For CYP3A4, all had wild type alleles, suggesting that the hosts are good metabolizers of lumefantrine. For CYP2C8 60% had wild type alleles, 35% heterozygous and 5% homozygous recessive alleles suggesting efficient metabolism of amodiaquine by the hosts. For pfmdr1 gene, at codon 86, 95% were wild type (N86) and 5% mutant (Y86). For codon 184, 36% were wild type (Y184) and 64% mutant (F184) while for codons 1034, 1042 and 1246, 100% (all) were wild type. The high prevalence of N86-F184-D1246 haplotype (NFD) suggest presence of parasites with reduced susceptibility to lumefantrine and not amodiaquine. Delayed clearance was observed in individuals with mutations in the pfmdr1 gene and not cytochrome 450 gene. Both synonymous and non-synonymous mutations were observed in the pfmdr1 at low prevalence. CONCLUSION: The outcome of this study indicates that the parasite's genetic factors rather than the host's are likely to drive resistance to ACT in Ghana.

Plasmodium falciparum Kelch Propeller Polymorphisms in Clinical Isolates from Ghana from 2007 to 2016.

The continuous surveillance of polymorphisms in the kelch propeller domain of Plasmodium falciparum from Africa is important for the discovery of the actual markers of artemisinin resistance in the region. The information on the markers is crucial for control strategies involving chemotherapy and chemoprophylaxis for residents and nonimmune travelers to the country. Polymorphisms in the kelch propeller domain of Ghanaian malaria parasites from three different ecological zones at several time periods were assessed. A total of 854 archived samples (2007 to 2016) collected from uncomplicated malaria patients aged ≤9 years old from 10 sentinel sites were used. Eighty-four percent had wild-type sequences (PF3D7_1343700), while many of the mutants had mostly nonsynonymous mutations clustered around codons 404 to 650. Variants with different amino acid changes of the codons associated with artemisinin (ART) resistance validated markers were observed in Ghanaian isolates: frequencies for I543I, I543S, I543V, R561P, R561R, and C580V were 0.12% each and 0.6% for R539I. Mutations reported from African parasites, A578S (0.23%) and Q613L (0.23%), were also observed. Three persisting nonsynonymous (NS) mutations, N599Y (0.005%), K607E (0.004%), and V637G (0.004%), were observed in 3 of the 5 time periods nationally. The presence of variants of the validated markers of artemisinin resistance as well as persisting polymorphisms after 14 years of artemisinin-based combination therapy use argues for continuous surveillance of the markers. The molecular markers of artemisinin resistance and the observed variants will be monitored subsequently as part of ongoing surveillance of antimalarial drug efficacy/resistance studies in the country.

Characterizing local-scale heterogeneity of malaria risk: a case study in Bunkpurugu-Yunyoo district in northern Ghana.

BACKGROUND: Bayesian methods have been used to generate country-level and global maps of malaria prevalence. With increasing availability of detailed malaria surveillance data, these methodologies can also be used to identify fine-scale heterogeneity of malaria parasitaemia for operational prevention and control of malaria. METHODS: In this article, a Bayesian geostatistical model was applied to six malaria parasitaemia surveys conducted during rainy and dry seasons between November 2010 and 2013 to characterize the micro-scale spatial heterogeneity of malaria risk in northern Ghana. RESULTS: The geostatistical model showed substantial spatial heterogeneity, with malaria parasite prevalence varying between 19 and 90%, and revealing a northeast to southwest gradient of predicted risk. The spatial distribution of prevalence was heavily influenced by two modest urban centres, with a substantially lower prevalence in urban centres compared to rural areas. Although strong seasonal variations were observed, spatial malaria prevalence patterns did not change substantially from year to year. Furthermore, independent surveillance data suggested that the model had a relatively good predictive performance when extrapolated to a neighbouring district. CONCLUSIONS: This high variability in malaria prevalence is striking, given that this small area (approximately 30 km × 40 km) was purportedly homogeneous based on country-level spatial analysis, suggesting that fine-scale parasitaemia data might be critical to guide district-level programmatic efforts to prevent and control malaria. Extrapolations results suggest that fine-scale parasitaemia data can be useful for spatial predictions in neighbouring unsampled districts and does not have to be collected every year to aid district-level operations, helping to alleviate concerns regarding the cost of fine-scale data collection.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine combinations for uncomplicated malaria in 10 sentinel sites across Ghana: 2015-2017.

