Natural vectors of Plasmodium knowlesi and other primate, avian and ungulate malaria parasites in Narathiwat Province, Southern Thailand.

To date, four species of simian malaria parasites including Plasmodium knowlesi, P. cynomolgi, P. inui and P. fieldi have been incriminated in human infections in Thailand. Although the prevalence of malaria in macaque natural hosts has been investigated, their vectors remain unknown in this country. Herein, we performed a survey of Anopheles mosquitoes during rainy and dry seasons in Narathiwat Province, Southern Thailand. Altogether 367 Anopheles mosquitoes were captured for 40 nights during 18:00 to 06:00 h by using human-landing catches. Based on morphological and molecular identification, species composition comprised An. maculatus (37.06%), An. barbirostris s.l. (31.34%), An. latens (17.71%), An. introlatus (10.08%) and others (3.81%) including An. umbrosus s.l., An. minimus, An. hyrcanus s.l., An. aconitus, An. macarthuri and An. kochi. Analyses of individual mosquitoes by PCR, sequencing and phylogenetic inference of the mitochondrial cytochrome genes of both malaria parasites and mosquitoes have revealed that the salivary gland samples of An. latens harbored P. knowlesi (n = 1), P. inui (n = 2), P. fieldi (n = 1), P. coatneyi (n = 1), P. hylobati (n = 1) and an unnamed Plasmodium species known to infect both long-tailed and pig-tailed macaques (n = 2). The salivary glands of An. introlatus possessed P. cynomolgi (n = 1), P. inui (n = 1), P. hylobati (n = 1) and coexistence of P. knowlesi and P. inui (n = 1). An avian malaria parasite P. juxtanucleare has been identified in the salivary gland sample of An. latens. Three other distinct lineages of Plasmodium with phylogenetic affinity to avian malaria species were detected in An. latens, An. introlatus and An. macarthuri. Interestingly, the salivary gland sample of An. maculatus contained P. caprae, an ungulate malaria parasite known to infect domestic goats. Most infected mosquitoes harbored multiclonal Plasmodium infections. All Plasmodium-infected mosquitoes were captured during the first quarter of the night and predominantly occurred during rainy season. Since simian malaria in humans has a wide geographic distribution in Thailand, further studies in other endemic areas of the country are mandatory for understanding transmission and prevention of zoonotic malaria.

Structural organization and sequence diversity of the complete nucleotide sequence encoding the Plasmodium malariae merozoite surface protein-1.

The merozoite surface protein-1 (MSP1) is a prime candidate for an asexual blood stage vaccine against malaria. However, polymorphism in this antigen could compromise the vaccine's efficacy. Although the extent of sequence variation in MSP1 has been analyzed from various Plasmodium species, little is known about structural organization and diversity of this locus in Plasmodium malariae (PmMSP1). Herein, we have shown that PmMSP1 contained five conserved and four variable blocks based on analysis of the complete coding sequences. Variable blocks were characterized by short insertion and deletion variants (block II), polymorphic nonrepeat sequences (block IV), complex repeat structure with size variation (block VI) and degenerate octapeptide repeats (block VIII). Like other malarial MSP1s, evidences of intragenic recombination have been found in PmMSP1. The rate of nonsynonymous nucleotide substitutions significantly exceeded that of synonymous nucleotide substitutions in block IV, suggesting positive selection in this region. Codon-based analysis of deviation from neutrality has identified a codon under purifying selection located in close proximity to the homologous region of the 38 kDa/42 kDa cleavage site of P. falciparum MSP1. A number of predicted linear B-cell epitopes were identified across both conserved and variable blocks of the protein. However, polymorphism in repeat-containing blocks resulted in alteration of the predicted linear B-cell epitope scores across variants. Although a number of predicted HLA-class II-binding peptides were identified in PmMSP1, all variants of block IV seemed not to be recognized by common HLA-class II alleles among Thai population, suggesting that diversity in this positive selection region could probably affect host immune recognition. The data on structural diversity in PmMSP1 could be useful for further studies such as vaccine development and strain characterization of this neglected malaria parasite.

Cryptic Plasmodium inui and Plasmodium fieldi Infections Among Symptomatic Malaria Patients in Thailand.

