Malaria Research for Tailored Control and Elimination Strategies in the Greater Mekong Subregion.

The malaria landscape in the Greater Mekong Subregion has experienced drastic changes with the ramp-up of the control efforts, revealing formidable challenges that slowed down the progress toward malaria elimination. Problems such as border malaria and cross-border malaria introduction, multidrug resistance in Plasmodium falciparum, the persistence of Plasmodium vivax, the asymptomatic parasite reservoirs, and insecticide resistance in primary vectors require integrated strategies tailored for individual nations in the region. In recognition of these challenges and the need for research, the Southeast Asian International Center of Excellence for Malaria Research has established a network of researchers and stakeholders and conducted basic and translational research to identify existing and emerging problems and develop new countermeasures. The installation of a comprehensive disease and vector surveillance system at sentinel sites in border areas with the implementation of passive/active case detection and cross-sectional surveys allowed timely detection and management of malaria cases, provided updated knowledge for effective vector control measures, and facilitated the efficacy studies of antimalarials. Incorporating sensitive molecular diagnosis to expose the significance of asymptomatic parasite reservoirs for sustaining transmission helped establish the necessary evidence to guide targeted control to eliminate residual transmission. In addition, this program has developed point-of-care diagnostics to monitor the quality of artemisinin combination therapies, delivering the needed information to the drug regulatory authorities to take measures against falsified and substandard antimalarials. To accelerate malaria elimination, this program has actively engaged with stakeholders of all levels, fostered vertical and horizontal collaborations, and enabled the effective dissemination of research findings.

Anopheles bionomics in a malaria endemic area of southern Thailand.

BACKGROUND: Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand. METHODS: Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection. RESULTS: A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) (87.16%, n = 5035), An. dirus s.l. (7.05%, n = 407) and An. barbirostris s.l. (2.86%, n = 165). Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), of the An. dirus (s.l.) collected, 100% were An. baimaii (n = 348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (n = 44) and 6.38% were An. sawadwongporni (n = 3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (Kruskal-Wallis H-test: χ2 = 80.89, P < 0.0001, n = 5666), with more An. barbirostris (s.l.) and An. maculatus (s.l.) caught earlier compared to An. minimus (s.l.) (P = 0.0001 and P < 0.0001, respectively) and An. dirus (s.l.) (P = 0.0082 and P < 0.001, respectively). The proportion of parous An. minimus (s.l.) captured by hour increased throughout the night (Wald Chi-square: χ2 = 17.31, P = 0.000, odds ratio = 1.0535, 95% confidence interval 1.0279-1.0796, n = 3400). Overall, An. minimus (s.l.) parity was 67.68% (2375/3509) with an intra-cluster correlation of 0.0378. A power calculation determined that an An. minimus (s.l.) parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster, at an α = 0.05, will provide 82% power to detect a significant difference following ivermectin MDA. CONCLUSIONS: The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity.

Epidemiological profiles of recurrent malaria episodes in an endemic area along the Thailand-Myanmar border: a prospective cohort study.

