Understanding Spatiotemporal Human Mobility Patterns for Malaria Control Using a Multiagent Mobility Simulation Model.

BACKGROUND: More details about human movement patterns are needed to evaluate relationships between daily travel and malaria risk at finer scales. A multiagent mobility simulation model was built to simulate the movements of villagers between home and their workplaces in 2 townships in Myanmar. METHODS: An agent-based model (ABM) was built to simulate daily travel to and from work based on responses to a travel survey. Key elements for the ABM were land cover, travel time, travel mode, occupation, malaria prevalence, and a detailed road network. Most visited network segments for different occupations and for malaria-positive cases were extracted and compared. Data from a separate survey were used to validate the simulation. RESULTS: Mobility characteristics for different occupation groups showed that while certain patterns were shared among some groups, there were also patterns that were unique to an occupation group. Forest workers were estimated to be the most mobile occupation group, and also had the highest potential malaria exposure associated with their daily travel in Ann Township. In Singu Township, forest workers were not the most mobile group; however, they were estimated to visit regions that had higher prevalence of malaria infection over other occupation groups. CONCLUSIONS: Using an ABM to simulate daily travel generated mobility patterns for different occupation groups. These spatial patterns varied by occupation. Our simulation identified occupations at a higher risk of being exposed to malaria and where these exposures were more likely to occur.

Antibody signatures of asymptomatic Plasmodium falciparum malaria infections measured from dried blood spots.

BACKGROUND: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar. METHODS: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections. RESULTS: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%). CONCLUSIONS: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure.

Pregnancy outcomes and risk of placental malaria after artemisinin-based and quinine-based treatment for uncomplicated falciparum malaria in pregnancy: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria in pregnancy, including asymptomatic infection, has a detrimental impact on foetal development. Individual patient data (IPD) meta-analysis was conducted to compare the association between antimalarial treatments and adverse pregnancy outcomes, including placental malaria, accompanied with the gestational age at diagnosis of uncomplicated falciparum malaria infection. METHODS: A systematic review and one-stage IPD meta-analysis of studies assessing the efficacy of artemisinin-based and quinine-based treatments for patent microscopic uncomplicated falciparum malaria infection (hereinafter uncomplicated falciparum malaria) in pregnancy was conducted. The risks of stillbirth (pregnancy loss at ≥ 28.0 weeks of gestation), moderate to late preterm birth (PTB, live birth between 32.0 and < 37.0 weeks), small for gestational age (SGA, birthweight of < 10th percentile), and placental malaria (defined as deposition of malaria pigment in the placenta with or without parasites) after different treatments of uncomplicated falciparum malaria were assessed by mixed-effects logistic regression, using artemether-lumefantrine, the most used antimalarial, as the reference standard. Registration PROSPERO: CRD42018104013. RESULTS: Of the 22 eligible studies (n = 5015), IPD from16 studies were shared, representing 95.0% (n = 4765) of the women enrolled in literature. Malaria treatment in this pooled analysis mostly occurred in the second (68.4%, 3064/4501) or third trimester (31.6%, 1421/4501), with gestational age confirmed by ultrasound in 91.5% (4120/4503). Quinine (n = 184) and five commonly used artemisinin-based combination therapies (ACTs) were included: artemether-lumefantrine (n = 1087), artesunate-amodiaquine (n = 775), artesunate-mefloquine (n = 965), and dihydroartemisinin-piperaquine (n = 837). The overall pooled proportion of stillbirth was 1.1% (84/4361), PTB 10.0% (619/4131), SGA 32.3% (1007/3707), and placental malaria 80.1% (2543/3035), and there were no significant differences of considered outcomes by ACT. Higher parasitaemia before treatment was associated with a higher risk of SGA (adjusted odds ratio [aOR] 1.14 per 10-fold increase, 95% confidence interval [CI] 1.03 to 1.26, p = 0.009) and deposition of malaria pigment in the placenta (aOR 1.67 per 10-fold increase, 95% CI 1.42 to 1.96, p < 0.001). CONCLUSIONS: The risks of stillbirth, PTB, SGA, and placental malaria were not different between the commonly used ACTs. The risk of SGA was high among pregnant women infected with falciparum malaria despite treatment with highly effective drugs. Reduction of malaria-associated adverse birth outcomes requires effective prevention in pregnant women.

No evidence of amplified Plasmodium falciparum plasmepsin II gene copy number in an area with artemisinin-resistant malaria along the China-Myanmar border.

