Nematode infection among ruminants in monsoon climate (Ban-Lahanam, Lao PDR) and its role as food-borne zoonosis / Infecção por nematóides em ruminantes nas monções (Ban-Lahanam, RPD do Laos) como zoonose transmitida por alimento

Trichostrongylids infection has gained significant public health importance since Trichostrongylus spp. infections have been reported in humans in Lao PDR. In this study, gastrointestinal nematodes were identified and the intensity of infections was determined in goats and cattle, which are animals greatly used for meat production in Lahanam Village, Lao PDR. The total number of goats and bovines was 23 and 29, respectively, pertaining to 32 households surveyed in the area. Feacal samples were randomly collected from 14 goats and 11 bovines. Ninety three percent (13/14) of goats and 36% (3/11) of cattle were infected, with an average of 1,728 and 86 eggs per gram of faeces (EPG), respectively. Coproculture showed Trichostrongylus spp. (goats 16%; bovines 48%), Haemonchus spp. (goats 69%; bovines 37%), Cooperia spp. (bovines 8%) and Oesophagostomum spp. (goats 15%; bovines 6%). After performing the necropsy on an adult goat, Trichuris spp. was also found. We confirmed the presence of Oesophagostomum spp., H. contortus and T. colubriformis by morphology and DNA sequencing analysis of the ITS region of rDNA. Due to interactions between humans and goats in Lahanam Village and high EPG results, the diagnosis of species and the intensity of gastrointestinal nematode infection in these animals are important public-health issues. Other ruminant parasites, such as Oesophagostomum and Haemonchus, found in caprines and bovines, are reported to be causes of zoonosis and their presence in humans should be investigated in future field surveys in this area.

Infecção por tricostrongilídeos ganhou significativa importância para a saúde pública, desde que infecções por Trichostrongylus spp. foram relatadas em humanos no Laos. Neste estudo, determinou-se a intensidade de verminoses gastrintestinais em caprinos e bovinos, importantes animais de produção na região de Lahanam Village, RPD do Laos. O número total de caprinos e bovinos foi 23 e 29, respectivamente, nas 32 famílias estudadas. Amostras de fezes foram coletadas, aleatoriamente, de 14 caprinos e 11 bovinos. Noventa e três por cento (13/14) dos caprinos e 36% (3/11) dos bovinos encontraram-se parasitados, com uma média de 1728 e 86 ovos por grama de fezes (OPG), respectivamente. Pela coprocultura, identificou-se Trichostrongylus spp. (caprinos 16%; bovinos48%), Haemonchus spp. (caprinos 69%; bovinos 37%), Cooperia spp. (bovinos 8%) e Oesophagostomum spp. (caprinos 15%; bovinos 6%). A necropsia de um caprino registrou também a presença de formas adultas de Trichuris spp. Morfologicamente e por análise do sequenciamento da região ITS do rDNA, foi confirmada a presença de Oesophagostomum spp., H. contortus e T. colubriformis. Devido às interações entre seres humanos e caprinos, em Lahanam Village, o alto OPG demonstrando o grau elevado de infecção por nematóides gastrintestinais nesses animais e a comprovação de espécies causadoras de zoonoses, aponta-se uma importante questão de saúde pública. Outros parasitos de ruminantes, como Oesophagostomum e Haemonchus, encontrados nos caprinos e bovinos estudados, são também relatados como agentes de zoonose, e sua presença em seres humanos deve ser investigada em futuras pesquisas de campo no local.

Parasitological monitoring of helminth control program in Northern Thailand.

