Distribution of the newly invasive New Guinea flatworm Platydemus manokwari (Platyhelminthes: Geoplanidae) in Thailand and its potential role as a paratenic host carrying Angiostrongylus malaysiensis larvae.

Invasive species constitute one of the most serious threats to biodiversity and ecosystems, and they potentially cause economic problems and impact human health. The globally invasive New Guinea flatworm, Platydemus manokwari (Platyhelminthes: Geoplanidae), has been identified as a threat to terrestrial biodiversity, particularly soil-dwelling native species (e.g. molluscs, annelids and other land planarians), and is listed among 100 of the world's worst invasive alien species. We report here, for the first time, P. manokwari occurrences in many locations throughout Thailand, using voluntary digital public participation from the social network portals associated with the Thailand Biodiversity Conservation Group and collections of living flatworm specimens. Mitochondrial cytochrome c oxidase subunit I (COI) sequences confirmed that all collected flatworms were P. manokwari and placed them in the "world haplotype" clade alongside other previously reported specimens from France, Florida (USA), Puerto Rico, Singapore, French Polynesia, New Caledonia, and the Solomon Islands. In addition, infective stage larvae (L3) of the nematode Angiostrongylus malaysiensis were found in the flatworm specimens, with a 12.4% infection rate (15/121 specimens examined). Platydemus manokwari occurrence in Thailand and its capacity to carry L3 of Angiostrongylus should be of concern to biodiversity conservation and human health practitioners, because this invasive flatworm species may be involved in the life cycle of angiostrongylid worms in Thailand.

Genetic differences among Haplorchis taichui populations in Indochina revealed by mitochondrial COX1 sequences.

Haplorchis taichui is an intestinal heterophyid fluke that is pathogenic to humans. It is widely distributed in Asia, with a particularly high prevalence in Indochina. Previous work revealed that the lack of gene flow between three distinct populations of Vietnamese H. taichui can be attributed to their geographic isolation with no interconnected river basins. To test the hypothesis that interconnected river basins allow gene flow between otherwise isolated populations of H. taichui, as previously demonstrated for another trematode, Opisthorchis viverrini, we compared the genetic structures of seven populations of H. taichui from various localities in the lower Mekong Basin, in Thailand and Laos, with those in Vietnam, using the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. To determine the gene flow between these H. taichui populations, we calculated their phylogenetic relationships, genetic distances and haplotype diversity. Each population showed very low nucleotide diversity at this locus. However, high levels of genetic differentiation between the populations indicated very little gene flow. A phylogenetic analysis divided the populations into four clusters that correlated with the country of origin. The negligible gene flow between the Thai and Laos populations, despite sharing the Mekong Basin, caused us to reject our hypothesis. Our data suggest that the distribution of H. taichui populations was incidentally associated with national borders.

Molecular identification of the strongyloid nematode Oesophagostomum aculeatum in the Asian wild elephant Elephas maximus.

The transmission of zoonoses by wildlife, including elephants, is a growing global concern. In this study, we screened for helminth infections among Asian wild elephants (Elephas maximus) of the Salakpra Wildlife Sanctuary, Kanchanaburi, Thailand. Elephant faecal samples (45) were collected from the sanctuary grounds during January through November 2013 and assayed individually using the tetranucleotide microsatellite technique. Microscopic examination indicated a high prevalence of strongylids (93.0%) and low prevalences of trichurids (2.3%) and ascarids (2.3%). To identify the strongylid species, small subunit (SSU) rDNA sequences were amplified from copro-DNA and compared with sequences in GenBank. The generated SSU-rDNA sequences comprised five distinct haplotypes that were closely related to Oesophagostomum aculeatum. A phylogenetic analysis that incorporated related nematodes yielded a tree separated into two main clades, one containing our samples and human and domestic animal hookworms and the other consisting of Strongyloides. The present results indicate that O. aculeatum in local elephants is a potential source of helminthiasis in human and domestic animals in this wild-elephant irrupted area.

