"Enterocytozoon bieneusi and Cryptosporidium: a cross-sectional study conducted throughout Thailand".

BACKGROUND: Enterocytozoon bieneusi and Cryptosporidium spp. are prevalent zoonotic parasites associated with a high burden among children. To date only limited molecular epidemiological data on E. bieneusi and Cryptosporidium spp. in humans living in Thailand has been published. METHODS: PCR-based tools were used to detect and characterize E. bieneusi and Cryptosporidium spp. The internal transcribed spacer (ITS) region of the rRNA gene was used to investigate E. bieneusi, and the small subunit (SSU) rRNA gene was used to investigate Cryptosporidium spp., and 697 fecal samples from villagers and school children in rural areas in Thailand were analyzed. RESULTS: The infection rates were 2.15% (15/697) for E. bieneusi and 0.14% (1/697) for Cryptosporidium spp. The prevalence of E. bieneusi was significantly high in Loei province. Sequence analysis indicated that the Cryptosporidium isolate was C. parvum. Nine E. bieneusi genotypes were identified, EbpC, Peru12, TMH6, TMH3, TMH7, H, D, and two novel genotypes TMLH1 and TMLH2. E. bieneusi prevalence was significantly higher in male participants than in female participants, and in children aged 3-15 years than in participants aged > 15 years. CONCLUSIONS: The prevalence, genotypes, and zoonotic potential of E. bieneusi were found to vary significantly high even in one country. Transmission routes and key animal carriers of E. bieneusi may be associated with differences in hygiene, sanitation, and cultural behaviors. Further molecular studies including longitudinal studies will be required to unveil epidemiological characteristics of these opportunistic intestinal protozoa in all over the countries.

Acidosis and acute kidney injury in severe malaria.

BACKGROUND: In severe falciparum malaria metabolic acidosis and acute kidney injury (AKI) are independent predictors of a fatal outcome in all age groups. The relationship between plasma acids, urine acids and renal function was investigated in adult patients with acute falciparum malaria. METHODS: Plasma and urinary acids which previously showed increased concentrations in proportion to disease severity in patients with severe falciparum malaria were quantified. Patients with uncomplicated malaria, sepsis and healthy volunteers served as comparator groups. Multiple regression and multivariate analysis were used to assess the relationship between organic acid concentrations and clinical syndromes, in particular AKI. RESULTS: Patients with severe malaria (n = 90), uncomplicated malaria (n = 94), non-malaria sepsis (n = 19), and healthy volunteers (n = 61) were included. Univariate analysis showed that both plasma and creatinine-adjusted urine concentrations of p-hydroxyphenyllactic acid (pHPLA) were higher in severe malaria patients with AKI (p < 0.001). Multiple regression analysis, including plasma or creatinine-adjusted urinary acids, and PfHRP2 as parasite biomass marker as independent variables, showed that pHPLA was independently associated with plasma creatinine (ß = 0.827) and urine creatinine (ß = 0.226). Principal component analysis, including four plasma acids and seven urinary acids separated a group of patients with AKI, which was mainly driven by pHPLA concentrations. CONCLUSIONS: Both plasma and urine concentrations of pHPLA closely correlate with AKI in patients with severe falciparum malaria. Further studies will need to assess the potential nephrotoxic properties of pHPLA.

Toxoplasma gondii infection: What is the real situation?

The prevalence of chronic Toxoplasma infections reported in the literature varies enormously. We hypothesize that one factor could be due to the different methods used in the evaluation of infections. Serological evidence of Toxoplasma infections in 450 pregnant women (PW) and 300 HIV-infected patients (HIV) were investigated by the Sabin-Feldman dye test and two other commercial ELISA kits (kit1 and kit2). Anti-Toxoplasma IgG antibodies obtained from the Sabin-Feldman dye test, ELISA kit1 and ELISA kit2 in the PW subjects were 14.7%, 29.6% and 38.7%, and in the HIV subjects were 13%, 34.7% and 36.3%, respectively. So there were significant differences in the seroprevalences when different diagnostic tests were used (P<0.05). Regarding Sabin-Feldman dye test as the gold standard for anti-Toxoplasma antibodies detection, we found that the sensitivity and specificity of the ELISA kit1 and kit2 was in the range of their specification. However as the two ELISA kits used in our study identified a much higher prevalence of Toxoplasma infections which indicated that false positive cases were being reported. Based on results obtained, it is therefore highly recommended that research workers should be aware that the reports of serological studies in terms of high positive results should be treated with some skepticism until additional precise diagnostic tools are developed.

