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Background: Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission., Methodology/principal Findings: Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44-94% for P. falciparum and from 23-72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37-100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398)., Conclusions/significance: Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling., Competing Interests: The authors have declared that no competing interests exist.

Background: Thailand's success in reducing malaria burden is built on the efficient "1-3-7" strategy applied to the surveillance system. The strategy is based on rapid case notification within 1 day, case investigation within 3 days, and targeted foci response to reduce the spread of Plasmodium spp. within 7 days. Autochthonous transmission is still occurring in the country, threatening the goal of reaching malaria-free status by 2024. This study aimed to assess the effectiveness of the 1-3-7 strategy and identify factors associated with presence of active foci., Methods: Data from the national malaria information system were extracted from fiscal years 2013 to 2019; after data cleaning, the final dataset included 81,012 foci. A Cox's proportional hazards model was built to investigate factors linked with the probability of becoming an active focus from 2015 to 2019 among foci that changed status from non-active to active focus during the study period. We performed a model selection technique based on the Akaike Information Criteria (AIC)., Results: The number of yearly active foci decreased from 2227 to 2013 to 700 in 2019 (68.5 %), and the number of autochthonous cases declined from 17,553 to 3,787 (78.4 %). The best Cox's hazard model showed that foci in which vector control interventions were required were 18 % more likely to become an active focus. Increasing compliance with the 1-3-7 strategy had a protective effect, with a 22 % risk reduction among foci with over 80 % adherence to 1-3-7 timeliness protocols. Other factors associated with likelihood to become or remain an active focus include previous classification as an active focus, presence of Plasmodium falciparum infections, level of forest disturbance, and location in border provinces., Conclusions: These results identified factors that favored regression of non-active foci to active foci during the study period. The model and relative risk map align with the national malaria program's district stratification and shows strong spatial heterogeneity, with high probability to record active foci in border provinces. The results of the study may be useful for honing Thailand's program to eliminate malaria and for other countries aiming to accelerate malaria elimination.

Background: Tak Province, at the Thai-Myanmar border, is one of three high malaria incidence areas in Thailand. This study aimed to describe and identify possible factors driving the spatiotemporal trends of disease incidence from 2012 to 2015., Methods: Climate variables and forest cover were correlated with malaria incidence using Pearson's r. Statistically significant clusters of high (hot spots) and low (cold spots) annual parasite incidence per 1000 population (API) were identified using Getis-Ord Gi* statistic., Results: The total number of confirmed cases declined by 76% from 2012 to 2015 (Plasmodium falciparum by 81%, Plasmodium vivax by 73%). Incidence was highly seasonal with two main annual peaks. Most cases were male (62.75%), ≥ 15 years (56.07%), and of Myanmar (56.64%) or Thai (39.25%) nationality. Median temperature (1- and 2-month lags), average temperature (1- and 2-month lags) and average relative humidity (2- and 3-month lags) correlated positively with monthly total, P. falciparum and P. vivax API. Total rainfall in the same month correlated with API for total cases and P. vivax but not P. falciparum. At sub-district level, percentage forest cover had a low positive correlation with P. falciparum, P. vivax, and total API in most years. There was a decrease in API in most sub-districts for both P. falciparum and P. vivax. Sub-districts with the highest API were in the Tha Song Yang and Umphang Districts along the Thai-Myanmar border. Annual hot spots were mostly in the extreme north and south of the province., Conclusions: There has been a large decline in reported clinical malaria from 2012 to 2015 in Tak Province. API was correlated with monthly climate and annual forest cover but these did not account for the trends over time. Ongoing elimination interventions on one or both sides of the border are more likely to have been the cause but it was not possible to assess this due to a lack of suitable data. Two main hot spot areas were identified that could be targeted for intensified elimination activities.

Background: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone ® , a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before., Results: One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC 50 s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project., Conclusions: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified.

