Risk factors and prevalence of taeniasis among the Karen people of Tha Song Yang District, Tak Province, Thailand.

Taeniasis remains a prevalent public health problem in Thailand. National helminthiasis surveys report only the incidence of Taenia spp. eggs. The ability to differentiate Taenia species using morphological and molecular techniques is vital for epidemiological surveys. This study detected taeniasis carriers and other helminthic infections by Kato's thick smear technique and identified the Taenia species by multiplex PCR. The study subjects were the ethnic Karen people in Tha Song Yang District, Tak Province, Thailand, bordering Myanmar. In total, 983 faecal samples from villagers were examined for helminthiases. Interview-based questionnaires were used to gather information on possible risk factors for infection. The prevalence of helminth infections was 42.7% (420/983), including single (37.3%, 367/983) and mixed infections (5.4%, 53/983). The most common infection (19.23%, 189/983) was Ascaris lumbricoides, whereas taeniasis carriers comprised 2.8% (28/983). Multiplex PCR of Cox1 was used for species identification of Taenia tapeworms, eggs, or both in 22 taeniasis carriers. Most of the parasites (20 cases) were Taenia solium, with two cases of Taenia saginata. Taenia saginata asiatica was not found in the villagers examined. The analysis of 314 completed questionnaires showed that a statistically significant (p < 0.05) risk of taeniasis was correlated with being male, a history of being allowed to forage during childhood, a history of seeing tapeworm proglottids, and a history of raw or undercooked pork consumption. Health education programmes must seek to reduce and prevent reinfection in these communities.

TITLE: Facteurs de risque et prévalence de la téniase chez les Karens du district de Tha Song Yang, province de Tak, Thaïlande. ABSTRACT: La téniase reste un problème de santé publique répandu en Thaïlande. Les enquêtes nationales sur les helminthiases ne rapportent que l'incidence des œufs de Taenia spp. La capacité de différencier les espèces de Taenia à l'aide de techniques morphologiques et moléculaires est vitale pour les enquêtes épidémiologiques. Cette étude a détecté des porteurs de téniase et d'autres infections helminthiques par la technique de frottis épais de Kato et a identifié les espèces de Taenia par PCR multiplex. Les sujets de l'étude étaient les Karens du district de Tha Song Yang, province de Tak, Thaïlande, à la frontière du Myanmar. Au total, 983 échantillons de matières fécales provenant de villageois ont été examinés pour les helminthiases. Des questionnaires basés sur des entretiens ont été utilisés pour recueillir des informations sur les facteurs de risque possibles d'infection. La prévalence des helminthes était de 42,7 % (420/983), dont des infections uniques (37,3 %, 367/983) et mixtes (5,4 %, 53/983). L'infection la plus courante (19,23 %, 189/983) était Ascaris lumbricoides, tandis que les porteurs de téniase représentaient 2,8 % (28/983). La PCR multiplexe de Cox1 a été utilisée pour l'identification des adultes ou des oeufs de Taenia, ou des deux, chez 22 porteurs de téniase. La plupart des parasites (20 cas) étaient Taenia solium, avec deux cas de Taenia saginata. Taenia saginata asiatica n'a pas été trouvé chez les villageois examinés. L'analyse de 314 questionnaires a montré qu'un risque statistiquement significatif (p < 0,05) de téniase était en corrélation avec le fait d'être un homme, et des antécédents d'avoir été autorisé à ramasser sa nourriture pendant l'enfance, d'avoir vu des proglottis de ténia et de consommation de porc cru ou insuffisamment cuit. Les programmes d'éducation sanitaire doivent chercher à réduire et à prévenir la réinfection dans ces communautés.

Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage.

