Scientific Findings of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

For a decade, the Southern and Central Africa International Center of Excellence for Malaria Research has operated with local partners across study sites in Zambia and Zimbabwe that range from hypo- to holoendemic and vary ecologically and entomologically. The burden of malaria and the impact of control measures were assessed in longitudinal cohorts, cross-sectional surveys, passive and reactive case detection, and other observational designs that incorporated multidisciplinary scientific approaches: classical epidemiology, geospatial science, serosurveillance, parasite and mosquito genetics, and vector bionomics. Findings to date have helped elaborate the patterns and possible causes of sustained low-to-moderate transmission in southern Zambia and eastern Zimbabwe and recalcitrant high transmission and fatality in northern Zambia. Cryptic and novel mosquito vectors, asymptomatic parasite reservoirs in older children, residual parasitemia and gametocytemia after treatment, indoor residual spraying timed dyssynchronously to vector abundance, and stockouts of essential malaria commodities, all in the context of intractable rural poverty, appear to explain the persistent malaria burden despite current interventions. Ongoing studies of high-resolution transmission chains, parasite population structures, long-term malaria periodicity, and molecular entomology are further helping to lay new avenues for malaria control in southern and central Africa and similar settings.

Policy Implications of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

The International Centers of Excellence for Malaria Research (ICEMR) were established by the National Institute of Allergy and Infectious Diseases more than a decade ago to provide multidisciplinary research support to malaria control programs worldwide, operating in endemic areas and contributing technology, expertise, and ultimately policy guidance for malaria control and elimination. The Southern and Central Africa ICEMR has conducted research across three main sites in Zambia and Zimbabwe that differ in ecology, entomology, transmission intensity, and control strategies. Scientific findings led to new policies and action by the national malaria control programs and their partners in the selection of methods, materials, timing, and locations of case management and vector control. Malaria risk maps and predictive models of case detection furnished by the ICEMR informed malaria elimination programming in southern Zambia, and time series analyses of entomological and parasitological data motivated several major changes to indoor residual spray campaigns in northern Zambia. Along the Zimbabwe-Mozambique border, temporal and geospatial data are currently informing investigations into a recent resurgence of malaria. Other ICEMR findings pertaining to parasite and mosquito genetics, human behavior, and clinical epidemiology have similarly yielded immediate and long-term policy implications at each of the sites, often with generalizable conclusions. The ICEMR programs thereby provide rigorous scientific investigations and analyses to national control and elimination programs, without which the impediments to malaria control and their potential solutions would remain understudied.

Detecting Plasmodium falciparum in community surveys: a comparison of Paracheck Pf® Test and ICT Malaria Pf® Cassette Test to polymerase chain reaction in Mutasa District, Zimbabwe.

