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The use of a metofluthrin-impregnated spatial repellent device (MSRD) is a new and effective method for preventing mosquito blood feeding. Indoor environmental factors such as room temperature and ventilation rate are thought to be important for MSRD activity. Measurements of room temperature and vaporization of metofluthrin from MSRD in typical rural metal-roof and thatched-roof houses in southeastern Malawi were conducted. The relationship between house structure and the number of collected Anopheline mosquitoes with and without MSRD treatment was also investigated. The difference between daytime and nighttime room temperature was significantly higher in metal-roof houses than in thatched-roof houses. The vaporization of metofluthrin from the MSRD was not accelerated by the high room temperature, but by the high indoor air flow by ventilation. The number of mosquito collections was significantly higher in thatched-roof houses than in metal-roof houses. MSRD-treated thatched-roof houses have a higher probability of mosquito infestation, but the vaporization of metofluthrin is also higher because of indoor air flow, resulting in a reduction in mosquito numbers. Metal-roof houses with closed eaves reduce the probability of mosquito invasion, and a longer predicted effectiveness occurs with MSRD because of the controlled release of metofluthrin through lower indoor air flow., Japanese Journal of Infectious Diseases, 75(3), pp. 288-295; 2022

Long-lasting insecticidal nets (LLINs) experience some operational problems that reduce their effectiveness, such as limited spaces for hanging, biting of mosquitoes outdoors, a shift of key biting time from midnight to dawn or dusk, and development of pyrethroid resistance in mosquitoes. The concept of spatial repellency may be a countermeasure to overcome the above issues. The effect of the combined use of metofluthrin-impregnated spatial repellent devices (MSRDs) and LLINs (OlysetR Plus) on malaria prevalence and vector mosquitoes were examined in malaria endemic villages in south-eastern Malawi. The intervention reduced the infection rate in children as well as the number of pyrethroid-resistant vector mosquitoes. To achieve effective malaria control, continued intervention using MSRDs with 2 strips per 10 m2 at 3-month intervals to reduce the density of malaria mosquitoes is recommended., Japanese journal of infectious diseases, 73(2), pp.124-131; 2020

Long-lasting insecticidal nets (LLINs) experience some operational problems that reduce their effectiveness, such as limited spaces for hanging, biting of mosquitoes outdoors, a shift of key biting time from midnight to dawn or dusk, and development of pyrethroid resistance in mosquitoes. The concept of spatial repellency may be a countermeasure to overcome the above issues. The effect of the combined use of metofluthrin-impregnated spatial repellent devices (MSRDs) and LLINs (Olyset

Background Border malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasites Plasmodium falciparum and Plasmodium vivax, recently a zoonotic parasite, Plasmodium knowlesi, has been identified in some countries of the region. The presence of this parasite poses a challenge to malaria control programmes, as it is maintained in a zoonotic reservoir of forest-dwelling macaque monkeys. Methods A cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species and P. knowlesi. A PCR targeting the 18S small subunit ribosomal RNA gene and circumsporozoite protein gene was used for Plasmodium species identification. Results Nine cases of P. knowlesi were detected by PCR in blood samples from the Laos side and three from the Vietnam side. All P. knowlesi infections were found in co-infection with P. vivax, with some triple infections of P. knowlesi, P. vivax and P. falciparum detected in Laos. Phylogenetic analysis of these parasites suggests that P. knowlesi is circulating in the Laos-Vietnam border region. Conclusion This report shows that P. knowlesi is transmited on both sides of the Vietnam-Laos border. Continued monitoring of the range and prevalence of P. knowlesi on both the sides of Laos-Vietnam border is of importance to the National Malaria Control Programmes of both countries.