The Angiostrongylus cantonensis lineage (Nematoda: Metastrongyloidea) consists of the closely related species A. cantonensis, Angiostrongylus malaysiensis, and Angiostrongylus mackerrasae. Various genetic markers have been used for species discrimination in molecular phylogenetic studies of this lineage. However, despite showing potential in other organisms, mitochondrial 12S and 16S ribosomal RNA (rRNA) genes have not been used for Angiostrongylus species discrimination. Therefore, this study assessed these genes' suitability for inter- and intraspecies discrimination in the A. cantonensis lineage. The ultimate aim was to provide a novel genetic marker to support existing phylogenies. Sixty adult Angiostrongylus spp. worms from four geographic locations in Thailand were identified morphologically before molecular identification with 12S and 16S rRNA genes. Neighbor-joining and maximum likelihood algorithms were used for phylogenetic analyzes, and sequence variation was calculated to determine whether the genes could be used to discriminate among species. Furthermore, sequence variation was compared among previously used genetic markers to evaluate the robustness of the 12S and 16S rRNA genes as markers. Using both markers, the A. cantonensis lineage formed a monophyletic clade with a clear separation between A. cantonensis, A. malaysiensis, and A. mackerrasae. From our representative A. cantonensis and A. malaysiensis specimens, the genetic distance between the two clades was 6.8% -7.9% and 7.9% -10.0% for 12S and 16S rRNA genes, respectively, which is sufficient interspecific genetic variation for species discrimination. Higher levels of genetic variation were observed for the 16S rRNA gene, with 12 haplotypes and an intraspecific variation ≤2.2%. Thus, as a genetic marker, the 16S rRNA gene is comparable to mitochondrial protein-coding genes, which are commonly used in intra-level Angiostrongylus spp. studies. In conclusion, mitochondrial 12S and 16S rRNA genes can discriminate among closely related species in the A. cantonensis lineage, and they represent novel genetic markers for supporting existing phylogenies and verifying the phylogenetic position of A. mackerrasae.

Fasciola gigantica, F. hepatica and Fasciola intermediate forms: geometric morphometrics and an artificial neural network to help morphological identification.

BACKGROUND: Fasciola hepatica and F. gigantica cause fascioliasis in both humans and livestock. Some adult specimens of Fasciola sp. referred to as "intermediate forms" based on their genetic traits, are also frequently reported. Simple morphological criteria are unreliable for their specific identification. In previous studies, promising phenotypic identification scores were obtained using morphometrics based on linear measurements (distances, angles, curves) between anatomical features. Such an approach is commonly termed "traditional" morphometrics, as opposed to "modern" morphometrics, which is based on the coordinates of anatomical points. METHODS: Here, we explored the possible improvements that modern methods of morphometrics, including landmark-based and outline-based approaches, could bring to solving the problem of the non-molecular identification of these parasites. F. gigantica and Fasciola intermediate forms suitable for morphometric characterization were selected from Thai strains following their molecular identification. Specimens of F. hepatica were obtained from the Liverpool School of Tropical Medicine (UK). Using these three taxa, we tested the taxonomic signal embedded in traditional linear measurements versus the coordinates of anatomical points (landmark- and outline-based approaches). Various statistical techniques of validated reclassification were used, based on either the shortest Mahalanobis distance, the maximum likelihood, or the artificial neural network method. RESULTS: Our results revealed that both traditional and modern morphometric approaches can help in the morphological identification of Fasciola sp. We showed that the accuracy of the traditional approach could be improved by selecting a subset of characters among the most contributive ones. The influence of size on discrimination by shape was much more important in traditional than in modern analyses. In our study, the modern approach provided different results according to the type of data: satisfactory when using pseudolandmarks (outlines), less satisfactory when using landmarks. The different reclassification methods provided approximately similar scores, with a special mention to the neural network, which allowed improvements in accuracy by combining data from both morphometric approaches. CONCLUSION: We conclude that morphometrics, whether traditional or modern, represent a valuable tool to assist in Fasciola species recognition. The general level of accuracy is comparable among the various methods, but their demands on skills and time differ. Based on the outline method, our study could provide the first description of the shape differences between species, highlighting the more globular contours of the intermediate forms.

Blastocystis subtype 5: Predominant subtype on pig farms, Thailand.