BACKGROUND: Microscopy and rapid diagnostic tests (RDTs) are the main techniques used to diagnose malaria. While microscopy is considered the gold standard, RDTs have established popularity as they allow for rapid diagnosis with minimal technical skills. This study aimed to compare the diagnostic performance of two Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs (Paracheck Pf® Test (Paracheck) and Malaria Pf™ ICT (ICT)) to polymerase chain reaction (PCR) in a community survey. METHODS: A cross-sectional study was conducted between October 2012 and December 2014 in Mutasa District, Manicaland Province, eastern Zimbabwe. Households were randomly selected using satellite imagery, and 224 households were visited. Residents present in the household on the date of the visit were recruited for the study. Participants of all age groups from the selected households were screened with Paracheck and ICT RDTs in parallel. Dried blood spots (DBS) and thin and thick smears were collected. Parasite DNA extracted from the DBS was subjected to nested PCR targeting the Plasmodium cytochrome b mitochondrial gene. Data analysis was performed using the Cohen's Kappa test to determine the interrater agreement and the sensitivity and specificity of the diagnostic test were reported. RESULTS: Results from a total of 702 participants were analysed. Most were females, 397 (57%), and the median age of participants was 21 years with an interquartile range of 9-39 years. Of those who were screened, 8 (1.1%), 35 (5.0%), and 21 (2.9%) were malaria parasite positive by microscopy, RDT and PCR, respectively. Paracheck and ICT RDTs had a 100% agreement. Comparing RDT and PCR results, 34 participants (4.8%) had discordant results. Most of the discordant cases were RDT positive but PCR negative (n = 24). Half of those RDT positive, but PCR negative individuals reported anti-malarials to use in the past month, which is significantly higher than reported anti-malarial drug use in the population (p < 0.001). The participant was febrile on the day of the visit, but relying on PfHRP2-based RDT would miss this case. Among the diagnostic methods evaluated, with reference to PCR, the sensitivity was higher with the RDT (52.4%) while specificity was higher with the microscopy (99.9%). The positive predictive value (PPV) was higher with the microscopy (87.5%), while the negative predictive values were similar for both microscopy and RDTs (98%). Overall, a strong correlated agreement with PCR was observed for the microscopy (97.9%) and the RDTs (95.2%). CONCLUSIONS: Paracheck and ICT RDTs showed 100% agreement and can be used interchangeably. As malaria transmission declines and Zimbabwe aims to reach malaria elimination, management of infected individuals with low parasitaemia as well as non-P. falciparum infection can be critical.

Steep Rebound of Chloroquine-Sensitive Plasmodium falciparum in Zimbabwe.

Removal of chloroquine from national malaria formularies can lead to the reversion of resistant Plasmodium falciparum to wild-type. We report a steep decline in chloroquine-resistant P falciparum within 10 years of national discontinuation of chloroquine monotherapy in Zimbabwe. Drug resistance surveillance is a vital component of malaria control programs, and the experience with chloroquine in Zimbabwe and elsewhere in sub-Saharan Africa is illustrative of the potentially rapid and dramatic impact of drug policy on antimalarial resistance.

Distinct Antibody Signatures Associated with Different Malaria Transmission Intensities in Zambia and Zimbabwe.

Antibodies to Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination.IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens-mainly vaccine candidates-have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance.

Inclusion of edaphic predictors for enhancement of models to determine distribution of soil-transmitted helminths: the case of Zimbabwe.

BACKGROUND: Reliable mapping of soil-transmitted helminth (STH) parasites requires rigorous statistical and machine learning algorithms capable of integrating the combined influence of several determinants to predict distributions. This study tested whether combining edaphic predictors with relevant environmental predictors improves model performance when predicting the distribution of STH, Ascaris lumbricoides and hookworms at a national scale in Zimbabwe. METHODS: Geo-referenced parasitological data obtained from a 2010/2011 national survey indicating a confirmed presence or absence of STH among school children aged 10-15 years was used to calibrate ten species distribution models (SDMs). The performance of SDMs calibrated with a set of environmental and edaphic variables was compared to that of SDMs calibrated with environmental variables only. Model performance was evaluated using the true skill statistic and receiver operating characteristic curve. RESULTS: Results show a significant improvement in model performance for both A. lumbricoides and hookworms for all ten SDMs after edaphic variables were combined with environmental variables in the modelling of the geographical distribution of the two STHs at national scale. Using the top three performing models, a consensus prediction was developed to generate the first continuous maps of the potential distribution of the two STHs in Zimbabwe. CONCLUSIONS: The findings from this study demonstrate significant model improvement if relevant edaphic variables are included in model calibration resulting in more accurate mapping of STH. The results also provide spatially-explicit information to aid targeted control of STHs in Zimbabwe and other countries with STH burden.

Individual- and Household-Level Risk Factors Associated with Malaria in Mutasa District, Zimbabwe: A Serial Cross-Sectional Study.