Border malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasitesA cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species andNine cases ofThis report shows that

Agarose gel (1.5%) electrophoresis of PCR products for the identification of Plasmodium species using DNA extracted from dried blood on the filter paper. M, 100Â bp size marker; QT65, QT250, QT835, XP677; human dried blood samples; NC, negative control; Pv (VIV1/VIV2), 18S rRNA of Plasmodium vivax; Pk (Pmk8/Pmk9) and Pk (knf1/knf3), 18S rRNA of Plasmodium knowlesi; Pk CSP, CSP of Plasmodium knowlesi. (PDF 649Â kb)

Phylogenetic tree constructed from the nucleotide sequences of the 18S rRNA gene of Plasmodium spp. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model. Number of substitutions per site is indicated by the scale bar. Bootstrap values were calculated for 1000 replications. Phylogenetic analysis was conducted by using MEGA7. Scale bar indicates nucleotide substitutions per site. S-2010 rRNA gene Human Laos are representative Laos specimen. QT-2010 rRNA gene Human Vietnam are representative Vietnam specimen. (PDF 649Â kb)

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002–2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

Background Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. Methods Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4–6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. Results Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. Conclusion These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

SUMMARYFour species of malaria parasite, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi infect humans living in the Khanh Phu commune, Khanh Hoa Province, Vietnam. The latter species also infects wild macaque monkeys in this region. In order to understand the transmission dynamics of the three species, we attempted to detect gametocytes of the three species in the blood of infected individuals, and sporozoites in the salivary glands of mosquitoes from the same region. For the detection of gametocyte-specific mRNA, we targeted region 3 of pfg377, pvs25, pmg and pks25 as indicators of the presence of P. falciparum, P. vivax, P. malariae and P. knowlesi gametocytes, respectively. Gametocyte-specific mRNA was present in 37, 61, 0 and 47% of people infected with P. falciparum (n = 95), P. vivax (n = 69), P. malariae (n = 6) or P. knowlesi (n = 32), respectively. We found that 70% of mosquitoes that had P. knowlesi in their salivary glands also carried human malaria parasites, suggesting that mosquitoes are infected with P. knowlesi from human infections.

Plasmodium falciparum SURFIN 4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN 4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN 4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN 4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima's D test, and Fu and Li's D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988-1989 still circulated 14 years later, suggesting low recombination of the analyzed surf 4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN 4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported., Parasitology International, 61(2), pp.317-323; 2012

A protein complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Clarification of the level of polymorphism and the evolutionary processes is important both for vaccine design and for a better understanding of the evolution of cell invasion in this parasite. In a previous study on 5 genes encoding RhopH1/Clag proteins, positive diversifying selection was detected in clag8 and clag9 but not in the paralogous clag2, clag3.1 and clag3.2. In this study, to extend the analysis of clag polymorphism, we obtained sequences surrounding the most polymorphic regions of clag2, clag8, and clag9 from parasites collected in Thailand. Using sequence data obtained newly in this study and reported previously, we classified clag2 sequences into 5 groups based on the similarity of the deduced amino acid sequences and number of insertions/deletions. By the sliding window method, an excess of nonsynonymous substitutions over synonymous substitutions was detected in the group 1 and group 2 clag2 and clag8 sequences. Population-based analyses also detected a significant departure from the neutral expectation for group 1 clag2 and clag8. Thus, two independent approaches suggest that clag2 is subject to a positive diversifying selection. The previously suggested positive selection on clag8 was also supported by population-based analyses. However, the positive selection on clag9, which was detected by comparing the 5 sequences, was not detected using the additional 34 sequences obtained in this study., Tropical Medicine and Health, 39(3), pp.77-82; 2011

