Asymptomatic Human Paragonimiasis among the Karen People in Tak Province, Thailand: A Case Report.

During the mobile clinic activities in Tak Province, Thailand, Paragonimus sp. eggs were found in a fecal sample of a 72-year-old Karen resident. Paragonimus DNA was amplified from the stool sample and identified to P. heterotremus. The patient did not have any symptoms. Apparent pulmonary lesion was not found on the chest X-ray. The patient admitted habitual consumption of semi-cooked or roasted waterfall crabs for several years. The waterfall crabs collected from stream near the village were found negative for Paragonimus metacercariae. In northern Thailand, paragonimiasis remains as one of the public health concerns and should be ruled out for asymptomatic pulmonary patients.

A SIMPLE AND EFFECTIVE MULTIPLEX PCR TECHNIQUE FOR DETECTING HUMAN PATHOGENIC TAENIA EGGS IN HOUSEFLIES.

Taenia solium, T. saginata, and T. asiatica are cestode pathogens causing taeniasis in humans. Houseflies can transfer Taenia eggs to food. However, houseflies are thought to carry only small numbers of Taenia eggs, sometimes fewer than 10. Although several PCR-based methods have been developed to detect Taenia DNA, these require more than 10 eggs for adequate detection. We developed a multiplex PCR method with high specificity for the discrimination among the eggs of the three Taenia species, T. solium, T. saginata, and T. asiatica, using 18S ribosomal DNA (rDNA) as a genetic marker. This technique was found to be highly sensitive, capable of identifying the Taenia species from only one egg. This multiplex PCR technique using 18S rDNA specific primers should be suitable to diagnose Taenia eggs.

Genetic differentiation of strongyloides stercoralis from two different climate zones revealed by 18S ribosomal DNA sequence comparison.

Over 70 countries in tropical and subtropical zones are endemic areas for Strongyloides stercoralis, with a higher prevalence of the parasite often occurring in tropical regions compared to subtropical ones. In order to explore genetic variations of S. stercoralis form different climate zones, 18S ribosomal DNA of parasite specimens obtained from Thailand were sequenced and compared with those from Japan. The maximum likelihood indicates that S. stercoralis populations from these two different climate zones have genetically diverged. The genetic relationship between S. stercoralis populations is not related to the host species, but rather to moisture and temperature. These factors may directly drive genetic differentiation among isolated populations of S. stercoralis.

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.

Impact of hookworm deworming on anemia and nutritional status among children in Thailand.

Hookworm infection is associated with anemia, especially among children and deworming can improve anemic status; however, little information is available about the degree to which anemia improves after deworming. We chose hookworm-endemic rural areas of Thailand, Nan Province in the north, Kanchanaburi Province in the west and Nakhon Si Thammarat Province in the south, to evaluate this problem. Subjects were selected by primary school-based stool egg examinations. Blood tests of 182 hookworm-positive primary school children, composed of 22 heavy, 65 moderate and 95 light infections, were compared with a control group of 57 children who were helminth-free both before and after receiving deworming medicine. Before deworming, the red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) and albumin levels of the hookworm-infected groups were significantly lower than the helminth-free control group. The Hb and Hct levels showed an inverse relationship with intensity of hookworm infection. After deworming, the Hb, Hct, total protein and albumin levels of the hookworm-infected children improved within 2 months to become comparable with the helminth-free control group. One year after deworming, the mean blood test results in the 2 groups were not significantly different from each other.

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.

High prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand.

OBJECTIVES: To determine the prevalence of CTX-M beta-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in a rural area of Thailand. METHODS: Bacteria in stool specimens were screened for extended-spectrum beta-lactamase (ESBL) production on McConkey agar with cefotaxime and confirmed by the double-disc synergy test. Genetic detection and genotyping of CTX-M-type ESBL was performed by PCR with bacterial DNA extracted from isolates. RESULTS: A markedly high number (82 of 141, 58.2%) of the specimens showed the presence of CTX-M beta-lactamase-producing Enterobacteriaceae, as confirmed by both phenotypic and genetic examinations. The majority of the CTX-M beta-lactamase-producing bacteria were Escherichia coli (85.1%). CONCLUSIONS: The study revealed the wide dissemination of CTX-M beta-lactamase-producing Enterobacteriaceae in the healthy population.