Blastocystis is a unicellular protist most commonly detected in humans and a variety of animals. The predominant mode of its transmission is the fecal-oral route, but its zoonotic potential is not completely understood. The objective of this study was to determine the presence and genetic diversity of Blastocystis on pig farms in Nakhon Pathom Province, Central Thailand. A total of 154 human and 90 pig stool samples were collected and analyzed. Nested PCR detected Blastocystis in 35.55% of the pig samples and 6.49% of the human samples. Subtyping based on regions of the small-subunit ribosomal RNA (SSU rRNA) gene identified three Blastocystis subtypes in pigs and humans: ST1, ST3, and ST5. Blastocystis ST5 was the predominant subtype, followed by ST1 and then ST3. All the sequences from the Blastocystis-positive samples from both pigs and humans were closely related. This study reveals a possibility of low host specificity of Blastocystis STs (ST1, ST3 and ST5) on pig farms in Thailand. We tentatively suggest that close contact with or exposure to pig stools may be a significant source of Blastocystis detected in pig handlers. Further studies are required to confirm the zoonotic transmission of this organism in Thailand, because pigs may play an important role in the transmission of Blastocystis.

Hurdles in the evolutionary epidemiology of Angiostrongylus cantonensis: Pseudogenes, incongruence between taxonomy and DNA sequence variants, and cryptic lineages.

Angiostrongylus cantonensis, the rat lungworm, is a zoonotic pathogen that is one of the leading causes of eosinophilic meningitis worldwide. This parasite is regarded as an emerging pathogen with a global range expansion out of southeastern Asia post-WWII. To date, molecular systematic/phylogeographic studies on A. cantonensis have mainly used two mitochondrial (mtDNA) markers, cytochrome c oxidase 1 (CO1) and cytochrome b (CYTB), where the focus has largely been descriptive in terms of reporting local patterns of haplotype variants. In order to look for more global evolutionary patterns, we herein provide a collective phylogenetic assessment using the six available whole mtDNA genome samples that have been tagged as A. cantonensis, A. malaysiensis, or A. mackerrasae along with all other GenBank CO1 and CYTB partial sequences that carry these species identifiers. The results reveal three important complications that researchers will need to be aware of, or will need to resolve, prior to conducting future molecular evolutionary studies on A. cantonensis. These three problems are (i) incongruence between taxonomic identifications and mtDNA variants (haplotypes or whole mtDNA genome samples), (ii) the presence of a CYTB mtDNA pseudogene, and (iii) the need to verify A. mackerrasae as a species along with other possible cryptic lineages, of which there is suggestive evidence (i.e., A. cantonensis could be a species complex). We provided a discussion of how these complications are hurdles to our understanding of the global epidemiology of angiostrongyliasis. We call for future studies to be more explicit in morphological traits used for identifications (e.g., provide measurements). Moreover, it will be necessary to repeat prior morphological and life-history studies while simultaneously using sequence data in order to assess possible associations between critical epidemiological data (e.g., biogeography, virulence/pathology, host species use) and specific lineages.

Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand.

Blastocystis is a common intestinal pathogen of humans and a variety of animals, with various host-specific subtypes. The aim of this study was to determine the prevalence and subtype distribution of Blastocystis in humans and domestic animals, Thailand. 113 stool samples were collected from pigs, goats, and cattle in Ayutthaya Province (AP; central Thailand) and 218 stool samples were collected from pigs, dogs, cats, chickens, and humans in Kanchanaburi Province (KP; western Thailand). Blastocystis was detected by nested PCR targeting the SSU rRNA gene. Subtypes were identified by DNA sequencing, and phylogenetic analysis was conducted. The overall prevalence of Blastocystis in animals was 76.1% (86/113) and 11.88% (12/101) in AP and KP, respectively, and the prevalence in humans was 12.82% (15/117) in KP. The prevalence of Blastocystis in the AP and KP pigs were 87.88% (29/33) and 20.37% (11/54), respectively. Blastocystis ST5 was the most abundant in pigs in both areas while Blastocystis ST10 and ST12 were most frequently found in cattle and goats. In addition, low percentage of Blastocystis ST1 and Blastocystis ST14 were found in pigs and goats, respectively. In this study, Blastocystis ST3, followed by ST2 and ST1 were predominantly found in humans. In conclusion, pigs may be a natural host of Blastocystis and this ST may be the pig-adapted ST in the studied areas, in this study.

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand.

