Yaowalark Sukthana

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.

Transmission cycles of Giardia duodenalis in dogs and humans in Temple communities in Bangkok—A critical evaluation of its prevalence using three diagnostic tests in the field in the absence of a gold standard

The prevalence and associated risk factors for Giardia duodenalis in canine and human populations in Temple communities of Bangkok, Thailand were determined by evaluating three common diagnostic methods utilised to detect Giardia, namely zinc sulphate flotation and microscopy, an immunofluoresence antibody test and nested polymerase chain reaction (PCR) based on the SSU-rDNA gene. The diagnostic sensitivity and specificity together with the negative and positive predictive values of each test were evaluated in the absence of a gold standard using a Bayesian approach. The median estimates of the prevalence of infection with G. duodenalis in dogs and humans in Thailand were 56.8% (95% PCI, 30.4%, 77.7%) and 20.3% (95% PCI, 7.3%, 46.3%) respectively. PCR and immunofluorescence antibody tests (IFAT) were the most accurate tests overall with diagnostic sensitivity and specificity of 97.4% (95% PCI, 88.5%, 99.9%) and 56.2% (95% PCI, 40.4%, 82.9%) for the PCR and 61.8% (95% PCI, 40.8%, 99.1%) and 94.7% (95% PCI, 87.4%, 99.1%) for IFAT respectively Three cycles, anthroponotic, zoonotic and dog-specific cycles of G. duodenalis were shown to be operating among the human and canine populations in these Temple communities in Bangkok, supporting the role of the dog as a potential reservoir for Giardia infections in humans.

Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2. Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations of drugs, resulting in sigmoid dose-response curves, similar to those obtained from conventional drug sensitivity assays. HRP2 therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use in drug sensitivity or bioassays.

Presence of zoonotic Enterocytozoon bieneusi in cats in a temple in central Thailand

Enterocytozoon bieneusi is a common opportunistic intestinal pathogen in humans and animals. To investigate the prevalence, genotype and host specificity of E. bieneusi, 111 dog faecal samples were collected from dairy cattle farms, and 95 and 80 faecal samples were collected from dogs and cats, respectively, in a temple in central Thailand. E. bieneusi was found in 25 (31.3%) cats by nested PCR, but not in dogs. Genotyping analysis targeting the internal transcribed spacer of the rRNA gene identified genotype D - and other novel genotypes very similar to genotype D - which is a zoonotic genotype reported in both HIV patients and villagers in rural communities in Thailand. This is the first study to find E. bieneusi genotype D in cats, and it may be that cats are found to play an important role in E. bieneusi zoonotic transmission to humans. The present study indicates that further molecular epidemiological investigations of E. bieneusi among cats are necessary to evaluate their possible role as reservoir hosts and the potential risk they represent to humans.

A high prevalence of Toxoplasma in Australian chickens.

A small survey was undertaken of commercially reared free-range chickens in Western Australia using serology and molecular detection. Eighteen out of 20 serum samples showed antibody responses with titers of 1:64 in 5 chickens and ≥ 1:128 in 13 chickens. DNA extracted from 22 out of 50 tissue samples, 10 brains and 12 spleens, were positive by nested PCR, and sequencing at the B1 locus on DNA from 3 brain and 3 spleen samples confirmed that 2 isolates were Toxoplasma gondii, Type I, and 4 Type II/III. The high prevalence of Toxoplasma infection found in commercial, free-range chickens raises public health issues with respect to both exposure in the workplace, during carcass processing, and subsequent transmission during food handling and/or consumption as food. The results of this study emphasize the need for more data on the incidence of Toxoplasma infection in domestic animals and humans in Australia.