BACKGROUND: Routine surveillance on the therapeutic efficacy of artemisinin-based combination therapy (ACT) has been ongoing in Ghana since 2005. The sixth round of surveillance was conducted between 2015 and 2017 to determine the therapeutic efficacy of artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) in 10 sentinel sites across the country. METHODS: The study was a one-arm, prospective, evaluation of the clinical, parasitological, and haematological responses to directly observed treatment with AS-AQ and AL among children 6 months to 9 years old with uncomplicated falciparum malaria. The WHO 2009 protocol on surveillance of anti-malaria drug efficacy was used for the study with primary outcomes as prevalence of day 3 parasitaemia and clinical and parasitological cure rates on day 28. Secondary outcomes assessed included patterns of fever and parasite clearance as well as changes in haemoglobin concentration. RESULTS: Day 3 parasitaemia was absent in all sites following treatment with AS-AQ whilst only one person (0.2%) was parasitaemic on day 3 following treatment with AL. Day 28 PCR-corrected cure rates following treatment with AS-AQ ranged between 96.7% (95% CI 88.5-99.6) and 100%, yielding a national rate of 99.2% (95% CI 97.7-99.7). Day 28 PCR-corrected cure rates following treatment with AL ranged between 91.3% (95% CI 79.2-97.6) and 100%, yielding a national rate of 96% (95% CI 93.5-97.6). Prevalence of fever declined by 88.4 and 80.4% after first day of treatment with AS-AQ and AL, respectively, whilst prevalence of parasitaemia on day 2 was 2.1% for AS-AQ and 1.5% for AL. Gametocytaemia was maintained at low levels (< 5%) during the 3 days of treatment. Post-treatment mean haemoglobin concentration was significantly higher than pre-treatment concentration following treatment with either AS-AQ or AL. CONCLUSIONS: The therapeutic efficacy of AS-AQ and AL is over 90% in sentinel sites across Ghana. The two anti-malarial drugs therefore remain efficacious in the treatment of uncomplicated malaria in the country and continue to achieve rapid fever and parasite clearance as well as low gametocyte carriage rates and improved post-treatment mean haemoglobin concentration.

Probing the composition of Plasmodium species contained in malaria infections in the Eastern region of Ghana.

BACKGROUND: Asymptomatic falciparum and non-falciparum malaria infections are major challenges to malaria control interventions, as they remain a source of continual infection in the community. This becomes even more important as the debate moves towards elimination and eradication. This study sought to quantify the burden of Plasmodium malaria infection in seven communities in the Eastern Region of Ghana. METHODS: The cross-sectional study recruited 729 participants aged 85 years old and below from 7 closely linked communities. Finger pricked blood was used to prepare thick and thin blood smears as well as spot filter paper and an histidine rich protein 2 (HRP2) rapid diagnostic test kit (RDT). Genomic DNA was extracted from the filter paper dry blood spot (DBS) and used in PCR to amplify the Plasmodium 18S rRNA gene using species specific PCR. RESULTS: 96.6% of the participants were identified as afebrile, with axillary temperatures below 37.5 °C. PCR identified 66% of the participants to harbor malaria parasites, with 9 P. malariae and 7 P. ovale mono-infections accounting for 2.2% and P. falciparum combined with either 36 P. malariae or 25 P. ovale infections, accounting for 13.3%. Parasite prevalence by microscopy (32%) was similar to the RDT positivity rate (33%). False positive RDT results ranged from 64.6% in children aged between 5 and 9 years to 10% in adults aged 20 years and above. No significant differences were observed in falciparum and non-falciparum parasite carriage at the community level, however young adults aged between 15 and 19 years had the highest prevalence (34.8% (16/46)) of P. falciparum and P. malariae parasite carriage whilst children aged between 5 and 9 years had the highest level (11.4% (14/123)) of P. ovale carriage. CONCLUSION: The high rate of misidentification of non-falciparum parasites and the total absence of detection of P. ovale by microscopy suggests that more sensitive malaria diagnostic tools including molecular assays are required to accurately determine the prevalence of carriers of non-falciparum parasites and low density P. falciparum infections, especially during national surveillance exercises. Additionally, malaria control interventions targeting the non-falciparum species P. malariae and P. ovale parasites are needed.