Malaria constitutes a major public health problem in Zimbabwe, particularly in the north and east bordering Zambia and Mozambique. In Manicaland Province in eastern Zimbabwe, malaria transmission is seasonal and unstable. Over the past decade, Manicaland Province has reported increased malaria transmission due to limited funding, drug resistance and insecticide resistance. The aim of this study was to identify risk factors at the individual and household levels to better understand the epidemiology of malaria and guide malaria control strategies in eastern Zimbabwe. Between October 2012 and September 2014, individual demographic data and household characteristics were collected from cross-sectional surveys of 1,116 individuals residing in 316 households in Mutasa District, one of the worst affected districts. Factors associated with malaria, measured by rapid diagnostic test (RDT), were identified through multilevel logistic regression models. A total of 74 participants were RDT positive. Sleeping under a bed net had a protective effect against malaria despite pyrethroid resistance in the mosquito vector. Multivariate analysis showed that malaria risk was higher among individuals younger than 25 years, residing in households located at a lower household density and in closer proximity to the Mozambique border. The risk factors identified need to be considered in targeting malaria control interventions to reduce host-vector interactions.

High-Resolution Plasmodium falciparum Malaria Risk Mapping in Mutasa District, Zimbabwe: Implications for Regaining Control.

In Zimbabwe, more than half of malaria cases are concentrated in Manicaland Province, where seasonal malaria epidemics occur despite intensified control strategies. The objectives of this study were to develop a prediction model based on environmental risk factors and obtain seasonal malaria risk maps for Mutasa District, one of the worst affected districts in Manicaland Province. From October 2012 to September 2015, 483 households were surveyed, and 104 individuals residing within 69 households had positive rapid diagnostic test results. Logistic regression was used to model the probability of household positivity as a function of the environmental covariates extracted from high-resolution remote sensing data sources. Model predictions and prediction standard errors were generated for the rainy and dry seasons. The resulting maps predicted elevated risk during the rainy season, particularly in low-lying areas bordering Mozambique. In contrast, the risk of malaria was low across the study area during the dry season with foci of malaria risk scattered along the northern and western peripheries of the study area. These findings underscore the need for strong cross-border malaria control initiatives to complement country-specific interventions.

Reduction in Malaria Incidence following Indoor Residual Spraying with Actellic 300 CS in a Setting with Pyrethroid Resistance: Mutasa District, Zimbabwe.

BACKGROUND: More than half of malaria cases in Zimbabwe are concentrated in Manicaland Province, where seasonal malaria epidemics occur despite intensified control strategies. Recently, high levels of pyrethroid and carbamate resistance were detected in Anopheles funestus, the major malaria vector in eastern Zimbabwe. In response, a single round of indoor residual spraying (IRS) using pirimiphos-methyl (an organophosphate) was implemented in four high burden districts of Manicaland Province from November 1, 2014 to December 19, 2014. The objective of this study was to evaluate the effect of this programmatic switch in insecticides on malaria morbidity reported from health care facilities in Mutasa District, one of the worst affected districts in Manicaland Province. METHODS: The number of weekly malaria cases for each health facility 24 months prior to the 2014 IRS campaign and in the subsequent high transmission season were obtained from passive case surveillance. Environmental variables were extracted from remote-sensing data sources and linked to each health care facility. Negative binomial regression was used to model the weekly number of malaria cases, adjusted for seasonality and environmental variables. RESULTS: From December 2012 to May 2015, 124,206 malaria cases were reported from 42 health care facilities in Mutasa District. Based on a higher burden of malaria, 20 out of 31 municipal wards were sprayed in the district. Overall, 87.3% of target structures were sprayed and 92.1% of the target population protected. During the 6 months after the 2014 IRS campaign, a period when transmission would have otherwise peaked, the incidence of malaria was 38% lower than the preceding 24 months at health facilities in the sprayed wards. CONCLUSIONS: Pirimiphos-methyl had a measurable impact on malaria incidence and is an effective insecticide for the control of An. funestus in eastern Zimbabwe.

Distribution of schistosomiasis and soil transmitted helminthiasis in Zimbabwe: towards a national plan of action for control and elimination.

