Co-occurrence of swine cysticercosis due to Taenia solium and Taenia hydatigena in ethnic minority villages at the Thai-Myanmar border.

As part of the international joint projects working towards the control of taeniosis/cysticercosis in Asia Pacific, epidemiological studies on Taenia solium cysticercosis have been carried out in high-incidence populations, such as minority groups in Thailand. To assess the epidemiology of cysticercotic infections in pigs in the hill-tribe minority villages (Karen) in Tak province, Thailand, we conducted serological screening and necropsies. The patterns of antibody response to T. solium antigens were then investigated using immunoblot assays. Of the 188 pig serum samples tested for antibody responses to partially purified low-molecular-weight antigens of T. solium cyst fluid, positive responses were detected in 37 samples (19.7%). Based on these results, 16 pigs (10 seropositive and 6 seronegative) were necropsied for investigation of cysticerci and intestinal parasites. All seropositive pigs were coinfected with both T. solium and Taenia hydatigena cysticerci, except one, which was infected with T. hydatigena alone. Three of the six seronegative pigs were confirmed to be infected with T. hydatigena. Pigs infected with T. solium showed much stronger antibody responses than those infected with T. hydatigena. Our results demonstrate the co-occurrence of two swine cysticercoses due to T. solium and T. hydatigena in the studied areas. This study also reveals the importance of direct confirmation of the presence of cysticerci by necropsy after serological screening. In addition to the prevalence of swine cysticercosis in these endemic areas, our findings also reveal potential implications for the development of serological diagnostic assays for swine cysticercosis.

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.

Gastrointestinal helminths and Taenia spp. in parenteral tissues of free-roaming pigs (Sus scrofa indicus) from hilltribe village at the western border of Thailand.

A serological survey of pig cysticercosis was conducted in a hill-tribe village at Thai-Myanmar border, Tak province of Thailand in 2012. Sixteen backyard pigs were examined for pig cysticercosis and gastrointestinal helminth infection. In addition to cysticerci of Taenia solium and Taenia hydatigena found outside the gut, nine other helminth species were found in guts: Echinostoma malayanum, Pseudanoplocephala crawfordi, Ascarops dentata, Physocephalus sexalatus, Gnathostoma doloresi, Ascaris suum, Globocephalus sp., Oesophagostomum dentatum and Bourgelatia diducta. The study presents a report for the first time of adult tapeworm, P. crawfordi infection in pigs from Thailand. For medical importance, E. malayanum, P. crawfordi, G. doloresi and A. suum have been confirmed as potentially zoonotic helminths and pigs may act as one of the reservoir hosts for human helminthiases. Pigs of both gender and all ages appeared to be exposed to the parasites equally and did not show any significant difference to these helminth species in richness and total intensity.

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.

Levels of infection with the lungworm Angiostrongylus cantonensis in terrestrial snails from Thailand, with Cryptozona siamensis as a new intermediate host.

Angiostrongylus cantonensis is primarily considered an emerging infectious agent of eosinophilic meningitis or meningoencephalitis with a worldwide distribution. Rodents and snails are important invasive hosts for transmission and expansion of A. cantonensis. The objective of this study was to investigate infection levels of A. cantonensis in snails, the most important natural intermediate host. Our study location was Mueang Kamphaeng Phet district, Kamphaeng Phet Province, and was undertaken between October and December 2012. A total of 2228 freshwater and terrestrial snails were collected, comprising 1119 Filopaludina spp., 409 Pomacea caniculata, 275 Achatina fulica and 425 Cryptozona siamensis. Angiostrongylus larvae were isolated by artificial digestion methods following Baermann's techniques. A low prevalence and intensity of A. cantonensis were observed in A. fulica, while higher numbers were found in C. siamensis. None of the Filopaludina spp. and Pomacea caniculata were infected with A. cantonensis. Molecular characterization was performed by analysing the 264 bp of cytochrome c oxidase subunit I (COI). Three COI sequences of Angiostrongylus were identical to A. cantonensis with 91-99% identity. Cryptozona siamensis has not previously been recorded as an intermediate host for A. cantonensis in Thailand. The infection of A. cantonensis identified in the natural intermediate hosts is new and important information to assist in the prevention and control of human angiostrongyliasis.

Multi-immunodot for rapid differential diagnosis of eosinophilic meningitis due to parasitic infections.