Nan Province, located in northern Thailand, is hyperendemic for parasite infections; the helminthic infection rate in 1,010 schoolchildren was 60.0% in 2001. Mass anthelmintic chemotherapy has been conducted with schoolchildren, and selective treatment has been given to people in the community, from 2002. The modified cellophane thick smear method was used to examine the prevalence and intensity of helminth infections in schoolchildren and community people once a year during the period 2002-2004. The prevalence of helminth infections decreased slowly from 60.0 to 40.3% in schoolchildren and from 70.8 to 60.0% in the older age population. Three parasite species were common: hookworm, Ascaris and Haplorchis, an intestinal trematode. Hookworm presented throughout the whole district. Ascaris infection occurred at high rates in some villages, while in some villages none was found. The villages where Ascaris infection was nil had high rates of Haplorchis infection, and vice versa. Most hookworm and Trichuris infections were of light intensity. Heavy intensity infection was found in 12.8-18.1% of Ascaris cases examined. Parasite infection rates in Chaloem Phra Kiat District can be classified as low prevalence.

Paragonimiasis prevalences in Saraburi Province, Thailand, measured 20 years apart.

Saraburi Province, Central Thailand has been a paragonimiasis-endemic area since 1956. This study compared the prevalences of human paragonimiasis in two villages near Chet Khot Waterfall, Kaeng Khoi District, investigated in 1984-1985 and 2005. The results from the 1980s showed 6.3% and 1% of villagers were positive for Paragonimus eggs in sputum and stool, respectively. In 2005, Paragonimus eggs were not found in feces or sputum. An IgG-ELISA for paragonimiasis was conducted on 33 serum samples collected in the 1980s, 23 collected in 2005 and 25 diagnosed with other parasitic infections. Ninety percent of the samples from the eighties were positive for paragoimiasis, and 43% from 2005 were positive, equivalent to 10.9% and 4.9% of the total population examined in the 1980s and 2005, respectively. Serodiagnosis is currently the best method for detecting paragonimiasis. The positive cases in the 1980s were age 10-60 years and in 2005 were age 34- 67-years-old. The prevalence and intensity of Paragonimus metacercariae in fresh Waterfall crabs collected from Chet Khot Waterfall were significantly lower in the 1980s than in 2005. The prevalence of paragonimiasis in this endemic area has decreased to the level that no egg-producing cases were detected. No infections were found in villagers age < 30 years, despite the high density of metacercariae in the crabs, indicating a change in the habit of eating raw food among the younger people.

Diphyllobothrium latum infection in a non-endemic country: case report / Infecção pelo Diphyllobothrium latum em país não endêmico: relato de caso

Diphyllobothriasis, which is rarely described in Brazil, was reported initially as a travelers’ disease and as an accidental infection in individuals who ate raw freshwater fish. This report aims to present the case of a 20-year-old patient with confirmed Diphyllobothrium latum infection.

A difilobotríase, raramente descrita no Brasil, foi referida inicialmente como doença de viajantes e como infecção acidental em indivíduos que se alimentam com peixe cru de água doce. Este relato objetiva apresentar o caso de uma paciente de 20 anos com infecção confirmada pelo Diphyllobothrium latum.

Molecular phylogenetic relationship of Paragonimus pseudoheterotremus.

A part of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear ribosomal DNA second internal transcribed spacer 2 (ITS2) of a newly described lung fluke, Paragonimus pseudoheterotremus, were sequenced and compared with P. heterotremus, the species with a similar morphology. Pairwise distance of COI sequences revealed a genetic difference between P. heterotremus and P pseudoheterotremus with a nucleotide difference of COI sequences between these two species of 10.6%. The constructed phylogenic tree with high bootstrap proportion suggested that P. pseudoheterotremus is a sister species of P. heterotremus.

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.

Detection of IgG antibodies of Brugian filariasis with crude male and female antigens of Dirofilaria immitis.