Background: In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation., Results: For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses., Conclusion: The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

Mefloquine (MQ) has been used for the treatment of malaria since the mid-1980s, first as monotherapy or as fixed combination with sulfadoxine-pyrimethamine (MSP) and since the mid-1990s in combination with artesunate. There is a renewed interested in MQ as part of a triple therapy for the treatment of multi-drug resistance P. falciparum malaria. The widespread use of MQ beyond south-East Asia has been constrained by reports of poor tolerability. Here we present the side effect profile of MQ for the treatment of uncomplicated malaria on the Thai-Myanmar/Cambodia borders. In total 19,850 patients received seven different regimens containing either 15 or 24-25 mg/kg of MQ, the latter given either as a single dose, or split over two or three days. The analysis focused on (predominantly) gastrointestinal and neuropsychiatric events as compared to the new fixed dose combination of MQ plus artesunate given as equal doses of 8 mg/kg MQ per day over three days. Gastrointestinal side effects were dose-dependent and associated with the severity of malaria symptoms. Serious neuropsychiatric side effects associated with MQ use were rare: for a single 25 mg/kg dose it was 11.9 per 10,000 treatments (95% confidence interval, CI, 4-285) vs. 7.8 (3-15) for the 15 mg/kg dose. The risk with 25 mg/kg was much higher when it was given as repeat dosing in patients who had failed treatment with 15 mg/kg MQ in the preceding month; (RR 6.57 (95% CI 1.33 to 32.4), p = 0.0077). MQ was best tolerated as 15 mg/kg or as 24 mg/kg when given over three days in combination with artesunate. We conclude that the tolerance of a single dose of MQ in the treatment of uncomplicated malaria is moderate, but can be improved by administering it as a split dose over three days., Competing Interests: The authors have declared that no competing interests exist.

Hemoglobin E (HbE) is one of the most common hemoglobin variants caused by a mutation in the β-globin gene, and found at high frequencies in various Southeast Asian groups. We surveyed HbE prevalence among 8 ethnic groups residing in 5 villages selected for their high period malaria endemicity, and 5 for low endemicity in northern Thailand, in order to uncover factors which may affect genetic persistence of HbE in these groups. We found the overall HbE prevalence 6.7%, with differing frequencies from 0% in the Pwo Karen, the Lawa, and the Skaw Karen to 24% in the Mon. All HbE genes were heterozygous (AE). Differences in HbE prevalence among the studied ethnic groups indirectly documents that ancestries and evolutionary forces, such as drift and admixture, are the important factors in the persistence of HbE distribution in northern Thailand. Furthermore, the presence of HbE in groups of northern Thailand had no effect on the in vitro infectivity and proliferation of Plasmodium falciparum, nor the production of hemozoin, a heme crystal produced by malaria parasites, when compared to normal red-blood-cell controls. Our data may contribute to a better understanding on the persistence of HbE among ethnic groups and its association with malaria.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and α-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and α-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.

Background: Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment failure is common. The objective of this study was to examine the duration of persistent parasite carriage following anti-malarial treatment in pregnancy., Methods: The data presented here are a collation from previous studies carried out since 1994 in the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed using the same unique methodology detailed in the Materials and Methods section. Screening for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick sample collected. Parasite DNA was extracted from the blood-spot samples using saponin lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2 and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or indeterminate. Factors associated with time to microscopy-detected recrudescence were analysed using multivariable regression techniques., Results: From December 1994 to November 2009, 700 women were treated for P. falciparum and there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR genotyping. Most of the recurrences, 85% (770/909), occurred after treatment with quinine monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean number of days to recurrence was significantly shorter in women with recrudescent infection, 24.5 (95%: 23.4-25.8), compared to re-infection, 49.7 (95%: 46.9-52.7), P<0.001. The proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63 from the start of treatment was 29.1% (124/428), 13.3% (57/428) and 5.6% (24/428). Recrudescent infections≥100 days after treatment occurred with quinine and mefloquine monotherapy, and quinine+clindamycin and artesunate+atovaquone-proguanil combination therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax infection increased the geometric mean interval to recrudescence by 1.28-fold (95% CI: 1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased 0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence) and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia., Conclusions: Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial treatment. Long intervals to recrudescence are more likely with the use of artemisinin-containing treatments and also observed with intercalated P. vivax infections treated with chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration of follow-up, preferably until delivery or day 63, whichever occurs last.

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.

Changes in blood cell parameters are already a well-known feature of malarial infections. To add to this information, the objective of this study was to investigate the varying effects that different levels of parasite density have on blood cell parameters. Patients diagnosed with malaria at Phobphra Hospital, Tak Province, Thailand between January 1st 2009 and January 1st 2012 were recruited as subjects for data collection. Blood cell parameters of 2,024 malaria-infected patients were evaluated and statistically analyzed. Neutrophil and platelet counts were significantly higher, however, RBC count was significantly lower in patients with P. falciparum infection compared to those with P. vivax infection (p<0.0001). Leukocyte counts were also significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia. In terms of differential leukocyte count, neutrophil count was significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia (p<0.0001). On the other hand, both lymphocyte and monocyte counts were significantly lower in patients with high parasitemia (p<0.0001). RBC count and Hb concentration, as well as platelet count were also significantly reduced (p<0.05) and (p<0.0001), respectively. To summarize, patients infected with different malaria parasites exhibited important distinctive hematological parameters, with neutrophil and eosinophil counts being the two hematological parameters most affected. In addition, patients infected with different malarial densities also exhibited important changes in leukocyte count, platelet count and hemoglobin concentration during the infection. These findings offer the opportunity to recognize and diagnose malaria related anemia, help support the treatment thereof, as well as relieve symptoms of severe malaria in endemic regions.