Seasonal variation and potential sources of Cryptosporidium contamination in surface waters of Chao Phraya River and Bang Pu Nature Reserve pier, Thailand.

Using molecular techniques, a longitudinal study was conducted with the aims at identifying the seasonal difference of Cryptosporidium contamination in surface water as well as analyzing the potential sources based on species information. One hundred forty-four water samples were collected, 72 samples from the Chao Phraya River, Thailand, collected in the summer, rainy and cool seasons and 72 samples from sea water at Bang Pu Nature Reserve pier, collected before, during and after the presence of migratory seagulls. Total prevalence of Cryptosporidium contamination in river and sea water locations was 11% and 6%, respectively. The highest prevalence was observed at the end of rainy season continuing into the cool season in river water (29%) and in sea water (12%). During the rainy season, prevalence of Cryptosporidium was 4% in river and sea water samples, but none in summer season. All positive samples from the river was C. parvum, while C. meleagridis (1), and C. serpentis (1) were obtained from sea water. To the best of our knowledge, this is the first genetic study in Thailand of Cryptosporidium spp contamination in river and sea water locations and the first report of C. serpentis, suggesting that humans, household pets, farm animals, wildlife and migratory birds may be the potential sources of the parasites. The findings are of use for implementing preventive measures to reduce the transmission of cryptosporidiosis to both humans and animals.

Toxoplasmosis and neosporosis among beef cattle slaughtered for food in Western Thailand.

Beef is a main type of meat consumed by Thais. The prevalences of anti-Toxoplasma gondii and anti-Neospora caninum antibodies were investigated among beef cattle slaughtered for food in western Thailand. A total of 389 blood samples obtained from beef cattle from 24 herds were collected at 3 slaughterhouses in 3 western provinces of Thailand: Kanchanaburi, Ratchaburi and Nakhon Pathom. An indirect immunofluorescent antibody test (IFAT) was performed using cut-off values of 1:128 for T. gondii and 1:200 for N. caninum. The antibodies to T. gondii were found in 100 samples (25.7%) and antibodies to N. caninum were found in 23 samples (5.9%) a significant difference (p < 0.001) in prevalences, indicating the cattle tested had a greater exposure to T. gondii than N. caninum, and they should be regarded as a potential source of T. gondii infection to humans. The low prevalence of neosporosis in this study is still a risk for morbidity among cattle, including abortions. This is the first study in Thailand finding both T. gondii and N. caninum antibodies among beef cattle.

Transplacental transmission in cattle: is Toxoplasma gondii less potent than Neospora caninum?

We compared the transplacental-transmission ability of Toxoplasma gondii and Neospora caninum in cattle. One uninfected pregnant heifer served as control, while three were inoculated with N. caninum K9WA strain and four with T. gondii RH strain at their midgestational period. Both infected groups showed clinical signs and antibodies either to N. caninum or T. gondii, while the control animal was normal. Two (50%) Toxoplasma dams aborted on days 6 and 11 postinoculation. T. gondii tachyzoites were found in various organs of those dams that had abortions but not in their fetuses. Two Neospora dams did not abort but gave birth to subclinically infected calves. The remaining two Toxoplasma dams and one from Neospora group became recumbent. Those two dams and their fetuses showed disseminated Toxoplasma DNA, but no Neospora DNA was found. Our findings suggest that maternal toxoplasmosis could be a cause of abortion and congenital toxoplasmosis in cattle, especially when they are infected by virulent strains.

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice.

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.

Is Sabin-Feldman dye test using T. gondii tachyzoites from animal inoculation still the best method for detecting Toxoplasma gondii antibodies?

