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.

First Record of Paramphistomes Fischoederius cobboldi and Paramphistomum epiclitum Detected in Bovine Rumen from a Local Market of Savannakhet Province, Lao PDR.

In the present study, we report on the occurrence of paramphistomes, Fischoederius cobboldi and Paramphistomum epiclitum, in Lao PDR with the basis of molecular data. Parasite materials were collected from bovines bred in Ban Lahanam area, Savannakhet Province, Lao PDR at Lahanam public market. Morphological observations indicated 2 different species of paramphistomes. The mitochondrial gene cox1 of the specimens was successfully amplified by PCR and DNA sequencing was carried out for diagnosis of 11 specimens. Pairwise alignment of cox1 sequences were performed and confirmed F. cobboldi and P. epiclitum infecting bovines in Laos. Although there were many limiting points, as the small number of worm samples, and the restricted access of the animal host materials, we confirmed for the first time that 2 species of paramphistomes, F. cobboldi and P. epiclitum, are distributed in Lao PDR. More studies are needed to confirm the paramphistome species present in Savannakhet and its hosts to clear the natural history of these parasites of ruminants in the region and measure the impact of this parasite infection in the life and health of the local people.

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.

Gastrointestinal parasites of dogs and cats in a refuge in Nakhon Nayok, Thailand.

We collected fecal samples from 500 dogs and 300 cats from an animal refuge in Nakhon Nayok Province, Thailand to test for gastrointestinal protozoa and helminths using a formalin-ether concentration technique. The overall prevalence of parasites in stool from dogs was 36.2% (181/500), 35.7% (177/500) had helminths and 2.8% (14/500) had protozoa. The helminths were: hookworm (30.6%), Trichuris vulpis (16.0%), Toxocara canis (6.6%), Hymenolepis diminuta (1.2%), Spirometra mansoni (0.6%), and Dipylidium caninum (0.2%). Giardia duodenalis (2.8%) was found in the stool of dogs. The overall prevalence of parasites in stool from cats was 44.3% (133/300), 43.3% (130/300) were helminths and 6.0% (18/300) were protozoa. The helminths were hookworm (34.7%), T. cati (9.7%), S. mansoni (4.0%), Platynosomum fastosum (2.7%), Strongyloides sp (0.7%), and Echinostoma sp (0.3%). Two species of protozoa, Isospora sp (5.7%) and G. duodenalis (0.3%) were found in the stool of cats. Two percent of dogs and 5.0% of cats had mixed protozoan and helminthic infections. Dogs with double, triple, and quadruple helminthic infections were found at rates of 22.0%, 2.8%, and 0.2%, respectively. Cats with double and triple helminthic infections were found at rates of 9.7% and 1.0%, respectively. Quadruple helminthic infections were not found in cats, and double protozoan infections were not found in either dogs or cats.

Genetic diversity of Taenia asiatica from Thailand and other geographical locations as revealed by cytochrome c oxidase subunit 1 sequences.

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.

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.

Abnormal Taenia saginata tapeworms in Thailand.

Sixty-eight residents of Ban Luang and Ban Pang Kae villages, in Nan Province, northern Thailand, visited our mobile field station in September 2006 and March 2007, seeking treatment for taeniasis. After treatment, 22 cases discharged tapeworm strobila in their fecal samples and 17 scolices were recovered. Among these, 3 were morphologically abnormal, with six suckers on the scolex. To confirm the species of these tapeworms, the mitochondrial cytochrome c oxidase subunit I (COI) gene was used as a molecular marker. The partial COI sequences (800 bp) of the abnormal tapeworms were identical to the sequences of Taenia saginata deposited in Genbank.

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.

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.

Haplorchis taichui as a possible etiologic agent of irritable bowel syndrome-like symptoms.