BACKGROUND: Schistosomiasis and STH are among the list of neglected tropical diseases considered for control by the WHO. Although both diseases are endemic in Zimbabwe, no nationwide control interventions have been implemented. For this reason in 2009 the Zimbabwe Ministry of Health and Child Care included the two diseases in the 2009-2013 National Health Strategy highlighting the importance of understanding the distribution and burden of the diseases as a prerequisite for elimination interventions. It is against this background that a national survey was conducted. METHODOLOGY: A countrywide cross-sectional survey was carried out in 280 primary schools in 68 districts between September 2010 and August 2011. Schistosoma haematobium was diagnosed using the urine filtration technique. Schistosoma mansoni and STH (hookworms, Trichuris trichiura, Ascaris lumbricoides) were diagnosed using both the Kato Katz and formol ether concentration techniques. MAIN FINDINGS: Schistosomiasis was more prevalent country-wide (22.7%) than STH (5.5%). The prevalence of S. haematobium was 18.0% while that of S. mansoni was 7.2%. Hookworms were the most common STH with a prevalence of 3.2% followed by A. lumbricoides and T. trichiura with prevalence of 2.5% and 0.1%, respectively. The prevalence of heavy infection intensity as defined by WHO for any schistosome species was 5.8% (range 0%-18.3% in districts). Only light to moderate infection intensities were observed for STH species. The distribution of schistosomiasis and STH varied significantly between provinces, districts and schools (p<0.001). Overall, the prevalence of co-infection with schistosomiasis and STH was 1.5%. The actual co-endemicity of schistosomiasis and STH was observed in 43 (63.2%) of the 68 districts screened. CONCLUSION AND RECOMMENDATIONS: This study provided comprehensive baseline data on the distribution of schistosomiasis and STH that formed the basis for initiating a national control and elimination programme for these two neglected tropical diseases in Zimbabwe.

Changes in the burden of malaria following scale up of malaria control interventions in Mutasa District, Zimbabwe.

BACKGROUND: To better understand trends in the burden of malaria and their temporal relationship to control activities, a survey was conducted to assess reported cases of malaria and malaria control activities in Mutasa District, Zimbabwe. METHODS: Data on reported malaria cases were abstracted from available records at all three district hospitals, three rural hospitals and 25 rural health clinics in Mutasa District from 2003 to 2011. RESULTS: Malaria control interventions were scaled up through the support of the Roll Back Malaria Partnership, the Global Fund to Fight AIDS, Tuberculosis and Malaria, and The President's Malaria Initiative. The recommended first-line treatment regimen changed from chloroquine or a combination of chloroquine plus sulphadoxine/pyrimethamine to artemisinin-based combination therapy, the latter adopted by 70%, 95% and 100% of health clinics by 2008, 2009 and 2010, respectively. Diagnostic capacity improved, with rapid diagnostic tests (RDTs) available in all health clinics by 2008. Vector control consisted of indoor residual spraying and distribution of long-lasting insecticidal nets. The number of reported malaria cases initially increased from levels in 2003 to a peak in 2008 but then declined 39% from 2008 to 2010. The proportion of suspected cases of malaria in older children and adults remained high, ranging from 75% to 80%. From 2008 to 2010, the number of RDT positive cases of malaria decreased 35% but the decrease was greater for children younger than five years of age (60%) compared to older children and adults (26%). CONCLUSIONS: The burden of malaria in Mutasa District decreased following the scale up of malaria control interventions. However, the persistent high number of cases in older children and adults highlights the need for strategies to identify locally effective control measures that target all age groups.

Evaluation of "Cyscope", a novel fluorescence-based microscopy technique for the detection of malaria.