Although the Sabin-Feldman dye test is the gold standard for detecting Toxoplasma antibodies in human, it is performed only in reference laboratories because live virulent T. gondii are used for the test. We collected 210 human serum samples and tested them by the dye test using in vivo tachyzoites (conventional method) then compared these results with three other methods: a dye test using cell culture-derived T. gondii tachyzoites and indirect immunofluorescent antibody tests (IFAT) using in vivo and in vitro tachyzoites. We found the conventional dye test detected the highest percent of cases (4.3%), followed by the IFAT using parasites from mice (3.8%), then the dye test and the IFAT using cell culture tachyzoites (both 2.8%). Agreement with the dye test when using mouse and cell culture derived tachyzoites was 96.7%. Using in vivo tachyzoites for the dye test and the IFAT gave 94.3% agreement, while using in vitro tachyzoites gave 94.8% agreement. When compared with the conventional dye test, the IFAT had 75% sensitivity and 100% specificity. The T. gondii tachyzoites obtained from cell culture had a lower virulence, as indicated by a three times longer survival period in the inoculated mice. We favor the conventional dye test as the gold standard for Toxoplasma antibody detection. In vitro tachyzoites can be used routinely in the dye test but false negative results may occur in some cases. The IFAT, using either in vivo or in vitro tachyzoites, are alternatives for laboratories where provision of live tachyzoites is limited.

Epidemiology of Toxoplasma gondii infection of stray cats in Bangkok, Thailand.

The objective of this study was to investigate the epidemiology of toxoplasmosis in stray cats in Bangkok. Sera were collected during 2006 and examined by Sabin-Feldman dye test. Five hundred sixty-four male and 926 female cats in and around monasteries from 50 districts were collected. Toxoplasma gondii was detected in 72 (4.8%) of 1,490 cats. The prevalence was significantly higher in females (5.6%) than in males (3.6%). Cats more than 5 years old had the highest infection rate (5.1%). Fifty-six percent (28/50) of areas were positive for T. gondii in cats. Our results show T. gondii is widespread in stray cats in Bangkok. It is essential to control the number of stray cats in order to reduce the transmission of toxoplasmosis to animals and humans.

PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok.

A survey of gastrointestinal parasites of dogs and humans from temple communities in Bangkok revealed that 58% of dogs and 3.4% of humans, among those sampled, were infected with hookworms utilising faecal flotation techniques and microscopy. A previously established polymerase chain reaction (PCR)-RFLP approach was utilised to determine the species of hookworms infecting dogs found positive for hookworm eggs. Single infections with Ancylostoma ceylanicum and Ancylostoma caninum were recorded in 77% and 9% of hookworm positive dogs, respectively and mixed infections with both species of Ancylostoma were recorded in 14% of dogs. A single-step PCR for the multiplex detection of Ancylostoma species and Necator americanus DNA in human faeces was developed and applied to characterise the species of hookworms in microscopy positive individuals. Single infection with N. americanus was recorded in five and A.ceylanicum infection in two, out of seven individuals positive for hookworm. This study demonstrates that humans are at risk of acquiring infection with A. ceylanicum in communities where this species of hookworm is endemic in dogs.

Seroprevalence of Toxoplasma gondii antibody in Vietnamese villagers.

Toxoplasmosis caused by Toxoplasma gondii is a protozoan infection found worldwide. It usually produces non-specific symptoms, but in pregnant women and immunocompomised individuals, it may cause severe and fatal illness. Many serological studies have been done in various parts of the world, but information is lacking for Vietnam. A seroprevalence study of T gondii antibodies in Vietnamese villagers (n = 650) was performed using the Sabin-Feldman dye test. The average seroprevalence was 4.19% (95% CI = 1.78-4.62), including 6.36% (95% CI = 3.22-11.09), 4.73% (95% CI = 1.92-9.50) and 1.09% (95% CI = 0.23-3.15) from Nghe An, Lao Cai and Tien Giang provinces, respectively. This study confirmed the low prevalence of toxoplasmosis in Vietnam similar to other countries in the region. Further studies are necessary in order to provide a complete picture for the country.

Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand.