The aim of this study is to clarify the clinical features of Haplorchis taichui infection in humans in Nan Province, Thailand, and to correlate the clinical features with irritable bowel syndrome (IBS)-like symptoms. In this study area, only H. taichui, but neither other minute intestinal flukes nor small liver flukes were endemic. The degree of infection was determined by fecal egg counts and also by collecting adult worms after deworming. The signs and symptoms of individual patients together with their hematological and biochemical laboratory data were gathered to evaluate the relationship between the clinical features and the severity of infection. Special emphasis was made to elucidate the possible similarities of the clinical features of H. taichui infection and IBS-like symptoms. The results showed useful clinical information and the significant (> 50%) proportion of haplorchiasis patients complained of abdominal pain, lassitude, and flatulence, which were the important diagnostic symptoms of IBS. This study has reported a possible link between H. taichui and IBS, and H. taichui might probably play a role in the etiology of these IBS-like symptoms.

Anchitrema sanguineum (Digenea: Anchitrematidae) accidentally found during colonoscopy of a patient with chronic abdominal pain: a case report.

In November 2007, a 46-year-old male Thai patient presented with chronic abdominal pain for over 3 years. Colonoscopy revealed a small parasite of about 2 x 1 mm in size attached to the cecum mucosa. The worm was removed endoscopically, fixed, and stained for morphological observations. The specimen was identified as Anchitrema sanguineum (Digenea: Anchitrematidae), a trematode first reported in a reptile, Chamaeleo vulgaris, from Egypt, and then sporadically found in the intestines of insectivorous bats and other mammals. The patient was treated with praziquantel but no more worms were found in his stool. His symptoms improved slightly but not cured completely. It remains unclear whether the chronic abdominal pain of the patient was caused by this trematode infection. Whatever is the pathogenicity of this trematode, this is the first human case of A. sanguineum infection in the literature.

Egg laying capacity of Haplorchis taichui (Digenea: Heterophyidae) in humans.

Quantitative fecal egg counts represented as the number of eggs per gram of feces (EPG) are generally a reliable parameter to estimate the worm burden of intestinal and hepatic parasitoses. Although Haplorchis taichui (Digenea: Heterophyidae) is one of the most common minute human intestinal flukes, little is known about the relationship between EPG and the actual worm burden in patients or the severity of the disease. In the present study, fecal samples were collected from 25 villagers in northern Thailand before and after praziquantel treatment. The EPG values of each participant were determined by the modified cellophane thick smear method, and adult worms were collected from the whole stool after the treatment. Eggs per day per worm (EPDPW) of H. taichui were estimated 82 from egg counts and expelled worms. The EPG was not well correlated with the worm burden, and a reverse correlation was observed between the EPDPW and the worm burden.

The role of domestic dogs in the transmission of zoonotic helminthes in a rural area of Mekong river basin.

Dogs have been bred since ancient times for companionship, hunting, protection, shepherding and other human activities. Some canine helminth parasites can cause significant clinical diseases in humans as Opisthorchis viverrini causing cholangiocarcinoma in Southeast Asian Countries. In this study, socio-cultural questionnaire, canine parasitological analysis, necropsy, parasite molecular confirmation and dog roaming data were evaluated in Savannakhet, Lao-PDR, a typical Mekong Basin area. Dog owners comprised 48.8% of the studied population, with 61.2% owning one dog, 25.1% 2 dogs, 8.5% 3 dogs and 1.8% owning more than 4 dogs. Data from GPS logger attached to dogs showed they walked from 1.4 to 13.3 km per day, covering an area of 3356.38m2 average, with a routine of accessing water sources. Thirteen zoonotic helminth species were observed. Causative agents of visceral and cutaneous larva migrans occurred in 44.1% and 70% of the samples respectively. Spirometra erinaceieuropaei was detected in 44.1% of samples. Importantly, O. viverrini was found in 8.8% of samples. Besides the known importance of dogs in the transmission of Ancylostoma spp., Toxocara spp. and S. erinaceieuropaei, the observed roaming pattern of dogs confirmed it as an important host perpetuating O. viverrini in endemic areas; their routine access to waterbodies may spread O. viverrini eggs in a favorable environment for the fluke development, facilitating the infection of fishes, and consequently infecting humans living in the same ecosystem. Therefore, parasitic NTDs control programs in humans should be done in parallel with parasite control in animals, especially dogs, in the Mekong River basin area.