A multi-dot enzyme-linked immunosorbent assay (ELISA) was developed for the rapid and simple differential diagnosis of eosinophilic meningitis due to helminth infections. Ultrafiltered, purified antigens of Parastrongylus (=Angiostrongylus) cantonensis, Gnathostoma spinigerum and Taenia solium metacestodes, the most common parasites that invade the central nervous system and cause eosinophilic pleocytosis, were dotted onto a single nitrocellulose membrane strip. Antigen-coated strips, when blocked with 5% skimmed milk and dried, were stable for at least 6 months at 4 degrees C. With peroxidase conjugated anti-human immunoglobulins and 4-chloro-1-naphthol as a substrate, antibodies in the corresponding patients' sera were clearly detected on the membrane strip as well-defined blue dots. Although cross-reactions between P. cantonensis and G. spinigerum antigens were observed with the use of partially purified antigens, the darkest dot correlated well with the infecting parasites in all cases. This fast, easy and economical multiple dot-blot ELISA method is useful for the differential diagnosis of eosinophilic meningitis caused by parasitic helminths, as semi-purified antigens can be easily obtained by ultrafiltration and used. Further improvements using highly specific parasite antigens may make this multi-immunodot test more suitable for wide-scale use in field studies and diagnostic laboratories.

[Imported gnathostomiasis acquired in Myanmar]. / Gnathostomose d'importation contractée au Myanmar.

We report a case of cutaneous gnathostomiasis acquired in Myanmar where this parasitic zoonosis was considered as non-endemic until a recent outbreak. Myanmar must be added to the list of countries where the infection can be acquired in Southeast Asia. Despite a treatment with ivermectin the patient relapsed after an apparent cure. A double-dose of ivermectin is now recommended for the treatment of gnathostomiasis.

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.

Three indigenous Thai medicinal plants for control of Aedes aegypti and Culex quinquefasciatus.

The potential larvicidal activity and insect growth regulator (IGR) properties of three selected indigenous medicinal Thai plants were tested against two species of mosquito with special reference to the late 3rd and early 4th instar larvae (L3 and L4, respectively). In case of larvicidal activity, Thevetia peruviana was the most potent, followed by Pueraria mirifica, and Butea superba was the least effective. In all cases, the late 3rd instar was more susceptible than the early 4th instar larvae, and the 48-hours exposure yielded more potent larvicidal activity than 24-hours exposure. However, at sublethal dosages, both P. mirifica and B. superba showed some dispersed effects interfering with ecdysis. A variety of toxic effects were observed and recorded in eight categories according to the stage of metamorphosis when death occurred. P. mirifica rendered the main deleterious effects in the pupa-adult period in both instar of Ae. aegypti and Cx. quinquefasciatus, whereas B. superba showed highest effect in black-pupa period of the late 3rd instar larval stage. The results were reversed for the early 4th instar larvae of both species of mosquito as the main effect appeared in the pupa-adult category. The overall results indicated that T. peruviana did not show any IGR properties; whereas, P. mirifica and B. superba seemed to exhibit the juvenile hormone type activity which resulted in abnormal death at various stages of development. B. superba was more promising than P. mirifica, and Ae. aegypti was about 2 times more susceptible than Cx. quinquefasciatus. In addition, L3 was always more susceptible than L4 with both mosquito species.

Imported cutaneous gnathostomiasis: report of five cases.

Gnathostomiasis has rarely been described outside endemic countries. We report on a series of 5 patients (4 females, 1 male, mean age 42.2 years) who returned to France from South-East Asia and presented with cutaneous gnathostomiasis. The cutaneous lesions appeared within a mean period of 62 d (range 10-150 d) after return. They consisted of creeping eruptions in 3 patients (in addition one also had papules, one had nodules and hepatitis, and one had hepatitis; all 3 had profound asthenia) and recurring migratory swellings in 2 patients. The mean eosinophil count was 1546/mm3 (range 398-3245/mm3). Diagnosis was based on positive serological tests in 3 patients and seroconversion in 2 patients, and was confirmed by identification of Gnathostoma hispidum in a biopsy specimen from one of the seropositive patients. Albandazole (1-4 courses) was given as treatment. Recurrences may occur up to 24 months after apparent cure without reinfection. Gnathostomiasis should be considered when patients return from tropical countries and present with migratory swellings or creeping eruption that does not respond to the usual treatment for cutaneous larva migrans. Serological tests may be negative initially and thus need to be repeated to check for seroconversion. Treatment may require multiple courses of albendazole and a prolonged period of follow-up is necessary before cure can be confirmed.

Medullary gnathostomiasis in a white patient: use of immunodiagnosis and magnetic resonance imaging.

A 48-year-old French diplomat presented with a sensory-motor paraparesis of rapid onset, leading to paraplegia. Successive magnetic resonance image scans showed lesions of the thoracic spinal cord that were at different levels from one examination to the next. Specific anti-gnathostome antibodies were detected by means of enzyme-linked immunosorbent assay and Western blot test in both plasma and cerebrospinal fluid. Albendazole treatment prevented disease progression, but only partial regression of the neurologic symptoms was obtained.

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.

Comparative studies on protein, isoenzyme and DNA restriction endonuclease profiles of Thai Paragonimus metacercariae.