BACKGROUND: Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. METHODS: To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 µg/ml, and anti-human IgG diluted at 1:4000. RESULTS: The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). CONCLUSIONS: Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.

Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand.

Enterocytozoon bieneusi is an opportunistic intestinal pathogen infecting humans and a variety of animals. Its mode of transmission and zoonotic potential are not completely understood. E. bieneusi has been frequently identified in pigs. The objective of our study was to investigate E. bieneusi in pigs and humans in Western and Central Thailand to determine its presence, genetic diversity, and zoonotic potential. A total of 277 human and 210 pig faecal samples were collected and analysed. E. bieneusi was found in 5.4% and 28.1% of human and pig samples, respectively, by nested PCR. Genotyping based on the internal transcribed spacer regions of the small subunit ribosomal RNA demonstrated three known genotypes (D, H, PigEb10) and eight novel genotypes (TMH1-8) in humans, and five known genotypes (D, EbpA, EbpC, H, O) and 11 novel genotypes (TMP1-11) in pigs. All known genotypes identified in humans and pigs had zoonotic potential. Further studies are needed to evaluate zoonotic risk of novel genotypes, as pigs may play an important role in the transmission of E. bieneusi.

Cryptic lineage diversity in the zoonotic pathogen Angiostrongylus cantonensis.

Delimitation of species is still a necessity among parasitic pathogens especially where morphological characters provide limited discernibility. Identification of cryptic lineages (independently evolving lineages that are morphologically similar) is critical as there could be lineage-specific traits that are of epidemiological importance. Angiostrongylus cantonensis is a zoonotic pathogen that can cause eosinophilic meningitis in humans. Recent reports of single marker sequence divergence hint at the potential for cryptic diversity in this lungworm. However, to definitively address if single marker divergence corresponds to independent evolving lineages, a multilocus approach is necessary. Using multilocus data, our goal was to determine if there were cryptic lineages within Thailand, a country plagued by several outbreaks and isolated cases of A. cantonensis infection. We analyzed the genetic structure of A. cantonensis samples collected from snails, Achatina fulica, across provinces in Thailand. Multilocus data (mitochondrial sequence data and 12 nuclear microsatellites) and individual based analyses were used to test for cryptic lineages. We found strong linkage disequilibrium patterns between mitochondrial haplotypes and nuclear-identified genetic clusters. There were clearly two divergent and independent clades. Moreover, within each clade, the data suggested additional substructure where individual provinces were likely to harbor unique genetic clusters. The results indicate there are at minimum two and possibly up to eight cryptic lineages within the assumed single species of A. cantonensis. Importantly, the two main clades do not show geographic affiliation and can be found in sympatry. With recent studies highlighting A. cantonensis strain diversity in pathogenicity and infectivity, it will be important to determine if these critical epidemiological traits are associated with specific lineages.

Cloning and expression of a 16-kDa recombinant protein from Angiostrongylus cantonensis for use in immunoblot diagnosis of human angiostrongyliasis.

Angistrongylus cantonensis is a zoonotic nematode parasite and causative agent of human angiostrongyliasis, which clinically presents as eosinophilic meningitis or meningoencephalitis. Diagnosis of the disease is problematic since parasitologic findings are infrequent, and infection determinations must be based on the clinical symptoms and serological tests with limited specificities and sensitivities. The aim of the present study was to identify and generate a novel recombinant protein from A. cantonensis and evaluate its efficacy in the diagnosis of human angiostrongyliasis when incorporated into a Western blot serodiagnostic system. A cDNA protein expression library from adult A. cantonensis was constructed, followed by immunoscreening with serum from confirmed infected patients to identify and isolate immunoreactive clones. One clone, designated fAC40, possessed a partial sequence encoding a LisH protein domain with a predicted molecular weight of 16 kDa and containing four predicted antigenic peptides. By incorporating recombinant fAC40 in Western immunoblot tests using a serum panel consisting of confirmed and clinically diagnosed cases of human angiostrongyliasis and other helminthic infections, fAC40 exhibited a sensitivity and specificity of 91.8 and 100 %, respectively, and a positive and negative predictive value of 100 and 97.19 %, respectively, in the diagnosis of angiostrongyliasis. Importantly, it was not reactive with antibodies from serum of patients infected with Gnathostoma spinigerum and Cysticercus cellulosae, infections that clinically present neurological symptoms similar to angiostrongyliasis. These data demonstrate that the 16-kDa recombinant protein from A. cantonensis possesses high potential as a candidate antigen for a more sensitive and specific serodiagnosis of human angiostrongyliasis.