The evaluation of the 29 and 31 kDa antigens in female Angiostrongylus cantonensis for serodiagnosis of human angiostrongyliasis.

The objective of this study was to evaluate the crude somatic extract of female Angiostrongylus cantonensis by ELISA and to evaluate the 29 kDa and the 31 kDa components of female worm by EITB for serodiagnosis of human angiostrongyliasis. Using ELISA, cross reaction occurred among all sera tested except capillariasis. The sensitivity, specificity, positive and negative predictive values of ELISA were found to be 100%, 66.8%, 27.1% and 100% respectively. Using EITB, The 29 kDa component cross-reacted with most of the heterologous sera while the 31 kDa component was recognized by 69.2% (18/26) sera from angiostrongyliasis patients, cross-reacted only with trichinellosis, trichuriasis and opisthorchiasis sera. This component may possibly used as reliable agents for angiostrongyliasis out of other tissue infected helminthiasis. The sensitivity, specificity, positive and negative predictive values of EITB for the 31.0 kDa component were 69.2%, 82.4%, 46.2% and 92.5% respectively.

Circumoval and larval microprecipitation reactions in experimental and human gnathostomiasis.

Mice, rats and cats were infected either orally or percutaneously with a number of early or advanced third-stage larvae (EL3 or AL3, respectively) of G. spinigerum. Sera obtained from these infected animals and 10 human gnathostomiasis cases were tested against various developmental stages of the parasite which were prepared and used while being alive (fresh) or dead (air-dried) for the circumoval and larval microprecipitation (COP and LMP) reactions. No precipitin reactions were observed in all sera tested against unembryonated eggs, embryonated eggs and first stage larvae neither air-dried nor fresh preparations. Sera were merely reactive giving various degrees of membranous or filamentous precipitates against the air-dried preparation of AL3.

Gnathostome infection in swamp eels, Fluta alba, in central Thailand.

To investigate the distribution of gnathostome worms in central Thailand, the infective larvae of Gnathostoma spp were examined from the flesh and liver of swamp eels, Fluta alba. Seven hundred and eighty-eight eels were purchased from markets in 11 provinces; Ang Thong (30), Ayutthaya (36), Chachoengsao (30), Lop Buri (30), Nakhon Nayok (437), Pathum Thani (30), Prachin Buri (48), Ratchaburi (53), Saraburi (30), Samut Prakan (30) and Suphan Buri (34). The highest rate of gnathostome infection was observed in swamp eels from Nakhon Nayok (68.7%). The infection rates in Ayutthaya, Ang Thong, Prachin Buri, Ratchaburi, Saraburi and Lop Buri were 33.3%, 26.7%, 25.0%, 18.9%, 13.3% and 10.0% respectively. Gnathostome larvae were not found in swamp eels from Chachoengsao, Pathum Thani, Samut Prakan and Suphan Buri. Among the 9,573 larvae recovered, almost all were the advanced third stage larvae of G. spinigerum, except one larva from Nakhon Nayok and two larvae from Ratchaburi which were identified as the advanced third stage larvae of G. vietnamicum and G. hispidum respectively. This study is the first report of swamp eels as natural intermediate hosts of G. vietnamicum and G. hispidum.

Strongyloides stercoralis infection and chronological changes of other soil-transmitted helminthiases in an endemic area of southern Thailand.

A field survey was conducted in 4 primary schools in Nakhon Si Thammarat Province, southern Thailand. By Sasa modified Harada-Mori cultivation method, 1.8% of the schoolchildren were found to be infected with Strongyloides stercoralis, and 25.1% had hookworm infection. By Kato's thick smear method, the overall prevalence of soil-transmitted helminths was 46.8%, being Trichuris trichiura 28.5%, hookworm 18.0%, and Ascaris lumbricoides 5.7%. Fecal examination, performed by Kato's thick smear and culture method, indicated that the prevalence of hookworm infection was 26.9%. The prevalence in the present study was very much lower than many previous reports in the past decade. This may indicate the partial success of the parasite control project in Thailand by mass treatment, improving the sanitation and personal hygiene of the people in the endemic area. In light infection with Trichuris, albendazole administered at a dosage of 200 mg daily for 3 days showed a 48.7% cure rate. When mebendazole was given at 100 mg twice daily for 3 days, its effectiveness was 88.5%. A lower cure rate was obtained (70.0%) in moderate to heavy infection.

