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.

A survey of Acanthamoeba in 100 public freshwater sources in 28 provinces across Thailand has identified 9 genotypes comprising T2/6, T3-T5, T9, T11, T12, T18 and a novel ‘T23’ among 131 isolates. Sequencing of the near complete 18S rRNA gene of Acanthamoeba of all isolates has shown that the most predominant genotype T4 found in 87 isolates (66.4%) contained 4 subtypes, i.e. T4A, T4B, T4C and T4F, while all isolates assigned to genotype T2/6 belonged to subtype B. Among intron-bearing genotypes, most isolates harbouring genotype T3 contained S516 introns, characterised by 3 distinct variants whilst all genotypes T4A and T5 were intronless. Identical 18S rRNA sequences of Acanthamoeba were identified across regions of the country and four isolates in this study shared the same sequences with those from remote nations, suggesting that some strains have reproductive success in diverse ecological niche. Nucleotide diversity of genotypes T2/6B, T3, T4, T9 and T11 in this study was significantly less than that among global isolates outside Thailand, implying that limited sequence diversity occurred within local populations. A remarkably higher level of nucleotide diversity in genotype T11 than those of other genotypes (0.041 vs. 0.012–0.024) could be due to cryptic subtypes. Recombination breakpoints have been detected within genotypes and subtypes as well as within isolates despite no evidence for sexual and parasexual cycles in the genus Acanthamoeba. Tajima’s D, Fu & Li’s D* and F* statistics revealed significantly negative deviation from neutrality across genotypes and subtypes, implying purifying selection in this locus. The 18S rRNA gene of the novel genotype ‘T23’ displayed 7.82% to 28.44% sequence differences in comparison with all known genotypes. Both Bayesian and maximum likelihood phylogenetic trees have placed genotype T23 as sister to the clade comprising genotypes T10, T12 and T14, all of these possess cyst structure belonging to morphological group III. Hence, Acanthamoeba bangkokensis sp. nov. is proposed for this novel genotype. It is likely that more genotypes of Acanthamoeba remain to be discovered while the evolution of the 18S rRNA gene of this pathogenic-free living amoeba seems to be ongoing.

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.

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Intestinal parasitic infections, including those caused by Entamoeba species, are a persistent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Entamoeba species and to analyze their genotypic diversity. Stool samples were collected from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive samples for E. histolytica and E. dispar were exclusively obtained from a few school classes, whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nuttalli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. dispar (n = 47) to investigate their diversity and potential modes of transmission. Five genotypes of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A were divergent, but several unique genotypes were significantly prevalent in limited classes, indicating that intra-classroom transmission has occurred. As it was unlikely that infection would have been limited within school classes if the mode of transmission of E. histolytica and E. dispar had been through the intake of contaminated drinking water or food, these results suggest a direct or indirect person-to-person transmission mode within school classes. Positive rates for three Entamoeba species were 2-fold higher in students who had siblings in the schools than in those without siblings, suggesting that transmission occurred even at home due to heavy contacts among siblings.