Blastocystis is a common intestinal pathogen of humans and a variety of animals, with various host-specific subtypes. The aim of this study was to determine the prevalence and subtype distribution of Blastocystis in humans and domestic animals, Thailand. 113 stool samples were collected from pigs, goats, and cattle in Ayutthaya Province (AP; central Thailand) and 218 stool samples were collected from pigs, dogs, cats, chickens, and humans in Kanchanaburi Province (KP; western Thailand). Blastocystis was detected by nested PCR targeting the SSU rRNA gene. Subtypes were identified by DNA sequencing, and phylogenetic analysis was conducted. The overall prevalence of Blastocystis in animals was 76.1% (86/113) and 11.88% (12/101) in AP and KP, respectively, and the prevalence in humans was 12.82% (15/117) in KP. The prevalence of Blastocystis in the AP and KP pigs were 87.88% (29/33) and 20.37% (11/54), respectively. Blastocystis ST5 was the most abundant in pigs in both areas while Blastocystis ST10 and ST12 were most frequently found in cattle and goats. In addition, low percentage of Blastocystis ST1 and Blastocystis ST14 were found in pigs and goats, respectively. In this study, Blastocystis ST3, followed by ST2 and ST1 were predominantly found in humans. In conclusion, pigs may be a natural host of Blastocystis and this ST may be the pig-adapted ST in the studied areas, in this study.

Blastocystis subtype 5: Predominant subtype on pig farms, Thailand.

Blastocystis is a unicellular protist most commonly detected in humans and a variety of animals. The predominant mode of its transmission is the fecal-oral route, but its zoonotic potential is not completely understood. The objective of this study was to determine the presence and genetic diversity of Blastocystis on pig farms in Nakhon Pathom Province, Central Thailand. A total of 154 human and 90 pig stool samples were collected and analyzed. Nested PCR detected Blastocystis in 35.55% of the pig samples and 6.49% of the human samples. Subtyping based on regions of the small-subunit ribosomal RNA (SSU rRNA) gene identified three Blastocystis subtypes in pigs and humans: ST1, ST3, and ST5. Blastocystis ST5 was the predominant subtype, followed by ST1 and then ST3. All the sequences from the Blastocystis-positive samples from both pigs and humans were closely related. This study reveals a possibility of low host specificity of Blastocystis STs (ST1, ST3 and ST5) on pig farms in Thailand. We tentatively suggest that close contact with or exposure to pig stools may be a significant source of Blastocystis detected in pig handlers. Further studies are required to confirm the zoonotic transmission of this organism in Thailand, because pigs may play an important role in the transmission of Blastocystis.

Immunohistochemical study of acute and chronic toxoplasmosis in experimentally infected mice.

Acute and chronic Toxoplasma infections were evaluated in mice using stage specific antibodies and immunocytochemistry. Mice with acute toxoplasmosis were less active, had erectile body hair and seldom took food or water resulting in weight loss. All mice died within 7 days post-inoculation. The immunohistochemical technique enhanced visualization of parasites allowing their distribution to be accurately followed. Following intraperitoneal infection, tachyzoites were initially identified on the surface of the liver and spleen. There was a rapid increase in the number of tachyzoites associated with invasion from the surrounding connective tissue into the organs with formation of inflammatory lesions in the liver. The focal inflammatory lesions showed increasing numbers of tachyzoites with the period post-inoculation. Similar increases in tachyzoites were observed for the spleen. In contrast, only a few individual tachyzoites were seen in the brain at the final time point. In chronic infections, the mice were asymptomatic but tissue cysts containing large numbers of bradyzoites were observed in all brains with the average number of 295 tissue cysts per half brain and the average cystic size of 46.02 +/- 5.08 microm. By histology and immunostaining, the tissue cysts were readily identifiable along with a mild inflammatory cell infiltration into the meninges and perivascular cuffing. Double immunocytochemical labelling confirmed the exclusive presence of tachyzoites during the acute phase and bradyzoites during the chronic phase.

Canine parasitic zoonoses in Bangkok temples.