INTRODUCTION: This study was designed to compare the detection of malaria parasites in peripheral blood smears using the Cyscope malaria rapid fluorescent microscopic technique and light microscopy of Giemsa-stained smears. METHODOLOGY: A total of 295 blood smears were collected from patients of all age groups presenting with clinical signs and symptoms of malaria to 10 City Health Clinics in Harare. For each patient two blood films were prepared. Microscopic examination was done independently in two laboratories, with one performing the Giemsa stain and the other the Cyscope method. After the tests were completed, the results were then matched and recorded without any alterations. RESULTS: An equal number of men and women were malaria positive and their ages ranged from five to 66 years. Concordance in the detection of parasites (positive or negative) was 98.6% (291/295). In all four cases of discordance, malaria parasites were detected using the Cyscope but not with conventional microscopy. The Cyscope gave a 100% sensitivity and a specificity of 98.6%. CONCLUSION: The Cyscope may be a valuable addition to diagnostics of malaria in resource-limited settings such as Zimbabwe.

Malaria epidemiology and control in Southern Africa.

The burden of malaria has decreased dramatically within the past several years in parts of sub-Saharan Africa, following the scale-up of interventions supported by the Roll Back Malaria Partnership, the President's Malaria Initiative and other partners. It is important to appreciate that the reductions in malaria have not been uniform between and within countries, with some areas experiencing resurgence instead. Furthermore, while interventions have greatly reduced the burden of malaria in many countries, it is also recognized that the malaria decline pre-dated widespread intervention efforts, at least in some cases where data are available. This raises more questions as what other factors may have been contributing to the reduction in malaria transmission and to what extent. The International Center of Excellence for Malaria Research (ICEMR) in Southern Africa aims to better understand the underlying malaria epidemiology, vector ecology and parasite genomics using three contrasting settings of malaria transmission in Zambia and Zimbabwe: an area of successful malaria control, an area of resurgent malaria and an area where interventions have not been effective. The Southern Africa ICEMR will capitalize on the opportunity to investigate the complexities of malaria transmission while adapting to intervention and establish the evidence-base to guide effective and sustainable malaria intervention strategies. Key approaches to attain this goal for the region will include close collaboration with national malaria control programs and contribution to capacity building at the individual, institutional and national levels.

Analysis of genetic polymorphism in select vaccine candidate antigens and microsatellite loci in Plasmodium falciparum from endemic areas at varying altitudes.

Plasmodium falciparum parasites obtained from symptomatic patients attending clinics in Bindura (altitude 1100 m), Chiredzi (600 m) and Kariba (<600 m), previously reported to differ in malaria endemicity were genotyped on the msp-1, msp-2 and glurp loci to examine the extent of parasite genetic diversity. While the parasites were monomorphic for msp-1 allele RO33 from the three locations, the K1 allele was over-represented in Kariba (p=0.02) and Mad20 alleles occurred at a higher frequency in Bindura. A similar PCR analysis for glurp and the two main allelic families of msp-2, i.e. IC/3D7 and FC-27 revealed minimal differences in the parasite population. A total of 8 msp-1 Block 2 and 11 msp-2 genotypes were identified from the three locations combined. On the glurp locus, 13 different genotypes ranging in size from 660 to 1160 bp were detected in parasites obtained from Bindura and Kariba. To gain further insight into P. falciparum genetic diversity in the three different geographical locations, parasites were examined for neutral microsatellite markers (C4M8, C13M30 and TA81). The number of microsatellite alleles ranged from 8 to 17 and the average expected heterozygosity (HE) for the three areas combined was 0.83 suggesting that the parasite population of Zimbabwe is genetically heterogeneous. These findings have implications in understanding the impact of genetic variation on immunity and possibly emergence of drug resistance.

High prevalence of molecular markers for resistance to chloroquine and pyrimethamine in Plasmodium falciparum from Zimbabwe.