Health Status of Immigrant Children and Environmental Survey of Child Daycare Centers in Samut Sakhon Province, Thailand.

Samut Sakhon is a Thai province popular among immigrants attracted to work in factories and the Thai food industry, especially people from Myanmar. Poor personal-hygiene behaviors, crowded accommodation and limited sanitation, result in health problems among immigrant workers. Various infectious diseases among this group are seen and managed by Samut Sakhon General Hospital. The impact of intestinal parasitic infections on public health is well known; they can spread from infected immigrant areas to uninfected areas via close contact and fecal-oral transmission from contaminated food and water. This study aimed to determine the prevalence of intestinal parasitic infections among 372 immigrant children at 8 child-daycare centers during their parents' work time, by physical examination, fecal examination, and examination of the environment around the centers. Physical examinations were generally unremarkable, except that head-lice and fingernail examinations were positive in two cases (0.8 %). The results showed intestinal parasitic infections to be highly prevalent, at 71.0 %. These infections comprised both helminths and protozoa: Trichuris trichiura (50.8 %), Enterobius vermicularis (25.2 %), Ascaris lumbricoides (15.3 %), hookworm (11.6 %), Giardia lamblia (10.2 %), Endolimax nana (3.5 %), Entamoeba coli (2.7 %), and Blastocystis hominis (0.5 %). The environmental survey found a small number of houseflies near the accommodation to be positive for helminthic eggs (0.2 %), including A. lumbricoides, E. vermicularis, hookworms, Taenia spp., and minute intestinal flukes. Regarding the high prevalence of intestinal parasitic infections among children, it has been conjectured whether they were infected, along with their parents, during their daily lives before or after settling in Thailand. Intestinal parasites among immigrant children may involve a significant epidemiological impact, since immigrant children can serve as carriers and transmitters of disease.

Wide dissemination of extended-spectrum ß-lactamase-producing Escherichia coli in community residents in the Indochinese peninsula.

Recent studies have reported a widespread distribution of extended-spectrum ß-lactamase (ESBL)-producing bacteria, not only in the nosocomial setting, but also in the community; some local communities in Southeast Asia have been reported to show a high prevalence of ESBL-producing bacteria. However, the details regarding the quantitative/qualitative state of ESBL-producing bacterial spread in Southeast Asia are currently unclear. Thus, the aim of this study was to assess the state of ESBL-producing bacterial spread in community residents from the Indochinese peninsula, as a representative region of Southeast Asia. In order to achieve this aim, local community residents in Laos and Vietnam were examined for fecal carriage of ESBL-producing Enterobacteriaceae, and the findings were compared with data from a previous study in Thailand which was conducted in the same manner as this study. Between 47.0%-70.2% of the Laotian and Vietnamese residents carried ESBL-producing CTX-M genotype Enterobacteriaceae. The most common sub-genotypes of CTX-M were CTX-M-1 (33.0%-47.5%) and CTX-M-9 (47.5%-64.1%), and these rates were similar among all three countries. Taken together, these results confirmed that ESBL-producing Enterobacteriaceae are widely disseminated in Indochinese countries, such as Thailand, Laos, and Vietnam.

Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study.