Understanding why some malaria-infected individuals are infective to mosquitoes while others are not, is of great importance when considering interventions to stop malaria transmission. Whether gametocytes are produced in every individual infected with Plasmodium falciparum remains unclear. Using a highly sensitive reverse transcription (RT)-PCR assay, we attempted to detect gametocyte-specific mRNA transcripts in isolates from Thai patients which newly adapted to continuous in vitro culture. We then compared the allelic types of the pfg377 gene between patient blood and culture-adapted parasites in order to determine whether the same parasite lines were producing gametocytes in vivo and in vitro. Transcripts of pfg377 were detected in all parasite isolates and in the corresponding cultured isolates, revealing that all patients had gametocytes circulating in their blood at the time of sampling. For isolates in continuous in vitro culture, there was a match between pfg377 allelic types detected by PCR from genomic DNA (and thus indicative of the dominant allelic type of asexual parasites) and those detected by RT-PCR of mRNA (gametocyte-specific), whereas in freshly isolated patient blood there were some differences between the asexual parasite allelic type and that of the gametocytes in the same infection. Seven isolates contained asexual stage parasites harbouring pfg377 alleles that were not detectable in gametocytes from the same infections, suggesting that some clones were not producing gametocytes at the time of sampling, or that they were below the level of detection., International Journal for Parasitology, 41(3-4), pp.317-323; 2011

Plasmodium falciparum is the main cause of human malaria and is one of the important pathogens causing high rates of morbidity and mortality. The total number of malaria patients in Vietnam has gradually decreased over the last decade. However, the spread of pathogens with drug resistance remains a significant problem. Defining the trend in genotypes related to drug resistance is essential for the control of malaria in Vietnam. We undertook a longitudinal survey of Plasmodium falciparum malaria in 2001, 2002, and 2005 to 2007. The pfcrt, pfmdr1, pfdhfr, and pfdhps genes were analyzed by sequencing; and correlations by study year, age, gender, and genotype were identified statistically. The ratio of the chloroquine resistance genotype pfcrt 76T was found to have decreased rapidly after 2002. High numbers of mutations in the pfdhfr and pfdhps genes were observed only in 2001 and 2002, while the emergence of parasites with a new K540Y mutation in the P. falciparum dihydropteroate synthetase (PfDHPS) was observed in 2002. For males and those in younger age brackets, a correlation between vulnerability to P. falciparum infection and strains with pfcrt 76K or strains with decreased numbers of mutations in pfdhfr and pfdhps was demonstrated. The parasites with pfcrt 76T exhibited a greater number of mutations in pfdhfr and pfdhps., Journal of clinical microbiology, 48(1), pp.70-77; 2010

The feasibility of identifying parasite DNA and specific mRNAs from wild-caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi DNA by conventional PCR and, furthermore, detected P. falciparum gametocyte-specific genes, pfg377 and pfs16 mRNA, P. knowlesi circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2) mRNA by reverse transcription-PCR. Using this technique, we were able to confirm the presence of P. vivax, P. falciparum and P. knowlesi in one particular wild-caught mosquito. These results indicate that P. knowlesi may be transmitted by the primary human malaria vector in forested areas in Vietnam. This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria., International journal for parasitology, 39(14), pp.1533-1537; 2009

Background Recent studies have described natural human infections of the non-human primate parasites Plasmodium knowlesi and Plasmodium cynomolgi. In Southeast Asia, mosquitoes of the Anopheles leucosphyrus group bite both humans and monkeys in the forest and thus offer a possible route for Plasmodium species to bridge the species barrier. In this study we analysed the species composition of malarial sporozoites infecting the salivary glands of Anopheles dirus in order to determine their potential role as bridge vectors of Plasmodium parasites from monkeys to humans. Methods Mosquitoes were collected in the forest and forest fringe area of Khanh Phu commune by human-baited landing collection. Anopheles species were determined on the basis of morphologic features. Sporozoite-infected salivary glands were applied to filter paper and dried in an ambient atmosphere, before storage in closed vials at 4–6 °C. Detection and identification of Plasmodium species in salivary glands were carried out by nested-PCR of the small subunit ribosomal RNA gene. Results Six species of Plasmodium parasites were detected by PCR, of which P. vivax was the most common, followed by P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. falciparum. Twenty-six of the 79 sporozoite infected mosquitoes showed multiple infections, most of which were a combination of P. vivax with one or more of the non-human primate Plasmodium species. Conclusions These results suggest that humans overnighting in this forest are frequently inoculated with both human and non-human primate malaria parasites, leading to a situation conducive for the emergence of novel zoonotic malaria. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0995-y) contains supplementary material, which is available to authorized users.