BACKGROUND: The emergence and spread of artemisinin resistance in Plasmodium falciparum poses a threat to malaria eradication, including China's plan to eliminate malaria by 2020. Piperaquine (PPQ) resistance has emerged in Cambodia, compromising an important partner drug that is widely used in China in the form of dihydroartemisinin (DHA)-PPQ. Several mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 (k13) are associated with artemisinin resistance and have arisen spread in the Great Mekong subregion, including the China-Myanmar border. Multiple copies of the plasmepsin II/III (pm2/3) genes, located on chromosome 14, have been shown to be associated with PPQ resistance. METHODS: The therapeutic efficacy of DHA-PPQ for the treatment of uncomplicated P. falciparum was evaluated along the China-Myanmar border from 2010 to 2014. The dry blood spots samples collected in the efficacy study prior DHA-PPQ treatment and from the local hospital by passive detection were used to amplify k13 and pm2. Polymorphisms within k13 were genotyped by capillary sequencing and pm2 copy number was quantified by relative-quantitative real-time polymerase chain reaction. Treatment outcome was evaluated with the World Health Organization protocol. A linear regression model was used to estimate the association between the day 3 positive rate and k13 mutation and the relationship of the pm2 copy number variants and k13 mutations. RESULTS: DHA-PPQ was effective for uncomplicated P. falciparum infection in Yunnan Province with cure rates > 95%. Twelve non synonymous mutations in the k13 domain were observed among the 268 samples with the prevalence of 44.0% and the predominant mutation was F446I with a prevalence of 32.8%. Only one sample was observed with multi-copies of pm2, including parasites with and without k13 mutations. The therapeutic efficacy of DHA-PPQ was > 95% along the China-Myanmar border, consistent with the lack of amplification of pm2. CONCLUSION: DHA-PPQ for uncomplicated P. falciparum infection still showed efficacy in an area with artemisinin-resistant malaria along the China-Myanmar border. There was no evidence to show PPQ resistance by clinical study and molecular markers survey. Continued monitoring of the parasite population using molecular markers will be important to track emergence and spread of resistance in this region.

An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance.

BACKGROUND: Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance. METHODS: Methods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification. RESULTS: The optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use. CONCLUSIONS: The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.

A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections.

BACKGROUND: Greater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density "asymptomatic" infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions. METHODS: A highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons. RESULTS: The lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards-equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16-22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season. CONCLUSIONS: The DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

A Single Mutation in K13 Predominates in Southern China and Is Associated With Delayed Clearance of Plasmodium falciparum Following Artemisinin Treatment.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and poses a threat to malaria control and elimination. Mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 have been associated with delayed parasite clearance following artemisinin treatment elsewhere in the region, but not yet in China. METHODS: Therapeutic efficacy studies of artesunate and dihydroartemisinin-piperaquine were conducted from 2009 to 2012 in the Yunnan Province of China near the border with Myanmar. K13 mutations were genotyped by capillary sequencing of DNA extracted from dried blood spots collected in these clinical trials and in routine surveillance. Associations between K13 mutations and delayed parasite clearance were tested using regression models. RESULTS: Parasite clearance half-lives were prolonged after artemisinin treatment, with 44% of infections having half-lives >5 hours (n = 109). Fourteen mutations in K13 were observed, with an overall prevalence of 47.7% (n = 329). A single mutation, F446I, predominated, with a prevalence of 36.5%. Infections with F446I were significantly associated with parasitemia on day 3 following artemisinin treatment and with longer clearance half-lives. CONCLUSIONS: Plasmodium falciparum infections in southern China displayed markedly delayed clearance following artemisinin treatment. F446I was the predominant K13 mutation and was associated with delayed parasite clearance.

Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Artemether-Lumefantrine Pharmacokinetics and Clinical Response Are Minimally Altered in Pregnant Ugandan Women Treated for Uncomplicated Falciparum Malaria.

Artemether-lumefantrine is a first-line regimen for the treatment of uncomplicated malaria during the second and third trimesters of pregnancy. Previous studies have reported changes in the pharmacokinetics and clinical outcomes following treatment with artemether-lumefantrine in pregnant women compared to nonpregnant adults; however, the results are inconclusive. We conducted a study in rural Uganda to compare the pharmacokinetics of artemether-lumefantrine and the treatment responses between 30 pregnant women and 30 nonpregnant adults with uncomplicated Plasmodium falciparum malaria. All participants were uninfected with HIV, treated with a six-dose regimen of artemether-lumefantrine, and monitored clinically for 42 days. The pharmacokinetics of artemether, its metabolite dihydroartemisinin, and lumefantrine were evaluated for 21 days following treatment. We found no significant differences in the overall pharmacokinetics of artemether, dihydroartemisinin, or lumefantrine in a direct comparison of pregnant women to nonpregnant adults, except for a statistically significant but small difference in the terminal elimination half-lives of both dihydroartemisinin and lumefantrine. There were seven PCR-confirmed reinfections (5 pregnant and 2 nonpregnant participants). The observation of a shorter terminal half-life for lumefantrine may have contributed to a higher frequency of reinfection or a shorter posttreatment prophylactic period in pregnant women than in nonpregnant adults. While the comparable overall pharmacokinetic exposure is reassuring, studies are needed to further optimize antimalarial efficacy in pregnant women, particularly in high-transmission settings and because of emerging drug resistance. (This study is registered at ClinicalTrials.gov under registration no. NCT01717885.).