Capillariasis: chronic watery diarrhea--not only from microorganisms.

A 54-year-old male Thai patient from Prachin Buri Province presented with a history of chronic watery diarrhea for many years. He passed stool five to ten times per day with occasionally colicky pain, abdominal distension, nausea and vomiting. He had visited hospitals and private clinics and received treatment but with no improvement. He presented to the Hospital for Tropical Diseases, Bangkok, Thailand, where on physical examination, he had moderate dehydration, weakness, abdominal distension and a gurgling abdomen. The eggs, larvae and adult worms of Capillaria philippinensis were found on stool examination. The patient was admitted and treated with Mebendazole for 20 days, whereupon his symptoms resolved. Two months previously, he had ingested a raw small fresh-water fish dish called "Phra-Pla Siw/Soi". Small fresh-water fish near the patient's home were collected and examined for Capillaria philippinensis larva. The results were negative for parasitic organisms.

Prevalence of strongyloides in Northern Thailand and treatment with ivermectin vs albendazole.

The stools of 697 cases were examined by agar plate technique at Tambon Makam Luang, Sun Pa Tong district, Chiang Mai; there were Strongyloides stercoralis 15.9%,Opisthorchis viverrini 5.1%, intestinal fluke 0.1%. Treatment with ivermectin 78 cases and albendazole 33 cases of strongyloidiasis gave cure rates at 98.7% and 78.7%, respectively. Alkaline phosphatase in some patients were increased at mild level after treatment. Side effects in ivermectin group were anorexia, nausia, diarrhea, diffuse itching and drowsiness; and in albendazole group were nausia and diarrhea. The efficacy of single dose and mild side effects suggest ivermectin as drug of choice for strongyloidiasis treatment.

Efficacy of benzimidazole carbamate on an intestinal fluke co-infected with nematodes.

The efficacy of a single dose of benzimidazole, drugs commonly used for the treatment of Ascaris and hookworm, was evaluated against one of the tiny-sized intestinal flukes, Haplorchis sp in the endemic area where mixed infections of roundworms and flatworms existed. At day 7 after treatment, albendazole (400 mg) induced 42.5% cure rate, mebendazole (500 mg) a cure rate of 32.4%, on the other hand, praziquantel (40 mg/kg) gave 94.6% cure rate and the placebo at 15.9%. At the single dose, benzimidazole could not completely expelled the haplorchid; but could reduce one third to two fifth of the infection, similar to the drugs efficacy against Trichuris infection.

Improved antigens for IgG-ELISA diagnosis of strongyloidiasis.

Two preparations of antigens for the diagnosis of strongyloidiasis were prepared from an extract of the infective larvae of Strongyloides stercoralis: a crude antigen (CA) and a molecular weight cut-off antigen (MWCOA). Both antigens were analysed by indirect ELISA against the sera of strongyloidiasis (26 cases), other helminthiases (167) and normal controls (30). The larvae were obtained from fecal culture by a modified polyethylene tube technique after screening tests by triple simple smears per case. The larvae were extracted with distilled water and further sonicated to obtain a supernatant, the CA. A part of the CA was separated for an antigen containing molecules of lower than 30 kDa by an ultrafree-MC centrifugal filter tube (PLTK): this was designed as the MWCOA. The CA gave 96.15% sensitivity and 40.12% (67/167) specificity at a cut-off value of 0.980 (5SD); false positives were produced by 19 of 20 different helminthiases. The MWCOA produced 96.15% sensitivity at cut-off value of 0.71 (4SD); the specificity of the test was 78.44% (131/167), higher than that of CA. False positives also appeared with 15 other helminthic infections. This study suggests that MWCOA is more specific than CA. A purified MWCOA will be necessary in order to reduce cross-reactivity and provide the suitable diagnosis of strongyloidiasis.