Sporozoites of Plasmodium falciparum are transmitted to human hosts by Anopheles mosquitoes. Thrombospondin-related adhesive protein (TRAP) is expressed in sporozoites and plays a crucial role in sporozoite gliding and invasion of human hepatocytes. A previous study showed that the TRAP gene has been subjected to balancing selection in the Gambian P. falciparum population. To further study the molecular evolution of the TRAP gene in Plasmodium falciparum, we investigated TRAP polymorphisms in P. falciparum isolates from Suan Phueng District in Ratchaburi Province, Thailand. The analysis of the entire TRAP coding sequences in 32 isolates identified a total of 39 single nucleotide polymorphisms (SNPs), which comprised 37 nonsynonymous and two synonymous SNPs. McDonald-Kreitman test showed that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. falciparum was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. falciparum and P. reichenowi. Furthermore, the rate of nonsynonymous substitution was significantly higher than that of synonymous substitution within Thai P. falciparum. These results indicate that the TRAP gene has been subject to diversifying selection in the Thai P. falciparum population as well as the Gambian P. falciparum population. Comparison of our P. falciparum isolates with those from another region of Thailand (Tak province, Thailand) revealed that TRAP was highly differentiated between geographically close regions. This rapid diversification seems to reflect strong recent positive selection on TRAP. Our results suggest that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of P. falciparum.

Background: Malaria in pregnancy remains a major health problem. Placental malaria infection may cause pathophysiological changes in pregnancy and result in morphological changes to placental villi. Quantitative histomorphological image analysis of placental biopsies was performed to compare placental villous architecture between active or treated placental malaria cases and controls., Methods: A total of 67 placentas were studied from three clinical groups: control patients who did not have malaria (n = 27), active (n = 14) and treated (n=26) malaria cases, including both Plasmodium falciparum and Plasmodium vivax infections. Image analysis of histological placental sections was performed using ImageJ software to measure the number and size (area) of terminal villi, perimeter measurement per villus and total perimeter per unit area, and number of capillaries per villus (vascularity). Histological features of placental malaria were scored and these results were correlated with malaria status and clinical outcomes., Results: Villous size correlated with vascularity (p <0.0001) but was inversely correlated with observed villi per unit area, (p = 0.0001). Significantly greater villous area and vascularity was observed in UK controls. Indices of histological malaria infection were significantly greater in active versus treated malaria cases. Active placental malaria cases showed significantly smaller villous area (p <0.0084), vascularity (p <0.0139) and perimeter (p <0.0006) than treated malaria cases or controls, but significantly more villi per unit area (p <0.0001). Villous size in treated malaria cases was significantly larger than active placental malaria cases (p <0.001) and similar to controls. There was a significant relationship between villous number and anaemia at the time of infection (p <0.0034), but not placental weight, birth weight or gestational age at delivery. No differences were found between histology or villous morphology comparing infections with P. falciparum or P. vivax., Conclusions: These results imply that villous size, perimeter and vascularity are acutely decreased during active placental malaria, decreasing the surface area available for gas exchange per villus. However the increased number of villi per unit area offsets this change and persists after treatment. Histopathological and villous architectural changes may be reversed by early detection and appropriate anti-malarial treatment.

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.