An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.

BACKGROUND: Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans. METHODS: A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR. RESULTS: Limits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80% humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR. CONCLUSIONS: This ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia.

Mobile phones improve case detection and management of malaria in rural Bangladesh.

BACKGROUND: The recent introduction of mobile phones into the rural Bandarban district of Bangladesh provided a resource to improve case detection and treatment of patients with malaria. METHODS: During studies to define the epidemiology of malaria in villages in south-eastern Bangladesh, an area with hypoendemic malaria, the project recorded 986 mobile phone calls from families because of illness suspected to be malaria between June 2010 and June 2012. RESULTS: Based on phone calls, field workers visited the homes with ill persons, and collected blood samples for malaria on 1,046 people. 265 (25%) of the patients tested were positive for malaria. Of the 509 symptomatic malaria cases diagnosed during this study period, 265 (52%) were detected because of an initial mobile phone call. CONCLUSION: Mobile phone technology was found to be an efficient and effective method for rapidly detecting and treating patients with malaria in this remote area. This technology, when combined with local knowledge and field support, may be applicable to other hard-to-reach areas to improve malaria control.

Comparison of deforestation and forest land use factors for malaria elimination in Myanmar.

Objectives: Within the remote region of Ann Township in Myanmar's Rakhine State, malaria prevalence has remained steady at ∼10% of the population from 2016-2019. Previous studies have linked areas of higher malaria prevalence in the region to heavily forested areas, however, little is known about how people live, work, and move through these areas. This work aims to disentangle landscape from land use in regard to malaria exposure. Methods: We investigated the roles of forest cover, forest loss, and land use activities with malaria prevalence through the combined use of land use surveys, malaria surveillance, and satellite earth observations. Results: Our results confirm previous research that linked areas of high forest cover with high malaria prevalence. However, areas experiencing high levels of deforestation were not associated with malaria prevalence. The land use factors that contribute most significantly to increased malaria risk remained those which put people in direct contact with forests, including conducting forest chores, having an outdoor job, and having a primary occupation in the logging and/or plantation industry. Conclusion: Malaria prevention methods in Myanmar should focus on anyone who lives near forests or engages in land use activities that bring them within proximity of forested landscapes, whether through occupation or chores.

An In Silico Analysis of Malaria Pre-Erythrocytic-Stage Antigens Interpreting Worldwide Genetic Data to Suggest Vaccine Candidate Variants and Epitopes.

Failure to account for genetic diversity of antigens during vaccine design may lead to vaccine escape. To evaluate the vaccine escape potential of antigens used in vaccines currently in development or clinical testing, we surveyed the genetic diversity, measured population differentiation, and performed in silico prediction and analysis of T-cell epitopes of ten such Plasmodium falciparum pre-erythrocytic-stage antigens using whole-genome sequence data from 1010 field isolates. Of these, 699 were collected in Africa (Burkina Faso, Cameroon, Guinea, Kenya, Malawi, Mali, and Tanzania), 69 in South America (Brazil, Colombia, French Guiana, and Peru), 59 in Oceania (Papua New Guinea), and 183 in Asia (Cambodia, Myanmar, and Thailand). Antigens surveyed include cell-traversal protein for ookinetes and sporozoites, circumsporozoite protein, liver-stage antigens 1 and 3, sporozoite surface proteins P36 and P52, sporozoite asparagine-rich protein-1, sporozoite microneme protein essential for cell traversal-2, and upregulated-in-infectious-sporozoite 3 and 4 proteins. The analyses showed that a limited number of these protein variants, when combined, would be representative of worldwide parasite populations. Moreover, predicted T-cell epitopes were identified that could be further explored for immunogenicity and protective efficacy. Findings can inform the rational design of a multivalent malaria vaccine.

Temporal and Spatial Differences between Symptomatic and Asymptomatic Malaria Infections in the Chittagong Hill Districts, Bangladesh.