BACKGROUND: In low malaria transmission areas, many people acquire multiple malaria infections within a single season. This study aimed to describe the pattern and epidemiological profile of malaria recurrence in a hypoendemic area of western Thailand and identify factors associated with having multiple malaria episodes. METHODS: An open cohort of 7000 residents in seven clusters along the Thai-Myanmar border was followed during a 6.5-year period (2011-mid 2017). Symptomatic and asymptomatic malaria infections were detected by passive case detection (PCD), weekly household visit, and mass blood surveys every 4-6 months. Malaria recurrence was defined as subsequent parasitaemic episodes occurred later than 7 days after receiving anti-malarial treatment. This study focused on analysis of recurrent episodes that occurred within 1 year after treatment. Numbers of malaria cases with single and multiple episodes were compared between clusters. Kaplan-Meier curve was performed to determine the intervals of recurrent episodes by Plasmodium species and age groups. The ordinal logistic model was used to determine factors associated with multiple malaria episodes, and to compare with single episodes, and those with no malaria infection. RESULTS: The cumulative incidence of malaria in the study area was 5.2% over the 6.5 years. Overall, 410 malaria patients were detected. Of these patients, 20% and 16% had multiple malaria episodes during the entire period and within 1 year after initial treatment, respectively. About 80% of repeated malaria episodes were caused by the same Plasmodium species as the primary infections. The median interval and interquartile range (IQR) between the first and second episode was 88 (43-175) days for all parasites, 56 (35-133) days for two Plasmodium falciparum episodes, and 90 (59-204) days for two Plasmodium vivax episodes. The interval between the episodes was increased with age. Factors significantly associated with multiple episodes of malaria infection included male sex, young age, Karen ethnicity, forest-related occupation, and having other malaria infected persons in the same house in the same period. CONCLUSIONS: People who have multiple malaria episodes may play an important role in maintaining malaria transmission in the area. Understanding epidemiological profiles of this group is important for planning strategies to achieve the elimination goal.

Prevalence of asymptomatic Plasmodium infections with sub-microscopic parasite densities in the northwestern border of Thailand: a potential threat to malaria elimination.

BACKGROUND: Asymptomatic infections with sub-microscopic Plasmodium serve as a silent reservoir of disease, critical to sustaining a low level of remanent malaria in the population. These infections must be effectively identified and targeted for elimination. The sensitivity of light microscopy, the traditional method used for diagnosing Plasmodium infections, is frequently insufficient for detecting asymptomatic infections due to the low density of parasitaemia. The objective of this study was to explore the current prevalence of asymptomatic sub-microscopic Plasmodium carriages to evaluate the parasite reservoir amongst residents from 7 hamlets in Tak Province in northwestern Thailand using a highly sensitive molecular method. METHODS: Malaria infection was screened in a real-world setting from 3650 finger-prick blood specimens collected in a mass cross-sectional survey using light microscopy and loop-mediated isothermal amplification (LAMP). LAMP results were later confirmed in a laboratory setting in Bangkok using nested PCR, restriction enzyme digestion and DNA sequencing. The association of malaria infection with demographic factors was explored. RESULTS: Parasite prevalence was 0.27% (10/3650) as determined by microscopy. Sub-microscopic infection prevalence was 2.33% (85/3650) by LAMP. Of these, 30.6% (26/85) were infected with Plasmodium falciparum, 52.9% (45/85) with Plasmodium vivax, 2.4% (2/85) with Plasmodium malariae, 4.7% (4/85) with mixed P. falciparum and P. vivax, and 9.4% (8/85) had parasite densities too low for species identification. Asymptomatic carriages (T < 37.5 °C) accounted for 95% (76/80) of all sub-microscopic cases with the highest prevalence occurring in the subjects 31-45 years of age (p ≤ 0.035). Participants working on plantations or as merchants had an increased infection risk. Evaluation by microscopy identified 10.53% (10/95) of all Plasmodium infected participants. CONCLUSION: Participants carrying asymptomatic Plasmodium infections with sub-microscopic parasite densities are considerable in this area. These findings provide the true disease burden and risk factors in this region. This information helps to direct policy makers towards better schemes and delivery of targeted interventions. Moreover, this is the first study to use LAMP in mass screening for sub-clinical and sub-microscopic infections in a field setting in Thailand. LAMP proves to be a sensitive and field-deployable assay suitable for national malaria control screening campaigns.

Imported Plasmodium falciparum and locally transmitted Plasmodium vivax: cross-border malaria transmission scenario in northwestern Thailand.