Crude antigens from male and female Dirofilaria immitis were used to detect antibody to Brugian filariasis in humans by indirect ELISA. Both antigens were tested with 42 cases of Brugian filariasis, 131 cases of 20 heterologous infections and 35 healthy controls. The results--using male and female antigens--showed sensitivity of 88.1% and 88.1%, and specificities of 64.1% and 51.8%, respectively. Cross-reaction from other helminthic infections using crude male antigen gave false-positives with 48 sera from 13 heterologous diseases at the threshold value of 0.180, while the female antigen gave 63 sera from 15 diseases, at 0.309. Serum antibodies from patients with other helminthic infections--gnathostomiasis, strongyloidiasis, hookworm infections, trichinellosis, capillariasis, angiostrongyliasis, ascariasis, trichuriasis, toxocariasis, neurocysticercosis, cystic echinococcosis, taeniasis and opisthorchiasis--resulted in false-positives with both male and female antigens. One each of sparganosis and paragonimiasis heterotremus sera cross-reacted with only crude female antigen and their OD values were close to the threshold value. Although crude male antigen showed better specificity than crude female antigen, both female and male worms are sources of antigens needed for further purification. This study provides baseline data for further serodiagnosis of Brugian filariasis using dirofilaria antigen.

Negative-ELISA using native and filtrated cystic fluid antigens to rule out cystic echinococcosis.

An increasing number of cases of echinococcosis in Thailand have been imported, probably native infections and medical transfers. Serodiagnosis is one diagnostic choice for interpreting infections before a further step is done. Due to limited antigen, indirect ELISA has been used as a negative screening test for IgG-detection to rule out echinococcosis. Native hydatid cystic fluid (HCF) antigen from Belgium was used for such testing, in which the ODs-ELISA of samples were compared with those of two positive controls. Subsequently, hydatid cyst fluid from a Thai patient was obtained and the filtrated cyst fluid antigen [(<30)-(>10) kDa, HCF30.10] was prepared to develop negative screening results for the serum samples. By using HCF, three of twenty-four samples resulted in higher ODs-ELISA than the controls. In an attempt to observe the cross-reactivity of this native antigen, IgG-antibodies from many helminthiases cross-reacted and showed high ODs-ELISA. The HCF30.10 Ag was used to develop the test and analyze IgG-antibodies from 5 positive controls (2 parasite-confirmed and 3 positive-serodiagnosed), 183 heterologous cases of 29 diseases and 50 healthy control sera. At a cut-off value of 0.484, the test had 100% sensitivity and 42% specificity. Only Malayan filariasis, onchocercosis, fascioliasis, amebiasis, giardiasis and blastocystosis gave true negatives. Antibodies from nematodiases strongly cross-reacted with HCF30.10 Ag. Nine of fifty (18%) healthy serum controls produced higher OD-values than the cut-off. The routine ELISA uses the HCF30.10 Ag to produce a negative result to echinococcosis, because limited cystic fluid antigen (Thai patient) for test improvement, a lot of cross-reactions and only two protoscolex-positive controls are available.

Paragonimiasis in Nan Province, northern Thailand.

Two cases of paragonimiasis were identified within the hill-tribe population living on the Thai-Laotian border of Nan Province, northern Thailand, where information on Paragonimus was then still limited. The patients were in the habit of eating improperly cooked crabs and freshwater prawns. A survey for natural intermediate hosts to complete the life cycle was in progress at that time, and the detection of paragonimiasis cases indicated that there was persistence of paragonimiasis in the endemic area of Nan Province.

The current situation regarding the establishment of national ethical guidelines for biomedical research in Thailand and its neighboring countries.

This study discusses the establishment of ethical guidelines for ethical review for biomedical research performed in Thailand, and to some extent, in neighboring countries. There are differences, from country to country, at national and institutional levels regarding guidelines for ethical review committees. Only a handbook issued by Mahidol University describes guidelines for human genetic research and on research dealing with reproductive technology. Both these areas require special consideration to avoid violating human dignity, rights, and confidentiality. This indicates that further efforts should be made to establish research guidelines and/or principles dealing with the human genome.

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%.

Cysticercosis: IgG-ELISA evaluations of peak1 antigen and <30 kDa antigen of delipidized extract of Taenia solium metacestodes.