Zoonotic potential of Enterocytozoon bieneusi among children in rural communities in Thailand

Enterocytozoon bieneusi is a common opportunistic intestinal pathogen worldwide. Genotype distribution of E. bieneusi differs by geography and host immunity. In order to investigate the prevalence, genotype characteristics, and host specificity of E. bieneusi in the community, we conducted a preliminary cross-sectional study among children in Western and Northern Thailand. Seventy-eight (78) and 102 stool samples were collected; the prevalence of E. bieneusi was 3.8% and 2.9% by nested PCR in Western and Northern Thailand, respectively. Three genotypes were identified: Genotype D predominated, followed by EbpC, and then novel genotype ETMK1. The first two genotypes have zoonotic potential. Analysis of the genetic proximity of the E. bieneusi ITS sequences from our study, compared with those published in genetic databases, showed that all positive samples were classified into Group 1, the largest group consisting of various host specificity. The present study demonstrates the possible zoonotic transmission of E. bieneusi in rural communities in Thailand. A large-scale investigation of both human and animal samples, as well as improvements in the available phylogenetic tools, will be required to elucidate transmission routes of E. bieneusi in this area.

Molecular identification of Cryptosporidium spp. in seagulls, pigeons, dogs, and cats in Thailand

Zoonotic Cryptosporidium spp., particularly C. meleagridis, C. canis, and C. felis, are enteric protozoa responsible for major public health concerns around the world. To determine the spread of this parasite in Thailand, we conducted molecular identification of Cryptosporidium spp. from animal samples around the country, by collecting and investigating the feces of seagulls (Chroicocephalus brunnicephalus and Chroicocephalus ridibundus), domestic pigeons (Columba livia domestica), dogs, and cats. Seagull and pigeon samples were collected at the seaside and on the riverside to evaluate their potential for waterborne transmission. Ten pigeon samples were combined into one set, and a total of seven sets were collected. Seventy seagull samples were combined into one set, and a total of 13 sets were collected. In addition, 111 dog samples were collected from cattle farms, and 95 dog and 80 cat samples were collected from a temple. We identified C. meleagridis in pigeons, Cryptosporidium avian genotype III in seagulls, C. canis in dogs, and C. felis in cats. In the temple, the prevalence was 2.1% (2/95) for dogs and 2.5% (2/80) for cats. No Cryptosporidium was found in dog samples from cattle farms. These are the first findings of C. meleagridis in domestic pigeons, and Cryptosporidium avian genotype III in seagulls. Our study invites further molecular epidemiological investigations of Cryptosporidium in these animals and their environment to evaluate the public health risk in Thailand.

Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

Geographical distribution of Toxoplasma gondii genotypes in Asia: A link with neighboring continents

Defining the pattern of genetic diversity of Toxoplasma gondii is important to understand its worldwide distribution. During the last decades, a large number of studies have been published on Toxoplasma genotypes circulating in Europe, in North and South America. Two continents are still largely unexplored, Africa and, to a less extent, Asia. In this last continent, an increasing number of publications reported genotypes circulating in diverse provinces of China, but very few data are available for other Asian countries. After a systematic database search, 47 papers related to T. gondii genotypes in Asia were analyzed. Genetic characterization of DNA was performed by microsatellite markers, or more usually by a multiplex PCR using 11 PCR-RFLP markers, allowing data comparison to draw a first global picture of the population structure of this parasite throughout Asia. Overall, 390 isolates or DNA extracts were completely typed by PCR-RFLP and/or microsatellite marker methods, revealing 36 different PCR-RFLP or equivalent microsatellite genotypes: 15 genotypes identified by a ToxoDB number and 21 atypical or unique genotypes. The most common genotype found in Asia is the genotype ToxoDB#9 (Chinese 1). The clonal types I, II and II variant, and III were also commonly found in Asia. The geographical distribution of these genotypes across Asia may reflect either a continuum with Europe for the western part of Asia (presence of Type II), or the circulation of strains through animal migration or human activities between Africa and the Southwestern part of Asia (Africa 1 genotype in Turkey or ToxoDB#20 both I Sri-Lanka and in Ethiopia or Egypt). Although there are some indications of a genetic population structure in Southeast Asian countries different from the rest of Asia, more studies in this tropical part of Asia will be necessary for a region which represent as well as Africa one of the missing links of the T. gondii genetic diversity.

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.