General proteins and 14 enzymes from metacercariae of Paragonimus heterotremus, P. siamensis and P. westermani were determined by vertical polyacrylamide gel electrophoresis. The isoenzyme profiles showed considerable interspecific polymorphism for general protein (PT), malate dehydrogenase (MDH), malic enzyme (ME) and tetrazolium oxidase (TO) while those of glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) showed similarity. The Pt-6 and To-I loci can be used as identification markers for these three species. The preliminary study of the molecular biology of Paragonimus heterotremus P. siamensis and P. westermani was based on analysis of metacercarial genomic DNA with restriction endonuclease Pst I. Agarose gel electrophoresis revealed restriction fragment length differences among the three species studied. The DNA restriction fragments were approximately 4-6 fragments, ranging from 5.35 to 14.67 kb. Among these. P westermani shared two homologous fragments with P. siamensis, ie, 5.35 and 7.22 kh, none with P. heterotremus, while P. heterotremus shared only one with P. siamensis, ie, 8.16 kb. Thus, the DNA restriction fragment length differences can be used to differentiate among these three species.

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.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.

Immunoblotting and enzyme linked-immunosorbent assay for diagnosis of toxoplasma infection in HIV Thai patients.

Toxoplasma gondii has been recognized as an important cause of morbidity and mortality among immunocompromised persons. The diagnosis of T. gondii infection is most often based on serological tests results. Serological diagnosis can be limited in AIDS patients because of depressed antibody responses. Fifty serum samples were used in this study to investigate serological evidence of toxoplasmosis in HIV positive Thai patients by Platelia kit, the commercial enzyme-linked immunosorbent assay (ELISA) in which the membrane protein p-30 is the predominant antigen and immunoblot technique (IB). Sera of HIV positive Thai patients with Toxoplasma infection recognized the same antigenic component, the 32 kDa antigenic band, as is recognized by Toxoplasma positive sera from immunocompetent patients and it may represent a specific marker for diagnosis of Toxoplasma infection in HIV positive Thai patients.

Human lung fluke Paragonimus heterotremus: differential diagnosis between Paragonimus heterotremus and Paragonimus westermani infections by EITB.

The specificity of three major polypeptides (35, 33 and 32.5 kD) from Paragonimus heterotremus antigens prepared from ether-extracted adult worms was tested against sera from heterologous infections as well as against P. westermani-infected sera. Only the 35 kD polypeptide was not present, its antigenic determinant being bound to the antibodies from all P. westermani-infected cases. Its cross-reactivity against various sera from heterologous helminthiases and other lung infections showed that it is not bound to these antigenic polypeptides. These major bands cannot be detected by Concanavalin A detector. Our research encourages the pattern (35, 33 and 32.5 kD) of immunoblot reactions for the diagnosis of P. heterotremus infections; the 35 kD antigen is specific for corresponding species and able to differentiate infections between both species of Paragonimus.

Use of delipidized antigens of Taenia solium metacestodes in IgG-ELISA for detection of neurocysticercosis.

The development of IgG-ELISA for detecting neurocysticercosis is aimed at the routine laboratory, and requires a particular antigen preparation, an acceptable number of serum samples to be tested (both homologous and heterologous) and patients with a diversity of helminthic infections to rule out cross-reactions. This study characterizes IgG-antibodies from cases of neurocysticercosis by assaying the sera against ether-delipidized antigens (5 microg/ml) prepared from metacestodes of Taenia solium. The test had a sensitivity of 90% and a specificity of 83%. IgG-antibodies from heterologous serum samples elicited a number of false positives (25/147) from six different helminthic infections, ie paragonimiasis, echinococcosis, opisthorchiasis, ascariasis, taeniasis and fascioliasis. In additional tests to detect antibody levels to these stage-related antigens, one of three serum samples from T. solium-infected cases gave negative at OD value of 0.187 while the others yielded 0.472 and 0.576. Conversely, assays of all serum samples from neurocysticercosis cases reacted against antigens from Echinococcus granulosus cystic fluid, Paragonimus heterotremus and Opisthorchis viverrini adult worms. In comparison, the antigens from these three species yielded higher mean OD values when assayed against the corresponding infected serum samples. Furthermore, neurocysticercosis cases yielded OD values that are separate and distinct from those of paragonimiasis cases.

Evaluation of Bithynia funiculata snail antigens by ELISA-serodiagnosis of human opisthorchiasis.

Four batches of crude somatic antigens from: (1) Opisthorchis viverrini adult worms, (2) Bithynia funiculata-whole body, (3) B. funiculata-head-foot, and (4) B. funiculata-visceral mass were assayed against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals, and 50 individuals infected with other helminthic infections, and their antibody levels determined. By IgG-ELISA, the antigenic reactive proteins were found in both the head-foot and the visceral mass of B. funiculata snails, but the whole snail antigens gave the best results. Furthermore, it was as good as when O. viverini antigens were used. Antibody levels of sera from patients with opisthorchiasis assayed against antigens from whole B. funiculata snails were significantly higher than those of the other two groups. The cut-off value for positivity at 0.228 gave 80.2% sensitivity and 81.2% specificity. Cross reactions were observed with sera from patients with paragonimiasis and strongyloidiasis. No cross reactions were found to occur with sera from healthy individuals.