The antigenicity of ether-delipidized Taenia solium metacestode extract (DLPAg) was investigated by IgG-ELISA. The antigen showed higher antigenicity than that of non-delipidized antigen (NDLPAg). Then the DLPAg was subjected to Sephacryl S-200 gel chromatography and a partially purified antigen (DLPP1Ag) was identified as the promised antigen by IgG-ELISA using 25 sera from cysticercosis cases, 177 cases of 24 heterologous infections, and healthy controls. Sensitivity was 52% and specificity was 91.8% at the cut-off value (X + 7SD), 0.399. Cross-reactivity occurred with 17 cases of eight diseases: cystic echinococcosis (7/11), taeniasis (1/16), gnathostomiasis (2/8), strongyloidiasis (1/12), angiostrongyliasis (1/12), paragonimiasis heterotremus (2/15), opisthorchiasis (1/9) and fascioliasis (2/7). When DLPP1Ag was fractionated through Ultra free centrifugal tube (retained 30 kDa) and Amicon (PM10), MWCOP1Ag (<30-10> kDa) was obtained; the antigen showed better results than DLPP1Ag with 88% sensitivity and 95.6% specificity at the cut-off value (X + 4SD), 0.264. Nine cases of six diseases cross-reacted with this antigen: cystic echinococcosis (2/11), gnathostomiasis (2/8), trichinellosis (2/12), toxocariasis (1/5), schistosomiasis (1/6), and fascioliasis (1/7). MWCOP1Ag gave higher sensitivity than that of DLPP1Ag but some cross-reactivity occurred.

Studies on concomitant antigens of Bithynia funiculata for detection of antibody to Opisthorchis viverrini: effect of different centrifugal speeds on antigen preparation.

Antigens derived from somatic extracts of Bithynia funiculata, an intermediate snail host of O. viverrini, have been demonstrated to be highly heterogeneous in molecular weight (MW). These antigens have been suggested to be of potential use for serodiagnosis. In this study, B. funiculata somatic antigens were extracted using five different centrifugal speeds, namely 10,000 (C1); 20,000 (C2); 30,000 (C3); 40,000 (C4) and 50,000 (C5) rpm, with the aim of removing some non-specific antigens and determining the optimal centrifugal speed to obtain the highest efficiency of the test for which they will be used. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five different centrifugal speed-prepared antigens. The sensitivity and specificity of all five antigens were compared by testing against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals and 50 individuals infected with other helminthic infections, using mean + 4SD of all healthy individuals as the cut-off value. The sensitivity of these antigens was 69.1, 84.0, 80.2, 84.0 and 70.4, respectively; while the specificity was 66.2, 76.2, 82.5, 86.2 and 71.2, respectively. Immunoreactive components of each antigen were analyzed by SDS-PAGE and Western blot technique. The assay showed that three pairs of antigens with MW of 29 and 30, 47 and 50, and 86 and 90 kDa of all five antigens, which have previously been shown to be highly immunogenic, still reacted with a pooled serum from patients with opisthorchiasis. However, the C4 antigens gave more distinct components. Our results showed that 40,000 rpm is the optimal speed for antigen preparation for use in the serological diagnosis of opisthorchiasis, as demonstrated by the most satisfactory results of both sensitivity and specificity in the indirect ELISA and Western blot technique.

Fish as the natural second intermediate host of Gnathostoma spinigerum.

Gnathostomiasis is a helminthic disease most frequently occurring in Thailand. Human infections are usually found to be caused by Gnathostoma spinigerum, although five species of the genus Gnathostoma exist in Thailand, and three of these are capable of infecting man. In Thailand, 47 species of vertebrates--fish (19), frogs (2), reptiles (11), birds (11) and mammals (4)--have been reported to serve naturally as the second intermediate (and/or paratenic) hosts of G. spinigerum. Of these, fish, especially swamp eels (Monopterus albus), were found to be the best second intermediate/paratenic hosts: they had the highest prevalence rate and the heaviest infection intensity. However, the scientific names of these fish have been revised from time to time. Therefore, for clarity and consistency, we have summarized the current scientific names of these 19 species of fish, together with their illustrations. We describe one additional fish species, Systomus orphoides (Puntius orphoides), which is first recorded as a naturally infected second intermediate host of G. spinigerum.