Gnathostomiasis in Thailand: a survey on intermediate hosts of Gnathostoma spp. with special reference to a new type of larvae found in Fluta alba.

To clarify current status of gnathostomiasis in Thailand, a survey on intermediate hosts has been carried out at various localities since 1987. It was found that Fluta alba (Fresh water eel) as well as Channa striata (snake-headed fish) might be important in playing a role of transmitting the infection either among humans or reservoir animals. During the three years from 1987 to 1989, larvae of Gnathostoma spinigerum were found in 80-100% of F. alba obtained from markets in Nakhon Nayok, with a maximum recovery of 2,582 larvae per eel. Among larvae found in these eels, five were peculiar in possessing four rows of hooklets with complicated branches at the base. Epithelial cells of the intestine of these larvae contained 1-2 nuclei. These observations indicate that the larvae are different from those of reported species of Gnathostoma from Thailand including G. spinigerum, suggesting a possibility of the advanced third-stage larvae of G. malaysiae.

A new type of advanced third-stage larvae of the genus Gnathostoma in freshwater eels, Fluta alba, from Nakhon Nayok, central Thailand.

Five advanced third-stage larvae of a newly identified type of genus Gnathostoma were collected from freshwater eels, Fluta alba, which were purchased at a market in Nakhon Nayok, central Thailand. The most remarkable characteristic of the newly identified larvae was the larger body size compared with any other larva of Gnathostoma spp. They were also distinguishable from other species by the shape of their hooklets, which branched in a complex manner at the base: this had not been previously observed in any other larval Gnathostoma. The newly described larvae had an average number of 44.5, 45.0, 49.0 and 55.1 hooklets on the head-bulb from the first to the fourth rows, respectively, which were comparable to those of larval G. spinigerum. However, the average number of nuclei in each intestinal cell was 2.21 and fewer than those of the larvae of G. spinigerum. These results suggest that the new type of larvae belong to either G. vietnamicum, G. malaysiae, or constitute a new species of the genus Gnathostoma.

Minimum effective doses of mebendazole in treatment of soil-transmitted helminths.

Three hundred and fifteen primary school children infected with soil-transmitted helminths were divided into 5 groups. Three groups were treated with 25, 50 and 75 mg mebendazole (MBZ) single dose. One group was given MBZ conventional dose of 100 mg twice daily for 3 days and another group was given albendazole (ABZ) standard dose of 400 mg single dose. Every trial lower MBZ dose 75 mg, 50 mg and 25 mg regimen were highly effective against Ascaris lumbricoides but only moderately effective against Trichuris trichiura and Necator americanus.

Neurocysticercosis: utilizing the cystic fluid antigen from Taenia solium metacestodes for diagnosis by IgG-ELISA.

Cystic fluid, which has antigenic properties of whole Taenia solium cysticerci, was used to discriminate neurocysticercosis cases and other parasitic infections, especially helminthiases. Twenty-one neurocysticercosis and several kinds of 22 different parasitic infections, including HIV cases (n=234) evaluated a 90.48% sensitivity and 86.32% specificity of indirect ELISA as follows: a low antigen concentration of 5 microg/ml. serum dilution of 1:400, conjugate dilution of 1:2,000 and a cut-off value of 0.349. Eight different helminthic infections (n = 25); echinococcosis (8/10), gnathostomiasis (6/8), strongyloidiasis (5/14), hookworm infection (1/18), angiostrongyliasis (2/25), opisthorchiasis (1/18), onchocercosis (1/3) and toxocariasis (1/6) were cross-reactive with this antigen. No serum antibody from other brain infections in the study gave a reaction with the antigen. In this study, the cystic fluid antigen gave high sensitivity of the test. However, the antigen contains various antigenic molecules able to bind with antibodies from several of the above helminthic sera, especially echinococcosis and gnathostomiasis. In Thailand, gnathostomiasis is one of the more famous tropical diseases but echinococcosis is quite rare. Cystic fluid antigen should be further investigated for its specific finding in diagnosis.