BACKGROUND: Cross-border malaria transmission is an important problem for national malaria control programmes. The epidemiology of cross-border malaria is further complicated in areas where Plasmodium falciparum and Plasmodium vivax are both endemic. By combining passive case detection data with entomological data, a transmission scenario on the northwestern Thai-Myanmar border where P. falciparum is likely driven by importation was described, whereas P. vivax is also locally transmitted. This study highlights the differences in the level of control required to eliminate P. falciparum and P. vivax from the same region. METHODS: Malaria case data were collected from malaria clinics in Suan Oi village, Tak Province, Thailand between 2011 and 2014. Infections were diagnosed by light microscopy. Demographic data, including migrant status, were correlated with concomitantly collected entomology data from 1330 mosquito trap nights using logistic regression. Malaria infection in the captured mosquitoes was detected by ELISA. RESULTS: Recent migrants were almost four times more likely to be infected with P. falciparum compared with Thai patients (OR 3.84, p < 0.001) and cases were significantly associated with seasonal migration. However, P. falciparum infection was not associated with the Anopheles mosquito capture rates, suggesting predominantly imported infections. In contrast, recent migrants were equally likely to present with P. vivax as mid-term migrants. Both migrant groups were twice as likely to be infected with P. vivax in comparison to the resident Thai population (OR 1.96, p < 0.001 and OR 1.94, p < 0.001, respectively). Plasmodium vivax cases were strongly correlated with age and local capture rates of two major vector species Anopheles minimus and Anopheles maculatus (OR 1.23, p = 0.020 and OR 1.33, p = 0.046, respectively), suggesting that a high level of local transmission might be causing these infections. CONCLUSIONS: On the Thai-Myanmar border, P. falciparum infections occur mostly in the recent migrant population with a seasonality reflecting that of agricultural activity, rather than that of the local mosquito population. This suggests that P. falciparum was mostly imported. In contrast, P. vivax cases were significantly associated with mosquito capture rates and less with migrant status, indicating local transmission. This highlights the different timelines and requirements for P. falciparum and P. vivax elimination in the same region and underlines the importance of multinational, cross-border malaria control.

Common asymptomatic and submicroscopic malaria infections in Western Thailand revealed in longitudinal molecular and serological studies: a challenge to malaria elimination.

BACKGROUND: Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals. RESULTS: Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers. CONCLUSIONS: Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.

Molecular Evolution of PvMSP3α Block II in Plasmodium vivax from Diverse Geographic Origins.

Block II of Plasmodium vivax merozoite surface protein 3α (PvMSP3α) is conserved and has been proposed as a potential candidate for a malaria vaccine. The present study aimed to compare sequence diversity in PvMSP3a block II at a local microgeographic scale in a village as well as from larger geographic regions (countries and worldwide). Blood samples were collected from asymptomatic carriers of P. vivax in a village at the western border of Thailand and PvMSP3α was amplified and sequenced. For population genetic analysis, 237 PvMSP3α block II sequences from eleven P. vivax endemic countries were analyzed. PvMSP3α sequences from 20 village-level samples revealed two length variant types with one type containing a large deletion in block I. In contrast, block II was relatively conserved; especially, some non-synonymous mutations were extensively shared among 11 parasite populations. However, the majority of the low-frequency synonymous variations were population specific. The conserved pattern of nucleotide diversity in block II sequences was probably due to functional/structural constraints, which were further supported by the tests of neutrality. Notably, a small region in block II that encodes a predicted B cell epitope was highly polymorphic and showed signs of balancing selection, signifying that this region might be influenced by the immune selection and may serve as a starting point for designing multi-antigen/stage epitope based vaccines against this parasite.

Microgeography and molecular epidemiology of malaria at the Thailand-Myanmar border in the malaria pre-elimination phase.