Chloroquine has been the first line drug of treatment for malaria in Zimbabwe until a recent adoption of an interim policy to treat using a combination of chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). We examined the prevalence of parasites with mutations associated with resistance to the drug combination in three areas that have been previously described to differ in malaria endemicity. Our results show that the parasite population from the three areas had a high prevalence of molecular markers of resistance to chloroquine and pyrimethamine. The prevalence of crt (K76T) was 64, 82, and 92% for Chiredzi, Kariba, and Bindura, respectively. On the dhfr locus, the presence of triple mutations (codons 51, 59, and 108) was approximately 50% for all the three locations. On the other hand, the prevalence of dhps mutations (codons 436, 437, and 540) was low accounting for less than 20% in all the areas. Studies reported here demonstrate widespread prevalence of molecular markers associated with chloroquine and pyrimethamine resistance and should be taken into consideration for further refinement of malaria control strategies in Zimbabwe. The design and implementation of successful control strategies would benefit from understanding the prevalence of mutations associated with drug resistance in parasite populations.

Antibody responses to Plasmodium falciparum vaccine candidate antigens in three areas distinct with respect to altitude.

Antibody levels against malaria antigens were measured among patients presenting with uncomplicated malaria at health centers from three locations in Zimbabwe (Bindura, Chiredzi and Kariba) that are distinct with regard to altitude and climatic conditions. Antibody levels were determined by ELISA using the antigens, apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), circumsporozoite surface protein (CSP), merozoite surface protein 1 (MSP-1) and Pfg27. For all the antigens tested, IgG and IgM levels were higher for Bindura (altitude 1100 m) compared to Kariba (<600 m, altitude) and Chiredzi (approximately 600 m, altitude) with the exception of IgG and IgM to AMA-1 and EBA-175 which were similar between Chiredzi and Bindura. Plasma samples were further analyzed for their functional activity by testing their ability to inhibit the growth of Plasmodium falciparum in culture. Our results, determined by microscopy and verified by the LDH assay revealed that plasma from the three locations had similar inhibitory activity against the growth of P. falciparum in vitro. Our data revealed that highest growth inhibition correlated with the highest levels of MSP-1 antibody values.

Association of house spraying with suppressed levels of drug resistance in Zimbabwe.

BACKGROUND: Public health strategies are needed to curb antimalarial drug resistance. Theoretical argument points to an association between malaria transmission and drug resistance although field evidence remains limited. Field observations, made in Zimbabwe, on the relationship between transmission and multigenic drug resistance, typified by chloroquine, are reported here. METHODS: Periodic assessments of the therapeutic response of uncomplicated falciparum malaria to chloroquine in two selectively sprayed or unsprayed health centre catchments, from 1995 - 2003. Cross-sectional analysis of in vivo chloroquine failure events for five sites in relation to natural endemicity and spraying history. RESULTS: During selective house spraying, the chloroquine failure rate for the sprayed catchment decreased, such that, after four years, the odds of chloroquine failure were 4x lower than before start of spraying in the area (OR 0.2, 95% CI 0.07 - 0.75, p = 0.010, n = 100). Chloroquine failure odds for the sprayed area became 4x lower than contemporaneous failure odds for the unsprayed area (OR 0.2 95% CI 0.08 - 0.65, p = 0.003, n = 156), although the likelihood of failure was not significantly different for the two catchments before selective spraying started (OR 0.5, 95% CI 0.21 - 1.32; p = 0.170, n = 88). When spraying ended, in 1999, the drug failure odds for the former sprayed area increased back 4 fold by 2003 (OR 4.2, 95%CI 1.49 - 11.78, p = 0.004, n = 146). High altitude areas with naturally lower transmission exhibited a 6x lower likelihood of drug failure than low-lying areas (OR 0.16 95% CI 0.068 - 0.353, -2 log likelihood change 23.239, p < 0.001, n = 465). Compared to sites under ongoing annual spraying, areas that were last sprayed 3-7 years ago experienced a 4-fold higher probability of chloroquine failure (OR 4.1, 95%CI 1.84 - 9.14, -2 log likelihood change 13.956, p < 0.001). CONCLUSION: Reduced transmission is associated with suppressed levels of resistance to chloroquine and presumably other regimens with multigenic drug resistance. It seems the adoption of transmission control alongside combination chemotherapy is a potent strategy for the future containment of drug-resistant malaria.