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP.

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.

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.

Sero-epidemiological survey of gnathostomiasis in Lao PDR.

While human gnathostomiasis cases have been reported sporadically in Lao PDR since 1975, little is known about the disease in this country. We aimed to investigate sero-prevalence of gnathostomiasis and Gnathostoma species in Lao PDR. One village each in the north, central and south regions of Lao PDR was selected as the study sites. Overall, 125 (29.8%) of 420 sera from the randomly selected participants were sero-positive by immunoblot technique, with anti-Gnathostoma IgG antibody against the 24 kDa fraction. The sero-prevalence was high in the central (47.1%) and south (38.6%), but low (3.6%) in the north. Risk factor analyses revealed that the consumption of raw/undercooked fish was significantly associated with Gnathostoma sero-positivity (95% CI 1.05-17.05, P=0.042). The sero-positivity significantly increased with the age of the participants. Several fish, swamp eels, and frogs collected from central and southern Lao were infected with G. spinigerum advanced 3rd-stage larvae. Channa limbata (red-tailed snakehead fish) was identified as a natural second intermediate host of G. spinigerum. Eggs of G. spinigerum were found in dog feces collected in the south. Gnathostomiasis is endemic in central and southern Laos, so that preventive measures should be introduced for people living in these regions.

Monophyly of Opisthorchis viverrini populations in the lower Mekong Basin, using mitochondrial DNA nad1 gene as the marker.

The liver fluke, Opisthorchis viverrini, causes serious public-health problems in the Lower Mekong Basin. This study aimed to clarify whether O. viverrini populations may be genetically divided into sub-specific taxa. We collected 6 populations of O. viverrini from different places in Cambodia, Lao PDR, and Thailand, along both sides of the Mekong River, and analyzed the population structure of these using the mitochondrial nad1 gene as a marker. The results of the DNA polymorphism measurements, by theta-w (thetaw) and -pi (thetapi) values, neutrality tests, and mismatch distribution, suggested that the population of O. viverrini has expanded under the influence of purifying selection and selective sweep. The analysis of molecular variance (AMOVA) test revealed no significant genetic differences among the O. viverrini populations on opposite sides of the Mekong River. O. viverrini haplotypes occurred in multiple populations, and no distinct geographical clade. The star-like haplotype network confirmed a demographic expansion of the O. viverrini population. Overall, the genetic data from these populations suggested that the postulated existence of an O. viverrini species complex should be rejected. The bio-geographical diversity of O. viverrini populations should be explored further, using other appropriate markers and a wider range of samples from geographically different areas.

The second outbreak of trichinellosis caused by Trichinella papuae in Thailand.

A human trichinellosis outbreak caused by Trichinella papuae occurred in the Uthai Thani Province of Thailand in September 2007. A total of 34 villagers suffering at least one of the symptoms suggestive of trichinellosis, or those who were asymptomatic but had a history of ingesting raw wild pig meat, were enrolled in the study. Twenty-two villagers had ingested undercooked pork from a hunted wild pig (Sus scrofa). One patient with a severe clinical picture was hospitalised and more than 80 non-encapsulated larvae were detected in the muscle biopsy. The larvae were identified as T. papuae by molecular analyses of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the expansion segment 5 (ES5) of the large subunit rRNA. Of the 34 suspected cases, 27 agreed to be subjected to haematological and serological tests. Immunoblot analysis using crude antigens from T. spiralis muscle larvae revealed anti-Trichinella IgG in 20 of the 26 serum samples (1 serum sample could not be analysed). All infected people were successfully treated with mebendazole; the one patient with severe symptomatology was treated successfully with prednisolone.