Osteopontin (OPN) knockout mice (OPN-KO mice) died ofPlasmodium chabaudi chabaudiinfection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.

Results: Urinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine. Methods. Urine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene. Background: Diagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis. Conclusions: The cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts., Malaria Journal, 13(1), 373; 2014

The effect of deoxyspergualin (DSG) on the K1 strain of human malarial parasite Plasmodium falciparum in vitro was studied to test a possible new antimalarial chemotherapy. Hypoxanthine labeled with tritium (3H) was used to assess macromolecular synthesis. The inhibitory effects of DSG on the parasite peaked after 72 h of incubation. Parasitemia without DSG treatment was 9%, whereas at a DSG concentration of more than 156 µg/ml it was less than 1%. The amount of [3H]hypoxanthine taken up decreased with increasing DSG concentration. DNA synthesis of malarial activity decreased with increasing DSG concentration. These findings provide more evidence for the effects of DSG on this malarial parasite. As in previous in vivo studies done with DSG, the in vitro findings showed that DSG may be a new antimalarial drug.

Biting density of anopheline mosquitoes was monitored in both the rainy and dry seasons at 6 outdoor stations in two villages of north Thailand by 3 human and 1 water buffalo bait. A total of 19 Anopheles was collected. The most predominant species were An. vagus and An. annularis. They were followed by An. hyrcanus group, An. minimus, An. aconitus, An. nivipes and An. kochi. The rest species were not abundant. The fauna of Anopheles changed seasonally. An. minimus, An. aconitus and An. barbirostris were abundant in the dry season, while An. maculatus sensu lato, An. kochi, An. tesselatus, An. vagus, An. annularis and An. hyrcanus group were the rainy season Anopheles. The 'crude feeding index' was relatively high in An. maculatus and An. minimus. They were followed by An. tesselatus and An. splendidus, while that of An. vagus and An. kochi was less than 1/10 of An. minimus. An. minimus was regarded as the most important vector. The biting density of An. minimus was higher at the peripheral area of villages with scarce dwellings and rich vegetation. At stations with dense dwellings and adjoining to an open field, the density of An. minimus was low, and the malaria risk was probably higher., 日本熱帯医学会雑誌, vol.23(3), pp.177-182; 1995

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 Ca2+-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 dN/dS 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., PLoS ONE, 8(3), e59192; 2013

Plasmodium falciparum SURFIN(4.1) is a type I transmembrane protein thought to locate on the merozoite surface and to be responsible for a reversible adherence to the erythrocyte before invasion. In this study, we evaluated surf(4.1) gene segment encoding extracellular region for polymorphism, the signature of positive selection, the degree of linkage disequilibrium, and temporal change in allele frequency distribution in P. falciparum isolates from Thailand in 1988-89, 2003, and 2005. We found that SURFIN(4.1) is highly polymorphic, particularly at the C-terminal side of the variable region located just before a predicted transmembrane region. A signature of positive diversifying selection on the variable region was detected by multiple tests and, to a lesser extent, on conserved N-terminally located cysteine-rich domain by Tajima's D test. Linkage disequilibrium between sites over a long distance (1.5 kb) was detected, and multiple SURFIN(4.1) haplotype sequences detected in 1988/89 still circulated in 2003. Few of the single amino acid polymorphism allele frequency distributions were significantly different between the 1988/89 and 2003 groups, suggesting that the frequency distribution of SURFIN(4.1) extracellular region remained stable over 14 years.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells. Experimental Parasitology 79, 383-390. CD36 is expressed in the endothelial cells of some human organs, but the ultrastructural localization of this molecule in the sinusoidal lining cells of human liver is not well established. We report the ultrastructural localization of CD36 in the liver using a novel murine monoclonal antibody against CD36, namely MO30, as a primary antibody. Immunocytochemistry by the postembedding method showed that CD36 was localized in endothelial cells of sinusoids and in hepatocyte microvilli protruding into the space of Disse. Moreover, in cultured human hepatoma (HepG2-A16) cells and C32 amelanotic melanoma cells, MO30 reacted with microvilli. Hence, CD36 expressed on these cells may be involved in recognition and/or entry of these cells by malaria sporozoites.