Intermittent preventive treatment (IPT) is increasingly used to reduce malaria morbidity and mortality in children and pregnant women. The efficacy of IPT depends on the pharmacokinetic and pharmacodynamic properties of the antimalarial drugs used. Healthy adult male volunteers whose occupation put them at high risk of malaria on the Northwest border of Thailand were randomized to receive a 3-day-treatment dose of dihydroartemisinin-piperaquine monthly (DPm) or every 2 months (DPalt) or an identical placebo with or without fat (6.4 g/dose) over a 9-month period. All volunteers were monitored weekly. One thousand adults were recruited. Dihydroartemisinin-piperaquine was well tolerated. There were 114 episodes of malaria (49 Plasmodium falciparum, 63 P. vivax, and 2 P. ovale). The protective efficacy against all malaria at 36 weeks was 98% (95% confidence interval [CI], 96% to 99%) in the DPm group and 86% (95% CI, 81% to 90%) in the DPalt group (for both, P < 0.0001 compared to the placebo group). As a result, the placebo group also had lower hematocrits during the study (P < 0.0001). Trough plasma piperaquine concentrations were the main determinant of efficacy; no malaria occurred in participants with a trough concentration above 31 ng/ml. Neither plasma piperaquine concentration nor efficacy was influenced by the coadministration of fat. DPm is safe to use and is effective in the prevention of malaria in adult males living in an area where P. vivax and multidrug-resistant P. falciparum malaria are endemic.

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Following the advent of mefloquine resistance in Plasmodium falciparum in Thailand in the 1990s, the combined treatment of falciparum malaria with artesunate and mefloquine was found to be highly effective in treating and curing the patients in the affected areas. Monitoring of the clinical-parasitological response and of the in vitro sensitivity of P. falciparum was systematically conducted in order to detect any signs of failure of this type of artemisinin-based combination treatment (ACT). In earlier observations the in vitro activity of artemisinin was found to be significantly enhanced when combined with retinol. The same applies to mefloquine. In order to check whether the synergism between artemisinin and mefloquine was maintained in the presence of retinol, the pharmacodynamic interaction of the three compounds was investigated in the western border area of Thailand. Successful parallel tests with mefloquine, artemisinin, retinol, mefloquine-artemisinin 5:1 as well as mefloquine-artemisinin (5:1) + retinol low, medium and high were obtained with 43 fresh parasite isolates. The retinol concentrations in the low, medium and high formulations corresponded to the 50th, 65th and 80th percentile of the physiological mean concentrations in the blood of healthy adults. The IC(50), IC(90) and IC(99) values for mefloquine alone showed a further increase over the data of 2008. In the combinations with artemisinin and retinol moderate synergism was observed at the IC(50), but synergism increased strongly at the IC(90) and the IC(99).

The increasing drug resistance of Plasmodium falciparum is a worldwide problem. The objective of the study was the assessment of the in vitro activity of artemisinin, mefloquine and quinine, in an area where P. falciparum is multi-drug resistant. The sensitivity tests were based on measuring the drug-dependent inhibition of schizont maturation. For the 43 successfully tested isolates the mean effective concentrations (IC(50) and IC(90)) for artemisinin were 0.0081 and 0.1372 μM, respectively. For mefloquine the IC(50) was 0.1260 μM and the IC(90) was 3.7345 μM. Quinine showed an IC(50) of 0.2155 μM and an IC(90) of 2.5040 μM. All tested drugs showed a significant reduction in the effectiveness, compared with the results of former years. This suggests a further rise of resistance of local P. falciparum, which is alarming especially for artemisinin and quinine.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency--the most common known enzymopathy--is associated with neonatal jaundice and hemolytic anemia usually after exposure to certain infections, foods, or medications. Although G6PD-deficient alleles appear to confer a protective effect against malaria, the link with clinical protection from Plasmodium infection remains unclear. We investigated the effect of a common G6PD deficiency variant in Southeast Asia--the G6PD-Mahidol(487A) variant--on human survival related to vivax and falciparum malaria. Our results show that strong and recent positive selection has targeted the Mahidol variant over the past 1500 years. We found that the G6PD-Mahidol(487A) variant reduces vivax, but not falciparum, parasite density in humans, which indicates that Plasmodium vivax has been a driving force behind the strong selective advantage conferred by this mutation.

A multidrug-resistant (MDR) clone of Plasmodium falciparum (C2A) from Thailand was adapted through serial passage to Aotus monkeys. During adaptation, the parasite showed resistance to a single 20 or 40 mg/kg oral dose of mefloquine (MQ). Infection was only cured when MQ was administered orally at 40 mg/kg once in combination with intravenous artesunic acid at 20 mg/kg for 3 days. Similarly, the parasite clone was found to be resistant to quinine, failing at 20 mg/kg orally for 5 days in combination with an experimental dihydrofolate reductase (DHFR) inhibitor (WR297608) at 10, 20, or 40 mg/kg orally for 3 days, and with atovaquone/proguanil at 25 mg/kg for 3 days. This new model will allow in vivo testing of new antimalarial compounds or their combinations against a currently circulating MDR P. falciparum strain.