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.

Diploscapter coronata infection in Thailand: report of the first case.

A 73-year-old Thai woman living in Mueang District, Saraburi Province, central Thailand presented with numerous hookworm-like nematodes, finally revealed as Diploscapter coronata, by fecal culture. The patient exhibited no significant clinical signs of the gastrointestinal or genitourinary systems, and was generally not ill as a result of this unusual infection. Less commonly, patients have presented with symptoms and signs of Diploscapter coronata infection. However, potentially serious consequences can occur where people have exposure to an environment that has been contaminated with infected feces, or more specifically, infective eggs; such conditions could lead to human infection with Diploscapter coronata worms. This was the first reported occurrence of human Diploscapter coronata infection in Thailand.

Host-finding behavior of Strongyloides stercoralis infective larvae to sodium cation, human serum, and sweat.

The host-finding behavior of Strongyloides stercoralis infective larvae was examined by in vitro agarose assay method. As human body fluid contains 0.85% (ca 0.15 molar) NaCl, various concentrations of sodium chloride, from 0.5M to 0.01M (7 steps), were examined. Many larvae were attracted at concentrations between 0.5 and 0.05M of sodium chloride. The concentration of 0.05M attracted the most larvae. The concentration of 0.02M of sodium chloride showed greatly reduced larval attraction compared with 0.05M. Therefore, the threshold concentration was determined as 0.05M. Then, 0.05M of chemicals were examined in a further experiment. Chloride compounds (NaCl, KCl, CaCl2, MgCl2) were investigated. These chemicals are components of human body fluids. Distilled water was used as the control in all experiments. Only sodium chloride attracted the larvae. Next, alkaline compounds were examined [NaOH, KOH, Ca(OH)2, and Mg(OH)2]. Larvae accumulated only at the NaOH site. The results suggested that the Na cation is important for larval attraction. A high pH value did not influence attraction at all. Next, human serum was tested. The human serum used was from normal serum to 1:32 diluted sera by distilled water (7 steps). Hierarchical attraction was seen according to serum concentration. Next, human sweat was collected from a limited zone of chest skin where only eccrine glands were distributed. Non-diluted sweat attracted the most larvae. Sweat might act as one of the most probable factors for infection by this skin-penetrating nematode.

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.

Soil-transmitted helminthiases and health behaviors among schoolchildren and community members in a west-central border area of Thailand.

The prevalence of soil-transmitted helminthic infections and health behaviors related to infections in schoolchildren and villagers of a community (4 hamlets) was studied in Hauy Kayeng subdistrict, Thong Pha Phum district, in the north of Kanchanaburi Province. The intestinal helminth infection rate of the schoolchildren was 15.6%. Hookworm infection was the most prominent (9.8%), followed by Trichuris trichiura (6.2%), and Ascaris lumbricoides (2.2%). The community showed higher prevalence rates and was infected with more types of intestinal helminths than the schoolchildren. Thirty-five point two percent (35.2%) of the residents were infected with soil-transmitted helminths, 30.5% with hookworm, 3.4% with A. lumbricoides and 2.2% with T. trichiura. Almost all hookworm cases (94.3%) were light intensity infections, while only 1.3% were heavy infections. Moreover, the hookworm infection rate in the community was found to be much higher when a stool culture method was used (39.1%). With this technique, 2.3% Strongyloides stercoralis infections were detected in the community population. Examination of the health behavior of the study samples showed that approximately 75% always defecated in a toilet. Schoolchildren who always wore shoes comprised 67%, which was lower than the community, at 85%.