BACKGROUND: Endemic malaria in Thailand continues to only exist along international borders. This pattern is frequently attributed to importation of malaria from surrounding nations. A microgeographical approach was used to investigate malaria cases in a study village along the Thailand-Myanmar border. METHODS: Three mass blood surveys were conducted during the study period (July and December 2011, and May 2012) and were matched to a cohort-based demographic surveillance system. Blood slides and filter papers were taken from each participant. Slides were cross-verified by an expert microscopist and filter papers were analysed using nested PCR. Cases were then mapped to households and analysed using spatial statistics. A risk factor analysis was done using mixed effects logistic regression. RESULTS: In total, 55 Plasmodium vivax and 20 Plasmodium falciparum cases (out of 547 participants) were detected through PCR, compared to six and two (respectively) cases detected by field microscopy. The single largest risk factor for infection was citizenship. Many study participants were ethnic Karen people with no citizenship in either Thailand or Myanmar. This subpopulation had over eight times the odds of malaria infection when compared to Thai citizens. Cases also appeared to cluster near a major drainage system and year-round water source within the study village. CONCLUSION: This research indicates that many cases of malaria remain undiagnosed in the region. The spatial and demographic clustering of cases in a sub-group of the population indicates either transmission within the Thai village or shared exposure to malaria vectors outside of the village. While it is possible that malaria is imported to Thailand from Myanmar, the existence of undetected infections, coupled with an ecological setting that is conducive to malaria transmission, means that indigenous transmission could also occur on the Thai side of the border. Improved, timely, and active case detection is warranted.

Submicroscopic and asymptomatic Plasmodium falciparum and Plasmodium vivax infections are common in western Thailand - molecular and serological evidence.

BACKGROUND: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels. METHODS: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals. RESULTS: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others. CONCLUSION: These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

The Plasmodium vivax merozoite surface protein 3ß sequence reveals contrasting parasite populations in southern and northwestern Thailand.

BACKGROUND: Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand. METHODOLOGY: We employed the P. vivax merozoite surface protein 3ß (PvMSP3ß) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3ß gene and examined its genetic diversity and molecular evolution between the parasite populations. PRINCIPAL FINDINGS: Of 58 isolates containing single PvMSP3ß alleles, 31 sequence types were identified. The overall haplotype diversity was 0.77 ± 0.06 and nucleotide diversity 0.0877±0.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3ß (HD=1.0). In contrast, the southern parasite population displayed a single PvMSP3ß allele (HD=0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas. CONCLUSION: Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax control measures such as drug policy and vaccine development.

Nested PCR detection of malaria directly using blood filter paper samples from epidemiological surveys.

BACKGROUND: Nested PCR is considered a sensitive and specific method for detecting malaria parasites and is especially useful in epidemiological surveys. However, the preparation of DNA templates for PCR is often time-consuming and costly. METHODS: A simplified PCR method was developed to directly use a small blood filter paper square (2 × 2 mm) as the DNA template after treatment with saponin. This filter paper-based nested PCR method (FP-PCR) was compared to microscopy and standard nested PCR with DNA extracted by using a Qiagen DNA mini kit from filter paper blood spots of 204 febrile cases. The FP-PCR technique was further applied to evaluate malaria infections in 1,708 participants from cross-sectional epidemiological surveys conducted in Myanmar and Thailand. RESULTS: The FP-PCR method had a detection limit of ~0.2 parasites/µL blood, estimated using cultured Plasmodium falciparum parasites. With 204 field samples, the sensitivity of the FP-PCR method was comparable to that of the standard nested PCR method, which was significantly higher than that of microscopy. Application of the FP-PCR method in large cross-sectional studies conducted in Myanmar and Thailand detected 1.9% (12/638) and 6.2% (66/1,070) asymptomatic Plasmodium infections, respectively, as compared to the detection rates of 1.3% (8/638) and 0.04% (4/1,070) by microscopy. CONCLUSION: This FP-PCR method was much more sensitive than microscopy in detecting Plasmodium infections. It drastically increased the detection sensitivity of asymptomatic infections in cross-sectional surveys conducted in Thailand and Myanmar, suggesting that this FP-PCR method has a potential for future applications in malaria epidemiology studies.

Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+)-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N)/d(S) ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.

Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010.