Mapping asymptomatic malaria infections, which contribute to the transmission reservoir, is important for elimination programs. This analysis compared the spatiotemporal patterns of symptomatic and asymptomatic Plasmodium falciparum malaria infections in a cohort study of ∼25,000 people living in a rural hypoendemic area of about 179 km2 in a small area of the Chittagong Hill Districts of Bangladesh. Asymptomatic infections were identified by active surveillance; symptomatic clinical cases presented for care. Infections were identified by a positive rapid diagnostic test and/or microscopy. Fifty-three subjects with asymptomatic P. falciparum infection were compared with 572 subjects with symptomatic P. falciparum between mid-October 2009 and mid-October 2012 with regard to seasonality, household location, and extent of spatial clustering. We found increased spatial clustering of symptomatic compared with asymptomatic infections, and the areas of high intensity were only sometimes overlapping. Symptomatic cases had a distinct seasonality, unlike asymptomatic infections, which were detected year-round. In a comparison of 42 symptomatic Plasmodium vivax and 777 symptomatic P. falciparum cases from mid-October 2009 through mid-March 2015, we found substantial spatial overlap in areas with high infection rates, but the areas with the greatest concentration of infection differed. Detection of both symptomatic P. falciparum and symptomatic P. vivax infections was greater during the May-to-October high season, although a greater proportion of P. falciparum cases occurred during the high season compared with P. vivax. These findings reinforce that passive malaria surveillance and treatment of symptomatic cases will not eliminate the asymptomatic reservoirs that occur distinctly in time and space.

Temporal Dynamics of Subclinical Malaria in Different Transmission Zones of Myanmar.

Countries in the Greater Mekong Subregion have committed to eliminate Plasmodium falciparum malaria by 2025. Subclinical malaria infections that can be detected by highly sensitive polymerase chain reaction (PCR) testing in asymptomatic individuals represent a potential impediment to this goal, although the extent to which these low-density infections contribute to transmission is unclear. To understand the temporal dynamics of subclinical malaria in this setting, a cohort of 2,705 participants from three epidemiologically distinct regions of Myanmar was screened for subclinical P. falciparum and P. vivax infection using ultrasensitive PCR (usPCR). Standard rapid diagnostic tests (RDTs) for P. falciparum were also performed. Individuals who tested positive for malaria by usPCR were followed for up to 12 weeks. Regression analysis was performed to estimate whether the baseline prevalence of infection and the count of repeated positive tests were associated with demographic, behavioral, and clinical factors. At enrollment, the prevalence of subclinical malaria infection measured by usPCR was 7.7% (1.5% P. falciparum monoinfection, 0.3% mixed P. falciparum and P. vivax, and 6.0% P. vivax monoinfection), while P. falciparum prevalence measured by RDT was just 0.2%. Prevalence varied by geography and was higher among older people and in those with outdoor exposure and travel. No difference was observed in either the prevalence or count of subclinical infection by time of year, indicating that even in low-endemicity areas, a reservoir of subclinical infection persists year-round. If low-density infections are shown to represent a significant source of transmission, identification of high-risk groups and locations may aid elimination efforts.

Mapping hypoendemic, seasonal malaria in rural Bandarban, Bangladesh: a prospective surveillance.

BACKGROUND: Until recently the Chittagong Hill tracts have been hyperendemic for malaria. A past cross-sectional RDT based survey in 2007 recorded rates of approximately 15%. This study was designed to understand the present epidemiology of malaria in this region, to monitor and facilitate the uptake of malaria intervention activities of the national malaria programme and to serve as an area for developing new and innovative control strategies for malaria. METHODS: This research field area was established in two rural unions of Bandarban District of Bangladesh north of Bandarban city, which are known to be endemic for malaria due to Plasmodium falciparum. The project included the following elements: a) a demographic surveillance system including an initial census with updates every four months, b) periodic surveys of knowledge attitude and practice, c) a geographic information system, d) weekly active and continuous passive surveillance for malaria infections using smears, rapid tests and PCR, f) monthly mosquito surveillance, and e) daily weather measures. The programme included both traditional and molecular methods for detecting malaria as well as lab methods for speciating mosquitoes and detecting mosquitoes infected with sporozoites. RESULTS: The demographic surveillance enumerated and mapped 20,563 people, 75% of which were tribal non-Bengali. The monthly mosquito surveys identified 22 Anopheles species, eight of which were positive by circumsporozoite ELISA. The annual rate of malaria was close to 1% with 85% of cases in the rainy months of May-October. Definitive clustering identified in the low transmission season persisted during the high transmission season. CONCLUSION: This demographically and geographically defined area, near to the Myanmar border, which is also hypoendemic for malaria, will be useful for future studies of the epidemiology of malaria and for evaluation of strategies for malaria control including new drugs and vaccines.