Prevalence of asymptomatic malaria parasitaemia following mass testing and treatment in Pakro sub-district of Ghana.

BACKGROUND: Global efforts to scale-up malaria control interventions are gaining steam. These include the use of Long-Lasting Insecticide Nets, Indoor Residual Spraying, Intermittent Preventive Treatment and Test, Treat and Track. Despite these, the drive for malaria elimination is far from being realistic in endemic communities in Africa. This is partly due to the fact that asymptomatic parasite carriage, not specifically targeted by most interventions, remains the bedrock that fuels transmission. This has led to mass testing, treatment and tracking (MTTT) as an alternative strategy to target asymptomatic individuals. We report the impact of MTTT on the prevalence of asymptomatic malaria parasitaemia over a one-year period in Ghana, hypothesizing that implementing MTTT could reduce the rate of asymptomatic parasitaemia. METHODS: A population of about 5000 individuals in seven communities in the Pakro sub-district of Ghana participated in this study. A register was developed for each community following a census. MTTT engaged trained community-based health volunteers who conducted house-to-house testing using RDTs every 4 months and treated positive cases with Artemisinin-based Combination Therapy. Between interventions, community-based management of malaria was implemented for symptomatic cases. RESULTS: MTTT Coverage was 98.8% in July 2017 and 79.3% in July 2018. Of those tested, asymptomatic infection with malaria parasites reduced from 36.3% (1795/4941) in July 2017 to 32.9% (1303/3966) in July 2018 (p = 0.001). Prevalence of asymptomatic parasitaemia among children under 15 years declined from 52.6% (1043/1984) in July 2017 to 47.5% (820/1728) in July 2018 (p = 0.002). Implementing MTTT significantly reduced asymptomatic parasitaemia by 24% from July 2017 to July 2018 after adjusting for age, ITN use and axillary temperature (OR = 0.76, CI = 0.67, 0.85 p ≤ 0.001). CONCLUSION: This study has demonstrated that implementing MTTT is feasible and could reduce the prevalence of asymptomatic malaria parasitaemia in children under 15 years of age. Furthermore, the use of community-based health volunteers could ensure high coverage at lower cost of implementation. TRIAL REGISTRATION: NCT04167566, Date 14/11/2019. Retrospective registration.

Detecting local risk factors for residual malaria in northern Ghana using Bayesian model averaging.

BACKGROUND: There is a need for comprehensive evaluations of the underlying local factors that contribute to residual malaria in sub-Saharan Africa. However, it is difficult to compare the wide array of demographic, socio-economic, and environmental variables associated with malaria transmission using standard statistical approaches while accounting for seasonal differences and nonlinear relationships. This article uses a Bayesian model averaging (BMA) approach for identifying and comparing potential risk and protective factors associated with residual malaria. RESULTS: The relative influence of a comprehensive set of demographic, socio-economic, environmental, and malaria intervention variables on malaria prevalence were modelled using BMA for variable selection. Data were collected in Bunkpurugu-Yunyoo, a rural district in northeast Ghana that experiences holoendemic seasonal malaria transmission, over six biannual surveys from 2010 to 2013. A total of 10,022 children between the ages 6 to 59 months were used in the analysis. Multiple models were developed to identify important risk and protective factors, accounting for seasonal patterns and nonlinear relationships. These models revealed pronounced nonlinear associations between malaria risk and distance from the nearest urban centre and health facility. Furthermore, the association between malaria risk and age and some ethnic groups was significantly different in the rainy and dry seasons. BMA outperformed other commonly used regression approaches in out-of-sample predictive ability using a season-to-season validation approach. CONCLUSIONS: This modelling framework offers an alternative approach to disease risk factor analysis that generates interpretable models, can reveal complex, nonlinear relationships, incorporates uncertainty in model selection, and produces accurate predictions. Certain modelling applications, such as designing targeted local interventions, require more sophisticated statistical methods which are capable of handling a wide range of relevant data while maintaining interpretability and predictive performance, and directly characterize uncertainty. To this end, BMA represents a valuable tool for constructing more informative models for understanding risk factors for malaria, as well as other vector-borne and environmentally mediated diseases.