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.

Nitrate levels in CSF and sera from 16 coma and 19 noncoma falciparum malaria patients were determined using nitric oxide colorometric assay. The medians (range lower, upper limits) of nitrate in sera of comatose and noncomatose patients were 0.28 (0.11, 1.24) and 0.23 (0.05, 0.87) microM, respectively. The medians of nitrate level in CSF of coma and noncoma cases were 0.09 (0.01, 0.28) and 0.15 (0, 1.18) microM, respectively. There was no difference of nitrate level in sera and CSF from comatose or noncomatose patients compared to that in normal sera and CSF. The amount of nitrate in sera and CSF of both groups was not significantly correlated with coma depth, parasitemia, parasite clearance time and time to recovery. Contrast to our in vitro study using immunoperoxidase staining, we found inducible nitric oside synthase production by brain endothelial cells during 4-24 hours of coculturing with late stage of P. falciparum infected red blood cells. These results suggests that malaria severity can not be differentiated by nitrate level in body fluid.

Wild caught zoophilic Anopheles and suspected malaria vector species collected in northwest Thailand were experimentally infected with local human malaria parasites using a membrane feeding. One week post-feeding a number of mosquitos were dissected for oocyst examination. The remainder were kept for another one week or more, and then the salivary glands were examined for the presence of sporozoites. The results revealed that An. vagus, An. kochi and An. annularis were susceptible to both Plasmodium falciparum and P. vivax whereas An. barbirostris and An. sinensis were susceptible to only P. vivax. The non-susceptibility to P. falciparum of these two mosquito species may indicate their poor vector status of this malaria species in the field.

Mature oocysts of Plasmodium falciparum and P. vivax from western Thailand were separated from the midguts of Anopheles dirus by collagenase digestion, and the number of sporozoites contained in each was counted. For 26 P. vivax oocysts, the mean count was 3, 688 (range 1, 954-5, 577) and for 14 P. falciparum, the mean count was 3, 385 (range 1, 359-4, 554); a single P. cynomolgi oocyst contained 7, 521. Counts were not significantly correlated with oocyst density, oocyst age, or identity of the examiner. There may have been strain differences in fecundity, particularly between P. falciparum lines maintained in vitro. Mosquitoes receiving a second, uninfected blood meal seven days after feeding on P. vivax-infected volunteers developed no additional sporozoites per oocyst, but had salivary glands 3.4 times as infected. By calculation, more than 20% of P. vivax sporozoites released from oocysts subsequently invade the salivary glands.

An indirect immunofluorescence test with fresh non-fixed infected blood as antigen was used to show that antibody in human sera from the Gambia recognized antigens on the surface of Plasmodium falciparum-infected human erythrocytes. Surface immunofluorescence was detected on 90% of erythrocytes infected with trophozoites and schizonts produced in continuous culture of isolates from the Gambia (FCR 3/K+), Brazil and Thailand. Fluorescence was equally strong with a Gambian parasite clone (FCR 3/K-) that lacked knobs, an ultrastructural modification of the erythrocyte membrane associated with parasite sequestration. Immunofluorescence could not be detected with an isolate from Uganda. The surface antigenicity of parasitized erythrocytes was eliminated by chymotrypsin and trypsin treatment. Fluorescence was specific for the surface of trophozoite- and schizont-infected cells on the condition that fresh erythrocytes were added to cultures every 4-5 days (subculture); if fresh erythrocytes were not added for over 2 weeks, a large percentage of non-infected erythrocytes also bound antibody. Normal erythrocytes incubated with media from these cultures also gave positive surface immunofluorescence. Thus, there are two types of antigenicity on erythrocytes: one expressed on infected erythrocytes and another passively absorbed from media to normal erythrocytes when parasites are not subcultured for long periods.

A study of the effect of sulphadoxine/pyrimethamine (Fansidar) on P. falciparum's gametocytes in peripheral blood was carried out in Western Thailand. One group of 77 patients with asexual form P. falciparum sensitive to Fansidar were followed weekly to detect the appearance and the duration of gametocytes in peripheral blood after Fansidar treatment on the basis of thick blood film examination. Another group of 14 patients with sexual form P. falciparum was not given any antimalarial treatment and also followed up weekly. No significant difference of average duration of detectable gametocytes was observed between the groups. The average number of days that gametocytes appeared after asexual form in patients receiving treatment was the same as in the untreated group. It is unlikely that Fansidar has the stimulating effect on gametocytogenesis as previously reported.