BACKGROUND: Drug and multidrug-resistant Plasmodium falciparum malaria has existed in Thailand for several decades. Furthermore, Thailand serves as a sentinel for drug-resistant malaria within the Greater Mekong sub-region. However, the drug resistance situation is highly dynamic, changing quickly over time. Here parasite in vitro drug sensitivity is reported for artemisinin derivatives, mefloquine, chloroquine and quinine, across Thailand. METHODS: Blood was drawn from patients infected with P. falciparum in seven sentinel provinces along Thai international borders with Cambodia, Myanmar, Laos, and Malaysia. In vitro parasite sensitivity was tested using the World Health Organization's microtest (mark III) (between 1994 and 2002) and the histidine-rich protein-2 (HRP2)-based enzyme-linked immunosorbent assay (in 2010). Following World Health Organization protocol, at least 30 isolates were collected for each province and year represented in this study. Where possible, t-tests were used to test for significant differences. RESULTS: There appears to be little variation across study sites with regard to parasite sensitivity to chloroquine. Quinine resistance appears to have been rising prior to 1997, but has subsequently decreased. Mefloquine sensitivity appears high across the provinces, especially along the north-western border with Myanmar and the eastern border with Cambodia. Finally, the data suggest that parasite sensitivity to artemisinin and its derivatives is significantly higher in provinces along the north-western border with Myanmar. CONCLUSIONS: Parasite sensitivity to anti-malarials in Thailand is highly variable over time and largely mirrors official drug use policy. The findings with regard to reduced sensitivity to artemisinin derivatives are supported by recent reports of reduced parasite clearance associated with artemisinin. This trend is alarming since artemisinin is considered the last defence against malaria. Continued surveillance in Thailand, along with increased collaboration and surveillance across the entire Greater Mekong sub-region, is clearly warranted.

Natural infection of Plasmodium falciparum induces inhibitory antibodies against gametocyte development in human hosts.

We identified naturally induced antibodies from malaria patients in Thailand and clarified the effect of the antibodies on gametocyte development. Fifty-nine percent of the Plasmodium falciparum-infected blood samples (17 of 29) fed to female Anopheles mosquitoes showed no oocyst infection. Seventeen percent of the samples (5 of 29) distorted the morphology and hampered the maturity of the gametocytes. A possible mechanism for the gametocyte inhibitory activity was shown by the binding of the plasma antibodies to live, immature, intraerythrocytic gametocytes during the incubation period. One hundred fifty-seven proteins specific to different gametocyte stages were explored to find the targets of the antisera that bound to the live gametocytes. However, no additional gametocyte transmission-blocking vaccine candidate was detected. Therefore, the development of alternative transmission-blocking vaccines in high-transmission areas should focus on the identification of more gametocyte antigens-inducing inhibitory antibodies that reduce gametocytemia.

Challenges and prospects for malaria elimination in the Greater Mekong Subregion.

Despite significant improvement in the malaria situation of the Greater Mekong Subregion (GMS), malaria control for the region continues to face a multitude of challenges. The extremely patchy malaria distribution, especially along international borders, makes disease surveillance and targeted control difficult. The vector systems are also diverse with dramatic differences in habitat ecology, biting behavior, and vectorial capacity, and there is a lack of effective transmission surveillance and control tools. Finally, in an era of heavy deployment of artemisinin-based combination therapies, the region acts as an epicenter of drug resistance, with the emergence of artemisinin resistant Plasmodium falciparum posing a threat to both regional and global malaria elimination campaigns. This problem is further exacerbated by the circulation of counterfeit and substandard artemisinin drugs. Accordingly, this Southeast Asian Malaria Research Center, consisting of a consortium of US and regional research institutions, has proposed four interlinked projects to address these most urgent problems in malaria control. The aims of these projects will help to substantially improve our understanding of malaria epidemiology, vector systems and their roles in malaria transmission, as well as the mechanisms of drug resistance in parasites. Through the training of next-generation scientists in malaria research, this program will help build up and strengthen regional research infrastructure and capacities, which are essential for sustained malaria control in this region.

Malaria in the Greater Mekong Subregion: heterogeneity and complexity.

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by 'border malaria' and 'forest malaria' with high transmission occurring along international borders and in forests or forest fringes, respectively. 'Border malaria' is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and Plasmodium vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination.