Angiostrongyliasis: analysis of antigens of Angiostrongylus costaricensis adult worms versus IgG from infected patients with Angiostrongylus cantonensis.

The possibility of cross-reactivity was previously investigated by indirect ELISA with sera from Angiostrongylus cantonensis infections, normal controls and A. costaricensis antigen. 5 microg/ml of crude antigen from both sexes of each species reacted with diluted serum samples (1:800) of each of 20 cases of angiostrongyliasis and normal controls, and further with anti-human IgG conjugate at 1:1,000. The mean absorbance values were evaluated as follows; normal controls showed a value of 0.033 using A. costaricensis antigen lower than (0.085) A. costaricensis antigen. Both mean values of angiostrongyliasis cases were rather close (0.491) using A. costaricensis antigen and the other antigen (0.518). The present study continued with a crude antigen of 13 A. costaricensis females and males. Serum samples were analyzed; 27 sera of angiostrongyliasis, 30 negative controls and 193 cases of other parasitic infections (91 cases of nematodiasis; 45 cases of cestodiasis; 47 cases of trematodiasis and 10 cases of HIV) and 7 cases of other brain infections. This antigen was evaluated for ELISA with a concentration of 5 microg/ml, serum dilution 1:400 and anti-human IgG conjugate at 1:2,000. The test gave sensitivity and specificity at cut-off value 0.261; 92.59% and 73% respectively. The antigen was cross-reactive with 30 cases from 9 out of 10 different kinds of nematodiasis (gnathostomiasis, strongyloidiasis, ascariasis, hookworm infections, trichinosis, toxocariasis, trichuriasis, onchocercosis and Wuchereria bancrofti infections. Five cases from 3 of 6 kinds of cestodiasis (neurocysticercosis, echinococcosis and Hymenolepis nana infections) and 18 cases of 4 out of 5 kinds of trematodiasis (Paragonimus heterotremus infections, opisthorchiasis, schistosomiasis and fascioliasis). One case of other brain infections was observed. The crude antigen of A. costaricensis showed a high percentage sensitivity with serum antibodies of angiostrongyliasis cases. Low specificity of the test was observed by reactions of those serum antibodies with various kinds of antigenic molecules. This study provides baseline data for further immunodiagnosis of human angiostrongyliasis.

Toxocara and Gnathostoma among stray canines in Bangkok.

Stomachs and intestines of 88 adult and 112 young stray dogs were obtained from the Rabies Control Subdivision, Bangkok, and examined especially for the presence of Gnathostoma spinigerum and Toxocara canis. Forty-five dogs were found positive for T. canis (overall prevalence 22.5%) but none were found infected with G. spinigerum. The prevalence of T. canis in young dogs was 37.5% (42 of 112) whereas in adult dogs was only 3.4% (3 of 88). The total number of T. canis recovered from the 45 positive dogs was 272 (averaging 6.0 worms/dog). This includes 268 worms from 42 young dogs (averaging 6.4 worms/dog) and four worms from three adult dogs (averaging 1.3 worms/dog). The average number of worms, according to sex and stage, per young dog were as follows: male worms 2.4 +/- 3.5 (range 0-15), female worms 2.8 +/- 3.5 (0-16), immature worms 1.2 +/- 2.5 (0-9), and all worms 6.4 +/- 8.2 (1-34). The maximum number of worms per young dog was 34 while the minimum was one, and 35.7% (15/42) of these young dogs harbored only one worm. The body length of the recovered T. canis were as follows: males measuring 3.0-12.0 cm (averaging 7.1 +/- 2.1 cm), females 4.1-18.2 cm (11.0 +/- 4.1 cm), and immature worms 0.7-3.7 cm (2.1 +/- 0.8 cm).