The number and distribution of γδ T cells in spleens from patients who died of cerebral malaria and from rhesus monkeys severely infected with Plasmodium coatneyi were examined by immunocytochemistry. γδ T cells were significantly increased in these spleens. In a rodent malaria model using Plasmodium chabaudi adami, an avirulent strain of murine malaria parasites, the degree of parasitemia appears to be modulated by the number of γδ T cells in the spleen. As parasitemia increases, these T cells increase in number. At some critical point, γδ T cells in collaboration with macrophages and αβ T cells apparently start to clear parasitized erythrocytes from the blood, leading to an abatement of the parasitemia, which is followed by a reduction in the number of γδ T cells. This γδ T cell phenomenon may be responsible for the self-limiting infection in mice.

We studied the brains of rhesus monkeys infected with the primate malaria parasite Plasmodium fragile. Electron microscopy showed that, in these animals, erythrocytes infected with P. fragile undergo sequestration and that parasitized red blood cells adhere to endothelial cells in the cerebral microvessels by means of knobs. Cerebral microvessels with sequestered parasitized red blood cells were shown by immunohistochemical analysis to possess the platelet glycoprotein CD36, thrombospondin, and intracellular adhesion molecule-1. The formation of rosettes also was observed in the cerebral microvessels. In a fashion similar to human cerebral malaria, P. fragile produced neurological symptoms in the animals. Thus, rhesus monkeys infected with P. fragile, like those monkeys infected with Plasmodium coatneyi, can be used as a primate model to study human cerebral malaria.

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas., PLoS One, 6(8), e23430; 2011

A cross-sectional study was undertaken to identify the prevalence of malaria infection, behavioral patterns of inhabitants and risk factors for malaria infection in a minority village in an area endemic for malaria in Vietnam. The target population were all the inhabitants of a village in Binh Phuoc Province, Vietnam. Interviews using a structured questionnaire and blood examination for malaria infection were conducted in house-to-house visits. Relationships between malaria infection and variables were examined using uni- and multivariate adjusted analysis. A total of 682 individuals from 159 households participated in both the interview and blood examinations. All households earned income through farming without forest activities at night, and the socio-economic status was generally not very low. The total prevalence of malaria infection was 6.2%, with a peak among 3- to 5-year-old children. Univariate analysis identified 3 - 5 year olds, a family size ofor =5 people, sleeping with3 people in a bed, and living in a wooden/bamboo house as factors associated with malaria infection. Multivariate adjusted analysis after variable selection identified age 3 - 5 years old, a family size ofor =5 people and living in a wooden/bamboo house were significantly related to malaria infection. Malaria in this area can be controlled by basic activities, such as early diagnosis and treatment and prevention using bednets, since risk factors for malaria infection did not include forest activities, but were young age, living in a wooden/bamboo house and belonging to a large family. Continuous and intensive expansion of existing malaria control activities are required.