Plasmodium knowlesi Malaria in humans and macaques, Thailand.

Naturally acquired human infections with Plasmodium knowlesi are endemic to Southeast Asia. To determine the prevalence of P. knowlesi malaria in malaria-endemic areas of Thailand, we analyzed genetic characteristics of P. knowlesi circulating among naturally infected macaques and humans. This study in 2008-2009 and retrospective analysis of malaria species in human blood samples obtained in 1996 from 1 of these areas showed that P. knowlesi accounted for 0.67% and 0.48% of human malaria cases, respectively, indicating that this simian parasite is not a newly emergent human pathogen in Thailand. Sequence analysis of the complete merozoite surface protein 1 gene of P. knowlesi from 10 human and 5 macaque blood samples showed considerable genetic diversity among isolates. The sequence from 1 patient was identical with that from a pig-tailed macaque living in the same locality, suggesting cross-transmission of P. knowlesi from naturally infected macaques to humans.

Evaluation of loop-mediated isothermal amplification (LAMP) for malaria diagnosis in a field setting.

We used the loop-mediated isothermal amplification (LAMP) method developed by our group for malaria diagnosis with genus-specific and species-specific primers for the four human malaria parasites at a field clinic in comparison with standard microscopy. Among 110 blood samples collected from the malaria clinic in Thailand, LAMP detected 59 of 60 samples positive by microscopy (sensitivity = 98.3%) and none of the 50 microscopy-negative samples (specificity = 100%). Negative predictive value (NPV) and positive predictive value (PPV) of LAMP were 98% and 100%, respectively. These results indicate that LAMP is an effective tool for malaria diagnosis at a field clinic in a field setting.

Sensitivity of Plasmodium vivax to chloroquine, mefloquine, artemisinin and atovaquone in north-western Thailand.

Excepting tropical Africa, where Plasmodium falciparum prevails, Plasmodium vivax is the most frequent cause of malaria in Asia and Latin America. First reliable reports of chloroquine resistance came in 1989 from the area of the distribution of the Chesson-strain of P. vivax. Since then, reports also came from other areas of the world. This study had the objective of measuring the sensitivity of P.vivax to chloroquine and potential alternative compounds in western Thailand. The study was performed in 2008 in Mae Sot, Tak Province, and followed the method of Tasanor. The IC(50) and IC(90) values for chloroquine were 167 nM and 5445 nM, those for mefloquine were 139 nM and 5282 nM, those for artemisinin were 32 nM and 466 nM, and those for atovaquone 30 nM and 650 nM. The values for chloroquine indicate the existing or imminent occurrence of specific resistance. High prevalence of mefloquine resistance precludes its alternative use. However, atovaquone, in combination with proguanil, may be a possible alternative.

Synergism between pyronaridine and retinol in Plasmodium vivax in vitro.

Estimates of the annual number of infections with Plasmodium vivax reach 391 million. So far the blood-schizontocidal therapy with chloroquine remained effective in most parts of the world, but reports about emerging resistance are increasing. The study had the objective of determining the pharmacodynamic interaction between pyronaridine and retinol in Plasmodium vivax, since pyronaridine is a potential alternative for chloroquine and an earlier study had shown strong synergism between pyronaridine and retinol in Plasmodium falciparum. The study was conducted at the Malaria Clinic of Mae Sot, Tak Province, Thailand, near the border to Myanmar. The in vitro observations followed the method of Tasanor. Successful tests were performed with 44 isolates. The mean IC(50), IC(90) and IC(99) values for pyronaridine were 9.8, 2069.6 and 162446.5 nM. The mean IC(50), IC(90) and IC(99) values for the combinations with retinol (corresponding to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults) were 1.7, 542.8 and 59379.5 nM for pyronaridine + retinol "low", for the combination with retinol "medium" they were 0.5, 313.7 and 58891.4 nM and for the combination with retinol "high" they were 0.2, 96.7 and 16754.3 nM. These results suggest strong synergism between the two substances.