The emergence of colistin-resistant Klebsiella pneumoniae (CRKP) is a major public health concern worldwide. In this study, the prevalence and molecular basis of colistin resistance in CRKP isolated from healthy individuals and patients in Lao PDR, Thailand, Nigeria and France were investigated. Stool samples were screened by culture for the presence of colistin-resistant Klebsiella spp. Whole-genome sequence analysis was used to decipher the molecular mechanism of colistin resistance in a blaNDM-1-positive in vitro-selected CRKP mutant. PCR amplification and sequencing of the mgrB genetic environment was performed for all CRKP isolates as well as control colistin-susceptible K. pneumoniae (CSKP) isolates recovered from the same stools. A total of 869 stool samples were screened for colistin-resistant Klebsiella spp., yielding 32 CRKP and 2 colistin-resistant Klebsiella oxytoca. Comparative whole-genome sequence analysis revealed that an in vitro-selected CRKP mutant had an insertion sequence in its mgrB gene, as well as missense mutations in other selected clones. Of the 34 colistin-resistant Klebsiella spp. isolates, 14 (41.2%; 13 CRKP and 1 K. oxytoca) from the four countries also had various defects in their mgrB genes, but no such defects were found in the CSKP controls (P<10(-4)). Few mutations were observed in pmrAB compared with mgrB among the CRKP isolates. The worldwide emergence of CRKP is a major public health concern. Detection and surveillance of such strains are warranted to prevent an uncontrollable pandemic. Inactivation of the PhoP/PhoQ regulator gene mgrB is associated with ≥40% of colistin resistance among the CRKP isolates observed in this study.

Triplex polymerase chain reaction assay for detection of major soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, in fecal samples.

Ascaris lumbricoides, Trichuris trichiura, and Necator americanus are medically important soil-transmitted helminths (STHs) occurring frequently worldwide including Thailand. Fecal examination using a microscope has been recommended as the gold standard for diagnosis of STH infections, but suffers from low sensitivity. Recently, highly sensitive and specific assays, such as multiplex quantitative PCR, has been established, but the high cost and need for special instruments are still barriers limiting their applications in routine diagnosis. Therefore, a conventional multiplex PCR assay, with its lower cost and greater simplicity, was developed, for the simultaneous detection of STHs in fecal samples. The multiplex PCR assay was species-specific to the three STHs, and could detect one copy of DNA target. Compared with microscopic examination of fecal samples, sensitivity and specificity of the multiplex PCR was 87% and 83%, respectively. This multiplex PCR assay provides an alternative method for routine diagnosis of STHs infection, and might be applied for epidemiological studies of STHs in endemic areas.

Zoonotic potential of Enterocytozoon bieneusi among children in rural communities in Thailand.

Enterocytozoon bieneusi is a common opportunistic intestinal pathogen worldwide. Genotype distribution of E. bieneusi differs by geography and host immunity. In order to investigate the prevalence, genotype characteristics, and host specificity of E. bieneusi in the community, we conducted a preliminary cross-sectional study among children in Western and Northern Thailand. Seventy-eight (78) and 102 stool samples were collected; the prevalence of E. bieneusi was 3.8% and 2.9% by nested PCR in Western and Northern Thailand, respectively. Three genotypes were identified: Genotype D predominated, followed by EbpC, and then novel genotype ETMK1. The first two genotypes have zoonotic potential. Analysis of the genetic proximity of the E. bieneusi ITS sequences from our study, compared with those published in genetic databases, showed that all positive samples were classified into Group 1, the largest group consisting of various host specificity. The present study demonstrates the possible zoonotic transmission of E. bieneusi in rural communities in Thailand. A large-scale investigation of both human and animal samples, as well as improvements in the available phylogenetic tools, will be required to elucidate transmission routes of E. bieneusi in this area.

Population structure of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Thailand based on PCR-RAPD markers.

Angiostrongylus cantonensis is the causative agent of angiostrongyliasis, which is widely distributed throughout the world. It can specifically infect many species of intermediate and definitive hosts. This study examined the genetic differentiation and population structure using the RAPD-PCR method of parasites obtained from 8 different geographical areas of Thailand. Based on 8 primers, high levels of genetic diversity and low levels of gene flow among populations were found. Using genetic distance and neighbor-joining dendrogram methods, A. cantonensis in Thailand could be divided into two groups with statistically significant genetic differentiation of the two populations. However, genotypic variations and haplotype relationships need to be further elucidated using other markers.

Prevalence of and risk factors associated with faecal carriage of CTX-M ß-lactamase-producing Enterobacteriaceae in rural Thai communities.