Dimorphic variations in the genotype of the precursor to Plasmodium falciparum major merozoite surface antigens or gp195, among wild isolates in a small malaria parasite population were examined using Southern blot hybridization techniques. Hybridization, with DNA fragment probes and oligonucleotide probes derived from variable blocks of known gp195 alleles against 18 wild isolates from Mae Sod district in Thailand, revealed the existence of seven gp195 alleles, two of which were newly identified in this study. In four out of 17 patients, two different alleles coexisted in the circulation. It was furthermore noted that the seven alleles did not occur at the same frequency, but rather several alleles predominated in the population of P. falciparum in this small malaria field., American Journal of Tropical Medicine and Hygiene, 44(3), pp.299-305; 1991

The detection of gametocytes in human peripheral blood is one of the most important measures in a malaria survey. We attempted to detect gametocytes of Plasmodium falciparum by reverse transcription polymerase chain reaction (RT-PCR) of dried blood on filter paper. On field samples analysis, the specific RT-PCR products for region 3 of pfg377 mRNA were observed in 67 of 131 falciparum malaria patients. The minimum detection level of RT-PCR-positive samples was 0.03 gametocytes/microl on quantitative real-time RT-PCR. Gametocyte positive rate was not dependent on sex or age. A higher frequency of gametocytes was found in single P. falciparum infection than in mixed species infection (P

We examined the role of osteopontin (OPN) in immunity against Plasmodium falciparum infection. We measured the mRNA levels for OPN and several cytokines in RNA preparations extracted from dried blood on filter paper obtained from falciparum malaria patients in Vietnam. Expression of OPN mRNA was detected in 134 of 161 patients. The expression of both interleukin-12 p40 and interferon-gamma mRNAs in the group positive for OPN mRNA was significantly higher than that in the group negative for OPN mRNA. The level of parasitemia in the OPN mRNA-positive group was much lower than that in the negative one. These results suggest that OPN might suppress multiplication of the parasites through T helper 1 cells-mediated immune responses.

Recrudescence of Plasmodium berghei NK65 infection was studied to examine factors affecting recrudescence. Treatment with a high dose of chloroquine did not prevent recrudescence, but an extended duration of treatment suppressed the frequency of recrudescence. Infection with a larger number of parasites also resulted in more frequent recrudescences. Recrudescent parasites were as sensitive to chloroquine as those before treatment. Splenectomized mice were administered carbon particles, infected, and treated with chloroquine. Recrudescence was significantly more frequent in these mice than in mice given a sham operation and PBS. The results do not suggest that merozoite stages escape the effect of chloroquine by ‘hiding’ in phagocytes, but that latent parasites such as dormant ring stages may cause recrudescence.

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii. A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-beta. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.

Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.

Circulating immune complex (CIC) is known to play a role in pathological glomerular alterations in malaria. However, the nature of the antigens comprising the CIC is still not fully understood. We report here the isolation of the antigen in CIC and its localisation in mice infected with Plasmodium berghei NK65. The antigen was successfully isolated from CIC extracted from the blood of mice infected with P. berghei, by using C1q-coated microplates. The molecular mass of the antigen separated from CIC bound to C1q was found to be 78 kDa. Furthermore, localisation of the antigen was examined by the fluorescent antibody technique and immunoelectron microscopy. The antigen was detected in the parasitised erythrocyte and the mesangial matrix by both methods. These results suggest that the 78 kDa protein might be associated with the glomerular alterations in malaria infection.

The pathogenesis of human cerebral malaria is suspected to be caused by blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). Examination of infected tissues indicate PRBC sequestration in microvessels is the result of PRBC knob attachment to endothelial cell surface cytoadherence receptors such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). In lieu of fresh human tissue, several animal models for human cerebral malaria have been developed, the Plasmodium coatneyi-infected rhesus monkey model being the most versatile. To further the understanding of noncerebral malarial complications during disease, we examined noncerebral tissues of infected rhesus monkeys. Our study demonstrated similar microvessel PRBC sequestration and the presence of cytoadherence ligands in noncerebral tissues. Immunohistochemical analysis showed CD36, TSP, and ICAM-1 cytoadherence proteins in several major organs.