BACKGROUND: Some nonhuman primate Plasmodium species including P. knowlesi and P. cynomolgi can cross-transmit from macaque natural hosts to humans under natural infection. This study aims to retrospectively explore other simian Plasmodium species in the blood samples of symptomatic malaria patients in Thailand. METHODS: A total of 5271 blood samples from acute febrile patients from 5 malaria endemic provinces and 1015 blood samples from long-tailed and pig-tailed macaques from 3 locations were examined for Plasmodium species by microscopy and species-specific polymerase chain reaction. The Plasmodium mitochondrial cytochrome oxidase 1 (COX1) gene was analyzed by amplicon deep sequencing as well as Sanger sequencing from recombinant plasmid clones to reaffirm and characterize P. inui and P. fieldi. RESULTS: Besides human malaria, P. knowlesi, P. cynomolgi, P. inui and P. fieldi infections were diagnosed in 15, 21, 19, and 3 patients, respectively. Most P. inui and all P. fieldi infected patients had simultaneous infections with other Plasmodium species, and seemed to be responsive to chloroquine or artemisinin-mefloquine. P. inui was the most prevalent species among macaque populations. Phylogenetic analysis of the COX1 sequences from human and macaque isolates reveals the genetic diversity of P. inui and suggests that multiple parasite strains have been incriminated in human infections. CONCLUSIONS: Both P. inui and P. fieldi could establish infection in humans under natural transmission. Despite occurring at a low prevalence and mostly co-existing with other Plasmodium species, P. inui infections in humans have a wide distribution in Thailand.

Plasmodium cynomolgi Co-infections among Symptomatic Malaria Patients, Thailand.

Among 1,180 symptomatic malaria patients, 9 (0.76%) infected with Plasmodium cynomolgi were co-infected with P. vivax (n = 7), P. falciparum (n = 1), or P. vivax and P. knowlesi (n = 1). Patients were from Tak, Chanthaburi, Ubon Ratchathani, Yala, and Narathiwat Provinces, suggesting P. cynomolgi is widespread in this country.

Multiple Novel Mutations in Plasmodium falciparum Chloroquine Resistance Transporter Gene during Implementation of Artemisinin Combination Therapy in Thailand.

Mutations in the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) are associated with drug susceptibility status of chloroquine and other antimalarials that interfere with heme detoxification process including artemisinin. We aim to investigate whether an increase in duration of artemisinin combination therapy (ACT) in Thailand could affect mutations in Pfcrt. The complete coding sequences of Pfcrt and dihydrofolate reductase (Pfdhfr), and size polymorphisms of the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) of 189 P. falciparum isolates collected during 1991 and 2016 were analyzed. In total, 12 novel amino acid substitutions and 13 novel PfCRT haplotypes were identified. The most prevalent haplotype belonged to the Dd2 sequence and no wild type was found. A significant positive correlation between the frequency of Pfcrt mutants and the year of sample collection was observed during nationwide ACT implementation (r = 0.780; P = 0.038). The number of haplotypes and nucleotide diversity of isolates collected during 3-day ACT (2009-2016) significantly outnumbered those collected before this treatment regimen. Positive Darwinian selection occurred in the transmembrane domains only among isolates collected during 3-day ACT but not among those collected before this period. No remarkable change was observed in the molecular indices for other loci analyzed when similar comparisons were performed. An increase in the duration of artesunate in combination therapy in Thailand could exert selective pressure on the Pfcrt locus, resulting in emergence of novel variants. The impact of these novel haplotypes on antimalarial susceptibilities requires further study.

Variation in intronic microsatellites and exon 2 of the Plasmodium falciparum chloroquine resistance transporter gene during modification of artemisinin combination therapy in Thailand.

The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt, we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p < 0.05). By contrast, the number of haplotypes and haplotype diversity of the merozoite surface proteins 1 and 2 of these parasite populations did not differ significantly between these periods. Therefore, the Pfcrt locus of P. falciparum in Thailand continues to evolve and could have been affected by selective pressure from modification of ACT regimen.

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.

Ecology of malaria parasites infecting Southeast Asian macaques: evidence from cytochrome b sequences.

Although malaria parasites infecting non-human primates are important models for human malaria, little is known of the ecology of infection by these parasites in the wild. We extensively sequenced cytochrome b (cytb) of malaria parasites (Apicomplexa: Haemosporida) from free-living southeast Asian monkeys Macaca nemestrina and Macaca fascicularis. The two most commonly observed taxa were Plasmodium inui and Hepatocystis sp., but certain other sequences did not cluster closely with any previously sequenced species. Most of the major clades of parasites were found in both Macaca species, and the two most commonly occurring parasite infected the two Macaca species at approximately equal levels. However, P. inui showed evidence of genetic differentiation between the populations infecting the two Macaca species, suggesting limited movement of this parasite among hosts. Moreover, coinfection with Plasmodium and Hepatocystis species occurred significantly less frequently than expected on the basis of the rates of infection with either taxon alone, suggesting the possibility of competitive exclusion. The results revealed unexpectedly complex communities of Plasmodium and Hepatocystis taxa infecting wild southeast Asian monkeys. Parasite taxa differed with respect to both the frequency of between-host movement and their frequency of coinfection.

Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine samples from symptomatic malaria patients in a low endemic area.