OBJECTIVES: To determine the prevalence of CTX-M ß-lactamase-producing Enterobacteriaceae and to study the risk factors associated with faecal carriage in asymptomatic rural Thai people. METHODS: In all, 417 stool samples were obtained from rural Thai people and screened for extended-spectrum ß-lactamases (ESBLs) using MacConkey agar supplemented with 2 mg/L cefotaxime. Results were confirmed using cefotaxime and ceftazidime with and without clavulanic acid. The bla(CTX-M) genes were identified and genotyped using PCR with bacterial DNA samples. Multivariate analysis was performed to investigate risk factors associated with the faecal carriage of CTX-M producers. RESULTS: The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae was 65.7%. The CTX-M-9 group (60.6%) was dominant, followed by the CTX-M-1 group (38.7%). Most of the bacteria were Escherichia coli (85.4%) and Klebsiella pneumoniae (4.7%). Of a total of 234 E. coli strains, 48.7% belonged to phylogenetic group A, 28.6% to group B1, 15.8% to group D and 6.8% to group B2. Most CTX-M producers were susceptible to carbapenems and amikacin, but resistant to tetracycline and gentamicin. In a multivariate logistic regression model, better education status (OR 2.245; 95% CI 1.297-3.884), history of hospitalization (OR 1.643; 95% CI 1.036-2.603) and the use of antibiotics within the last 3 months (OR 1.883; 95% CI 1.221-2.903) were independently associated with faecal carriage. CONCLUSIONS: Faecal carriage of CTX-M-type ESBL-producing Enterobacteriaceae among asymptomatic individuals in rural Thailand remains alarmingly high, and previous antibiotic use and a history of hospitalization may contribute to its dissemination.

Degradation of human matrix metalloprotease-9 by secretory metalloproteases of Angiostrongylus cantonensis infective stage.

Angiostrongylus cantonensis infection is the major cause of eosinophilic meningitis. Successful migration and evasion of the immune system by infective-stage larvae (L3) rely heavily on secreted proteases, which activate human pro-matrix metalloprotease (MMP-9) into active MMP-9. This study showed that the proteases in excretory-secretory (ES) products of A. cantonensis third stage larvae degraded recombinant and native human proMMP-9 in a dose- and time-dependent manner. Protease inhibitory assays showed that metalloproteases were the key enzymes involved in the degradation of human proMMP-9. To assess the effects of ES products on inflammation, ES products were incubated with THP-1 human monocytic cells, which showed induction of MMP-2 and not MMP-9 production. These results indicated that degradation of human MMP-9 was due to metalloproteases present in ES of A. cantonensis L3, which may be involved in suppressing the host's immune response to allow parasite migration to the host central nervous system.

Analysis of risk factors for a high prevalence of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in asymptomatic individuals in rural Thailand.

The prevalence of and risk factors associated with extended-spectrum ß-lactamase (ESBL)-producing micro-organisms have not been well studied in healthy individuals. The aim of this study was to determine this in healthy individuals in Thailand. Stool samples and questionnaires obtained from 445 participants from three provinces in Thailand were analysed. The antimicrobial susceptibility of the isolates was assessed using phenotypic and genotypic methods. PCR analysis was performed to detect and group the bla(CTX-M) genes. The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae in the three provinces was as follows: 29.3 % in Nan (43/147), 29.9 % in Nakhon Si Thammarat (43/144) and 50.6 % in Kanchanaburi (78/154) (P<0.001). Of the 445 samples, 33 (7.4 %), 1 (0.2 %) and 127 (28.5 %) isolates belonged to the bla(CTX-M) gene groups I, III and IV, respectively. Escherichia coli was the predominant member of the Enterobacteriaceae producing CTX-M-type ESBLs (40/43, 39/43 and 70/78 isolates in Nan, Nakhon Si Thammarat and Kanchanaburi, respectively). No statistically significant association was observed between the presence of ESBL-producing bacteria and gender, age, education, food habits or antibiotic usage. However, the provinces that had the highest prevalence of ESBL-producing Enterobacteriaceae also had the highest prevalence of use and purchase of antibiotics without a prescription. Thus, this study revealed that faecal carriage of ESBL-producing Enterobacteriaceae is very high in asymptomatic individuals in Thailand, with some variations among the provinces. This high prevalence may be linked to antibiotic abuse.