The basis of recrudescence, the reappearance of malaria parasites after chemotherapy or after failure of immune suppressions of the parasites, was studied in cultures of Plasmodium falciparum. When the cultured parasites were exposed eight times over a 4-day period to 5% D-sorbitol, which destroyed infected RBCs containing trophozoites or schizonts, they showed recrudescence several days after cessation of treatment. Pyrimethamine-sensitive parasites were cleared by 10(-6) M pyrimethamine; pyrimethamine-resistant parasites also were cleared by 10(-4) M pyrimethamine. Both groups of parasites underwent recrudescence in the same manner as those exposed to 5% D-sorbitol. These recrudescent parasites were found to be as susceptible to these treatments as were the parasites before treatment. Therefore, our results strongly suggest that a subpopulation of the parasites escapes the effect of drugs by a mechanism other than drug resistance.

Sequestered parasitized erythrocytes were found in microvessels of subcutaneous tissues in a comatose patient with cerebral malaria even though the blood smears were negative after quinine treatment. This situation reflects the continued presence of sequestered parasites in the brain and suggests that negative parasitemia in peripheral blood does not necessarily mean the end point of malarial treatment has been reached. Our findings suggest that biopsy of subcutaneous tissue from severe malaria patients may be useful for determining the severity and prognosis of malaria patients.

Cerebral malaria is a major cause of mortality in patients with Plasmodium falciparum infection (Macpherson et al. 1985; Aikawa 1988; Howard and Gilladoga 1989). Blockage of cerebral vessels by erythrocyte sequestration appears to play a major role in the pathogenesis of cerebral malaria (Aikawa 1988; Aikawa et al. 1990; Riganti et al. 1990; Pongponratn et al. 1991). One of the mechanisms suggested to be involved in this sequestration process is formation of rosettes, clusters of uninfected erythrocytes around trophozoiteand schizont-infected erythrocytes. Rosettes of this type have been observed in an in vitro system that utilizes human erythrocytes infected with P. falciparum (Handunnetti et al. 1989; Udomsangperch et al. 1989). From a practical standpoint, use of an in vitro system is limited in terms of its applicability to the study of therapeutic interactions in a human infection. Clearly, an animal model that exhibits features similar to human in vivo and in vitro infections would be useful. P. coatneyi is a P.falciparum-like primate malaria parasite that induces both rosette formation (Udomsangpetch et al. 1991) and the adherence of infected erythrocytes to the endothelial cells of microvessels, leading to vascular obstruction (Aikawa et al. 1992). This article describes the ultrastructural features of rosette formation in an animal model that closely resembles the human system. A P. coatneyi-infected rhesus monkey was bled at 2 weeks after inoculation, when parasitemia was 220000 cells/ram 3. Whole blood (0.5 ml) was collected in heparin and washed in RPMI-1640 containing 10% heat-inactivated AB + human serum. The parasites in the collected blood ranged in stage from ring to trophozoite. These trophozoites readily formed rosettes in vitro at a high percentage. After the washing step, the presence of rosettes was confirmed by light microscopy using a wetmounted drop of cell suspension. For electron microscopy, 100 gl packed cells was

The N-terminal part of p190, the precursor to the major merozoite surface antigens of Plasmodium falciparum, contains the T and B cell epitopes and tripeptide repeats. The p190 gene exhibits allelic dimorphism, but the tripeptide repeat-encoding region is the only exception to the dimorphic variations of the gene. To date, sequences available to document variations in the epitopes are very limited. Thus, in this study, the extent of the variation in these regions was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty-five gene clones were obtained from 19 isolates from the Mae Sod district in Thailand and their sequences were compared with those reported elsewhere. Results reveal 3 sequences types in the tripeptide-encoding region, and each contains a novel repetitive consensus nucleotide sequence. On the other hand, almost all nucleotide substitutions in block III are dimorphic. Identification of linkages of the dimorphic substitutions has led us to postulate 6 potential crossover sites, where intragenic recombinations of p190 alleles could occur. Sequences of T and B cell epitopes are highly conserved among these wild isolates and those from geographically diverse culture-adapted parasites.