BACKGROUND: Definite diagnosis of malaria relies on microscopy detection of blood stages of parasites in peripheral blood and requires blood sample collection. The nested PCR method has shown to be more sensitive and superior to microscopy in detecting co-infections of Plasmodium species in circulation while Plasmodium falciparum DNA can be identified in urine and saliva specimens of patients, albeit at a lower sensitivity. METHODS: Matched blood, saliva and urine samples were collected from 100 microscopy-positive and 20 microscopy-negative febrile patients who attended a malaria clinic in Tak Province, northwestern Thailand for nested PCR analysis targeting the small subunit ribosomal RNA gene of human malaria. Both P. falciparum and Plasmodium vivax have been known to circulate at a comparable rate in the study area. RESULTS: Comparing with microscopy results, nested PCR of saliva samples had a sensitivity of 74.1% for P. falciparum detection and 84% for P. vivax detection while 44.4% and 34.0% of the corresponding values were observed for urine samples. Both nested PCR results of saliva and urine samples had a specificity of 100% for identification of P. falciparum and P. vivax when compared with nested PCR results from blood. Co-infections of both species were found in four, 26 and 8 patients by microscopy and nested PCR of blood and saliva samples, respectively. Although the positive rates of nested PCR of saliva samples for P. falciparum increased with parasite density, no tendency occurred in results from nested PCR of saliva samples for P. vivax as well as those of urine samples. CONCLUSIONS: Saliva and urine samples could be alternative noninvasive sources of DNA for molecular detection of both P. falciparum and P. vivax. Further improvement of the detection method will offer an opportunity to use these samples for diagnosis of malaria.

Differential prevalence of Plasmodium infections and cryptic Plasmodium knowlesi malaria in humans in Thailand.

BACKGROUND: A case of human infection with Plasmodium knowlesi has been recently discovered in Thailand. To investigate the prevalence of this malaria species, a molecular-based survey was performed. METHODS: Blood samples from 1874 patients were tested for Plasmodium species by microscopy and nested polymerase chain reaction. P. knowlesi was characterized by sequencing the merozoite surface protein 1 gene (msp-1). RESULTS: Of all Plasmodium species identified, P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi contributed 43.52%, 68.08%, 1.37%, 1.03%, and 0.57%, respectively. Mixed-species infections were more common in northwestern and southwestern regions bordering Myanmar (23%-24%) than in eastern and southern areas (3%-5%). In northwestern and southwestern regions, mixed-species infections had a significantly higher prevalence in dry than in rainy seasons (P < .001). P. knowlesi was found in 10 patients, mostly from southern and southwestern areas-9 were coinfected with either P. falciparum or P. vivax. Most of the P. knowlesi Thai isolates were more closely related to isolates from macaques than to isolates from Sarawak patients. The msp-1 sequences of isolates from the same area of endemicity differed and possessed novel sequences, indicating genetic polymorphism in P. knowlesi infecting humans. CONCLUSIONS: This survey highlights the widespread distribution of P. knowlesi in Thailand, albeit at low prevalence and mostly occurring as cryptic infections.

Malaria and Hepatocystis species in wild macaques, southern Thailand.

Southeast Asian macaques are natural hosts for a number of nonhuman primate malaria parasites; some of these can cause diseases in humans. We conducted a cross-sectional survey by collecting 99 blood samples from Macaca fascicularis in southern Thailand. Giemsa-stained blood films showed five (5.1%) positive samples and six (6.1%) isolates had positive test results by polymerase chain reaction. A phylogenetic tree inferred from the A-type sequences of the small subunit ribosomal RNA gene confirmed Plasmodium inui in five macaques; one of these macaques was co-infected with P. coatneyi. Hepatocystis, a hemoprotozoan parasite transmitted by Culicoides, was identified in an isolate that was confirmed by analysis of mitochondrial cytochrome b sequences. All malaria-infected monkeys lived in mangrove forests, but no infected monkeys were found in an urban area. These findings indicate regional differences in malaria distribution among these macaques, as well as differences in potential risk of disease transmission to humans.

Positive selection on the Plasmodium falciparum sporozoite threonine-asparagine-rich protein: analysis of isolates mainly from low endemic areas.

The sporozoite threonine-asparagine-rich protein (STARP) of Plasmodium falciparum is an attractive target for a pre-erythrocytic stage malaria vaccine because both naturally acquired and experimentally induced anti-STARP antibodies can block sporozoite invasion of hepatocytes. To explore the extent of sequence variation, we surveyed nucleotide polymorphism across the entire gene, encompassing 2 exons and an intron, of 124 P. falciparum-infected blood samples from Thailand and 10 from 4 other endemic areas. In total 24 haplotypes were identified despite low-level nucleotide diversity at this locus. The mean number of nonsynonymous substitutions per nonsynonymous site (d(N)) significantly exceeded that of synonymous substitutions per synonymous site (d(S)), suggesting that the STARP gene has evolved under positive selection, probably from host immune pressure. The preponderance of conservative amino acid exchanges and a strongly biased T-nucleotide toward the third position of codons in repeat arrays have reflected simultaneous constraints on this molecule, probably from its respective unknown function and nucleotide composition. Sequence conservation in the STARP locus among clinical isolates from different disease endemic areas would not compromise vaccine incorporation.