Spatio-temporal clustering analysis and its determinants of hand, foot and mouth disease in Hunan, China, 2009-2015.

BACKGROUND: Hand, foot and mouth disease (HFMD) is one of the highest reported infectious diseases with several outbreaks across the world. This study aimed at describing epidemiological characteristics, investigating spatio-temporal clustering changes, and identifying determinant factors in different clustering areas of HFMD. METHODS: Descriptive statistics was used to evaluate the epidemic characteristics of HFMD from 2009 to 2015. Spatial autocorrelation and spatio-temporal cluster analysis were used to explore the spatial temporal patterns. An autologistic regression model was employed to explore determinants of HFMD clustering. RESULTS: The incidence rates of HFMD ranged from 54.31/10 million to 318.06/10 million between 2009 and 2015 in Hunan. Cases were mainly prevalent in children aged 5 years and even younger, with an average male-to-female sex ratio of 1.66, and two epidemic periods in each year. Clustering areas gathered in the northern regions in 2009 and in the central regions from 2010 to 2012. They moved to central-southern regions in 2013 and 2014 and central-western regions in 2015. The significant risk factors of HFMD clusters were rainfall (OR = 2.187), temperature (OR = 4.329) and humidity (OR = 2.070). The protect factor was wind speed (OR = 0.258). CONCLUSIONS: The HFMD incidence from 2009 to 2015 in Hunan showed a new spatiotemporal clustering tendency, with the shifting trend of clustering areas toward south and west. Meteorological factors showed a strong association with HFMD clustering, which may assist in predicting future spatial-temporal clusters.

Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum.

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine combinations in the treatment of uncomplicated malaria in two ecological zones in Ghana.

BACKGROUND: Case management based on prompt diagnosis and adequate treatment using artemisinin-based combination therapy (ACT) remains the main focus of malaria control in Ghana. As part of routine surveillance on the therapeutic efficacy of ACT in Ghana, the efficacy of amodiaquine-artesunate (AS-AQ) and artemether-lumefantrine (AL) were studied in six sentinel sites representing the forest and savannah zones of the country. METHODS: Three sites representing the two ecological zones studied AS-AQ whilst the other three sites studied AL. In each site, the study was a one-arm prospective evaluation of the clinical, parasitological, and haematological responses to directly observed therapy for uncomplicated malaria with either AS-AQ or AL among children aged 6 months and 9 years. The WHO 2009 protocol for monitoring anti-malarial drug efficacy was used for the study between July 2013 and March 2014. RESULTS: Per-protocol analyses on day 28 showed an overall PCR-corrected cure rate of 100% for AS-AQ and 97.6% (95% CI 93.1, 99.5) for AL: 97.2% (95% CI 92.0, 99.4) in the forest zone and 100% in the savannah zone. Kaplan-Meier survival analysis showed similar outcomes. Prevalence of fever decreased by about 75% after the first day of treatment with each ACT in the two ecological zones. No child studied was parasitaemic on day 3, and gametocytaemia was generally maintained at low levels (<5%). Post-treatment mean haemoglobin concentrations significantly increased in the two ecological zones. CONCLUSIONS: Therapeutic efficacy of AS-AQ and AL remains over 90% in the forest and savannah zones of Ghana. Additionally, post-treatment parasitaemia on day 3 is rare suggesting that artemisinin is still efficacious in Ghana.

Impact of MBL and MASP-2 gene polymorphism and its interaction on susceptibility to tuberculosis.