Fecal samples were collected from 204 humans and 229 dogs from 20 different temples in Bangkok, as well as communities in the surrounding temple ground areas. Human and dog stool samples were examined for intestinal parasites including Giardia using zinc sulfate flotation and microscopy. Hookworms were the most common parasite in dogs (58.1%) followed by Trichuris (20.5%), Isospora (10%), Giardia (7.9%), Toxocara (7.4%), Dipylidium caninum (4.4%) and Spirometra (3.1%). Blastocystis hominis (5.9%) was the most common parasite in humans followed by hookworms (3.4%), Giardia (2.5%), Strongyloides (2%) and Cryptosporidium (1.5%). All samples microscopy-positive for Giardia were genotyped. The majority of Giardia isolated from the dog population was placed in Assemblage A, followed by Assemblages D, B and C, respectively, while human isolates were placed in Assemblages A and B. Therefore, dogs in temple communities posed a potential zoonotic risk to humans for transmission of hookworms, Giardia (especially Assemblage A genotypes) and Toxocara canis.

Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis.

BACKGROUND: Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum. METHODS: LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand. RESULTS: Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697). CONCLUSIONS: This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.

Seroprevalence of Toxoplasma gondii Infection in Refugee and Migrant Pregnant Women along the Thailand-Myanmar Border.

Toxoplasma gondii primary infection in pregnancy is associated with poor obstetric outcomes. This study aimed to determine the seroprevalence of Toxoplasma infection in pregnant migrant and refugee women from Myanmar attending antenatal care in Thailand. A random selection of 199 residual blood samples from first antenatal screen in 2014-2015 was tested for Toxoplasma IgG and IgM antibodies. Seroprevalence of Toxoplasma infection was 31.7% (95% confidence interval = 25.6-38.4). Avidity testing in the three positive IgM cases indicated all were past infections. Multiparity (≥ 3 children) was significantly associated with higher Toxoplasma seropositivity rates. Seroprevalence of T. gondii infection in this pregnant population is similar to the only other report from Myanmar, where multiparity was also identified as a significant association. Toxoplasma infection is important in pregnant women. Nevertheless, in this marginalized population, this infection may be given less priority, due to resource constraints in providing the most basic components of safe motherhood programs.

Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand.

Enterocytozoon bieneusi is an opportunistic intestinal pathogen infecting humans and a variety of animals. Its mode of transmission and zoonotic potential are not completely understood. E. bieneusi has been frequently identified in pigs. The objective of our study was to investigate E. bieneusi in pigs and humans in Western and Central Thailand to determine its presence, genetic diversity, and zoonotic potential. A total of 277 human and 210 pig faecal samples were collected and analysed. E. bieneusi was found in 5.4% and 28.1% of human and pig samples, respectively, by nested PCR. Genotyping based on the internal transcribed spacer regions of the small subunit ribosomal RNA demonstrated three known genotypes (D, H, PigEb10) and eight novel genotypes (TMH1-8) in humans, and five known genotypes (D, EbpA, EbpC, H, O) and 11 novel genotypes (TMP1-11) in pigs. All known genotypes identified in humans and pigs had zoonotic potential. Further studies are needed to evaluate zoonotic risk of novel genotypes, as pigs may play an important role in the transmission of E. bieneusi.

Toxoplasmosis: beyond animals to humans.

The parasitic zoonosis toxoplasmosis, which was poorly understood before the advent of the HIV epidemic, has become a major clinical problem worldwide. Humans acquire toxoplasmosis from cats, from consuming raw or undercooked meat and from vertical transmission to the foetus through the placenta during pregnancy. Studies of the unique environmental factors in various communities indicate the important roles that eating habits and culture have on the transmission of this infection. The socioepidemiological aspects of toxoplasmosis are thought to be important contributing factors for the spread of this disease. Preventative measures should consider the cultures and beliefs of people in various communities more than solving poverty and giving orthodox health education.

Cultivation of Giardia duodenalis in Mongolian gerbils.

The Mongolian gerbil (Meriones unguiculatus) is susceptible to infection with Giardia duodenalis trophozoites. Each animal was orally infected with 0.5 ml Diamond's TYIS-33 culture medium containing 10(6) trophozoites. Cysts were then collected and concentrated by sucrose gradient centrifugation. G. duodenalis cysts were first observed in feces on day 5 post-infection. The characteristic of G. duodenalis infection in gerbils was intermittent cyst release. The range in the number of cysts released per gerbil for a 4-hour collection period was 0-1.5 x 10(3).