School-based health education for the control of soil-transmitted helminthiases in Kanchanaburi province, Thailand.

Soil-transmitted helminthiases are major parasitic diseases that cause health problems world-wide. School-based health education is one of several basic interventions currently recommended by the World Health Organization for the control of these infections. A 3-year programme of health education for the control of soil-transmitted helminths (STH) has recently been completed in four primary schools in the Hauykayeng subdistrict of Thong Pha Phum district, in the Kanchanaburi province of Thailand. Overall, the percentage of the schoolchildren found infected with STH increased between the start of year 1 of the intervention (16.6%) and the end of year 2 (23.8%) but showed signs of falling by the end of year 3 (19.4%). Although none of these year-on-year changes in overall prevalence was statistically significant, some significant trends were detected when the six school grades (i.e. age-groups) were considered separately. The grade showing the highest prevalence of STH infection changed, from grade 6 (representing the oldest children investigated) at the start of year 1 (when grade-1 children were excluded from the survey) to grade 1 (representing the youngest children) at the ends of year 2 and year 3. By the end of year 3, the children in grades 5 and 6 had significantly lower prevalences of infection than the grade-1 subjects. The prevalence of STH infection in the grade-1 children was significantly higher than that in any of the older grades at the end of year 2 and significantly higher than that in grades 3-6 at the end of year 3. These results indicate that the health education had a greater impact on the children in the higher grades (who, presumably had better levels of understanding and practised better, personal, infection prevention) than on the younger children. Although school-based interventions can serve as a useful entry point for parasite control, more effort, including anthelminthic treatment, may be required among the youngest children. The activities need to be sustainable and supported by appropriate school-health policies.

Diagnostic values of IgG4 in human gnathostomiasis.

The diagnostic values of immunoglobulin G subclass antibodies from patients with gnathostomiasis were assessed by immunoblot technique. Antigen was prepared from crude extracts of Gnathostoma spinigerum advanced third-stage larvae obtained from naturally infected eels. The sera were obtained from 14 parasite-confirmed gnathostomiasis cases, 63 patients with other helminthic infections and 13 healthy controls. Nine prominent IgG4 reactive bands appeared with molecular weights of 94, 51, 47, 43, 38, 24, 21, 20 and 15 kDa. The diagnostic sensitivity of each of the nine reactive bands ranged from 100% to 64.3% in 14 parasite-confirmed gnathostomiasis cases. All (100%) confirmed cases recognized the 21 kDa antigenic band, but not other helminthic infections or parasite-free control. Recognition of 21 kDa antigen in G. spinigerum advanced third-stage larvae crude extracts is the most specific diagnostic marker for human gnathostomiasis, with 100% sensitivity and specificity. The 20 and 24 kDa protein bands were additional diagnostic bands for confirming diagnosis of infection where the 21 kDa band was faint. No specific binding of IgG1, IgG2, or IgG3 antibodies was observed in any sera from confirmed gnathostomiasis cases.

Effect of ivermectin on experimental gnathostomiasis in rabbits.

The efficacy of ivermectin against advanced third stage larvae of Gnathostoma spinigerum was investigated in rabbits. Six experimentally infected rabbits were treated at a single dose, 0.2 mg/kg of ivermectin subcutaneously, four weeks after infection. Another two rabbits were treated, once and three times, with 2 mg/kg at varying numbers of days after infection. Five rabbits served as untreated controls. All rabbits were sacrificed about the 28th week after infection. The reductions of worm load in the treated groups were 74.2% and 84.2% respectively. The immune response, detected by ELISA, was similar in treated and control rabbits.