BACKGROUND: Mannose-binding lectin (MBL) and MBL-associated serine proteases 2 (MASP-2) are important proteins in the lectin pathway of the immune system. Polymorphism of MBL and MASP-2 genes may affect the serum concentration of MBL and MASP-2. This study explores the association between MBL and MASP-2 gene polymorphism and their interactions and the susceptibility to tuberculosis (TB). METHOD: A total of 503 patients with TB and 419 healthy controls were recruited to participate in this case-control study. PCR-SSP technology was applied to genotype rs7096206 of MBL genes and rs2273346 and rs6695096 of MASP-2 genes. Demographic data and some exposure information were also obtained from study participants. Unconditional logistic regression analysis was used to identify association between the various factors and TB whilst Marginal Structural Linear Odds Models were used to estimate the interactions. RESULTS: Both genotype GC at rs7096206 of MBL genes and genotype TC at rs2273346 and rs6695096 of MASP-2 genes were more prevalent in the TB patient group than the healthy control group (P<0.05, OR 1.393, 1.302 and 1.426 respectively). The relative excess risk of interaction (RERI) between rs7096206 of MBL genes and rs2273346 and rs6695096 of MASP-2 genes was 0.897 (95% CI: 0.282, 1.513) and 1.142 (95% CI: 0.755, 1.530) respectively (P<0.05). CONCLUSION: Polymorphisms of MBL (rs7096206) and MASP-2 (rs2273346 and rs6695096) were associated with the susceptibility of TB, and there were gene-gene interactions among them.

Impact of tea drinking upon tuberculosis: a neglected issue.

BACKGROUND: Tuberculosis (TB) is a global public health issue posing serious harm to the human health. Many studies have suggested that smoking and excessive alcohol consumption are risk factors for TB. Laboratory evidence suggests that EGCG in tea leaves can arrest the growth of tubercle bacillus. Can drinking tea lead to decreased susceptibility of TB in humans? METHODS: A total of 574 TB patients and 582 healthy controls were recruited to participate in this case-control study. Self-designed questionnaire was used to collect data. Unconditioned logistic regression analysis was conducted to identify the associations between tea drinking and TB. RESULTS: Tea drinking has a negative association with TB, with OR = 0.583(0.423, 0.804) and P < 0.05. Drinking black tea, oolong and green tea are all negative association with TB, with OR being 0.683(0.517, 0.902), 0.674(0.508, 0.894) and 0.534(0.349, 0.817) respectively and P < 0.05. Trend χ (2) test indicated a decreasing risk for TB with increased tea consumption, with P < 0.05. CONCLUSION: There is a significance negative association between tea drinking and TB. Promoting the consumption of tea as the daily drink among populations, particularly those with high TB risk, may reduce the incidence of TB in the populations.

Gender and regional disparities of tuberculosis in Hunan, China.

INTRODUCTION: Major efforts have been made to improve the health care system in Hunan province, China. The aims of this study were to assess whether and to what extent these efforts have impacted on gender and regional disparities of Tuberculosis (TB) incidence in recent years, especially for less developed areas. METHODS: We obtained data from the 2005-2009 China Information System for Disease Control and Prevention (CISDCP)to conduct this study in Hunan province. Counties within the province were divided into four regions according to quartiles based on the 2007 per capita GDP. Index of Disparity (ID) and Relative Index of Inequality (RII) were used to measure the disparities of TB incidence in relation to gender and region. Bootstrap technique was used to increase the precision. RESULTS: The average annual incidence of TB was 111.75 per 100,000 in males and 43.44 per 100 000 in females in Hunan. The gender disparity was stable, with ID from 42.34 in 2005 to 43.92 in 2009. For regional disparity, ID, RII (mean) and RII (ratio) decreased significantly from 2005 to 2009 in males (P < 0.05) but remained stable among the female population. CONCLUSIONS: As interventions such as introduction of the New Rural Cooperative Scheme put in place to reduce health disparities in China, regional disparity in relation to incidence of TB decreased significantly, but the gender disparity remains in the Hunan province.

Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.

Introduction: Antimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined. Methods: Archived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing. Results: In all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95). Discussion: The observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.