Direct Comparison of Standard and Ultrasensitive PCR for the Detection of Plasmodium falciparum from Dried Blood Spots in Bagamoyo, Tanzania.

Ultrasensitive PCR used in low-transmission malaria-endemic settings has revealed a much higher burden of asymptomatic infections than that detected by rapid diagnostic tests (RDTs) or standard PCR, but there is limited evidence as to whether this is the case in higher transmission settings. Using dried blood spots (DBS) collected among 319 schoolchildren in Bagamoyo, Tanzania, we found good correlation (Pearson's R = 0.995) between Plasmodium falciparum parasite densities detected by a DNA-based 18s rRNA real-time PCR (qPCR) and an RNA-based ultrasensitive reverse transcriptase (RT)-PCR (usPCR) for the same target. Whereas prevalence by usPCR was higher than that found by qPCR (37% versus 32%), the proportion of additionally detected low-density infections (median parasite density < 0.050 parasites/µL) represented an incremental increase. It remains unclear to what extent these low-density infections may contribute to the infectious reservoir in different malaria transmission settings.

Prevalence and seroprevalence of Plasmodium infection in Myanmar reveals highly heterogeneous transmission and a large hidden reservoir of infection.

Malaria incidence in Myanmar has significantly reduced over recent years, however, completeness and timeliness of incidence data remain a challenge. The first ever nationwide malaria infection and seroprevalence survey was conducted in Myanmar in 2015 to better understand malaria epidemiology and highlight gaps in Annual Parasite Index (API) data. The survey was a cross-sectional two-stage stratified cluster-randomised household survey conducted from July-October 2015. Blood samples were collected from household members for ultra-sensitive PCR and serology testing for P. falciparum and P. vivax. Data was gathered on demography and a priori risk factors of participants. Data was analysed nationally and within each of four domains defined by API data. Prevalence and seroprevalence of malaria were 0.74% and 16.01% nationwide, respectively. Prevalent infection was primarily asymptomatic P. vivax, while P. falciparum was predominant in serology. There was large heterogeneity between villages and by domain. At the township level, API showed moderate correlation with P. falciparum seroprevalence. Risk factors for infection included socioeconomic status, domain, and household ownership of nets. Three K13 P. falciparum mutants were found in highly prevalent villages. There results highlight high heterogeneity of both P. falciparum and P. vivax transmission between villages, accentuated by a large hidden reservoir of asymptomatic P. vivax infection not captured by incidence data, and representing challenges for malaria elimination. Village-level surveillance and stratification to guide interventions to suit local context and targeting of transmission foci with evidence of drug resistance would aid elimination efforts.

Efavirenz-Based Antiretroviral Therapy Reduces Artemether-Lumefantrine Exposure for Malaria Treatment in HIV-Infected Pregnant Women.

BACKGROUND: The choice of malaria treatment for HIV-infected pregnant women receiving efavirenz-based antiretroviral therapy must consider the potential impact of drug interactions on antimalarial exposure and clinical response. The aim of this study was to investigate the effects of efavirenz on artemether-lumefantrine (AL) because no studies have isolated the impact of efavirenz for HIV-infected pregnant women. METHODS: A prospective clinical pharmacokinetic (PK) study compared HIV-infected, efavirenz-treated pregnant women with HIV-uninfected pregnant women in Tororo, Uganda. All women received the standard 6-dose AL treatment regimen for Plasmodium falciparum malaria with intensive PK samples collected over 21 days and 42-days of clinical follow-up. PK exposure parameters were calculated for artemether, its active metabolite dihydroartemisinin (DHA), and lumefantrine to determine the impact of efavirenz. RESULTS: Nine HIV-infected and 30 HIV-uninfected pregnant women completed intensive PK evaluations. Relative to controls, concomitant efavirenz therapy lowered the 8-hour artemether concentration by 76% (P = 0.013), DHA peak concentration by 46% (P = 0.033), and day 7 and 14 lumefantrine concentration by 61% and 81% (P = 0.046 and 0.023), respectively. In addition, there were nonsignificant reductions in DHA area under the concentration-time curve0-8hr (35%, P = 0.057) and lumefantrine area under the concentration-time curve0-∞ (34%, P = 0.063) with efavirenz therapy. CONCLUSIONS: Pregnant HIV-infected women receiving efavirenz-based antiretroviral therapy during malaria treatment with AL showed reduced exposure to both the artemisinin and lumefantrine. These data suggest that malaria and HIV coinfected pregnant women may require adjustments in AL dosage or treatment duration to achieve exposure comparable with HIV-uninfected pregnant women.