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

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 101, 102, and 103 per 10 µl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 102 per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting

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

Prevalence and Subtype Distribution of Blastocystis Isolated from School-Aged Children in the Thai-Myanmar Border, Ratchaburi Province, Thailand

Blastocystis is one of the most common enteric protozoa that inhabits the intestinal tract of humans and different animals. Moreover, it has a worldwide geographic distribution. Its main mode of transmission is via the fecal-oral route. At present, 26 subtypes are widely distributed across both humans and animals. The current study aimed to determine the prevalence and subtype distribution of Blastocystis among school-aged children living on the Thai-Myanmar border, Ratchaburi province, Thailand. In total, 508 samples were collected from children at six schools. The prevalence of Blastocystis infection was amplified and sequenced in the 600 bp barcode region of the small-subunit ribosomal RNA (SSU rRNA). The overall prevalence of Blastocystis infection was 3.35% (17/508). ST3 (11/17) was the most predominant subtype, followed by ST1 (5/17) and ST2 (1/17). A phylogenetic tree was constructed based on the Tamura92+G+I model using the maximum-likelihood algorithm. Surprisingly, all sequences of the ST3-positive samples were closely correlated with the cattle-derived sequence. Meanwhile, all sequences of the Blastocystis ST1-positive samples were closely correlated with the human-derived sequence. Nevertheless, further studies should be conducted to validate the zoonotic transmission of Blastocystis. Based on our findings, personal hygiene and sanitation should be improved to promote better health in children in this area

Microbiome analysis of thai traditional fermented soybeans reveals short-chain fatty acid-associated bacterial taxa

Abstract Thua Nao is a Thai traditional fermented soybean food and low-cost protein supplement. This study aimed to evaluate the bacterial community in Thua Nao from northern Thailand and assess potentially active short-chain fatty acids (SCFAs)-related bacteria. Sixty-five Thua Nao consisting of 30 wet and 35 dried samples were collected from six provinces: Chiang Rai, Chiang Mai, Mae Hong Son, Lampang, Lamphun, and Phayao. Bacterial diversity was significantly higher in the wet samples than in the dried samples. The dominant phyla were Firmicutes (92.7%), Proteobacteria (6.7%), Actinobacteriota (0.42%), and Bacteroidota (0.26%). The genus Bacillus (67%) was the most represented in all samples. Lactobacillus, Enterococcus, and Globicatella were enriched in the wet samples. Assessment of the SCFA-microbiota relationships revealed that high butyrate and propionate concentrations were associated with an increased Clostridiales abundance, and high acetate concentrations were associated with an increased Weissella abundance. Wet products contained more SCFAs, including acetate (P = 2.8e−08), propionate (P = 0.0044), butyrate (P = 0.0021), and isovalerate (P = 0.017), than the dried products. These results provide insight into SCFA-microbiota associations in Thua Nao, which may enable the development of starter cultures for SCFA-enriched Thua Nao production

Agreement (Kappa) between the detection of <i>N</i>. <i>fowleri</i> by LAMP and PCR in water samples.

<p>*<i>P</i> <0.001</p><p>Agreement (Kappa) between the detection of <i>N</i>. <i>fowleri</i> by LAMP and PCR in water samples.</p

Categories of water samples tested in this study with number <i>N</i>. <i>fowleri</i>-positive by LAMP and PCR.

<p>*<i>P</i> value for McNemar's test = 0.25</p><p>Categories of water samples tested in this study with number <i>N</i>. <i>fowleri</i>-positive by LAMP and PCR.</p

Details of the primer set targeting <i>N</i>. <i>fowleri</i> virulence-related protein used for amplification in the LAMP assay.

<p>Details of the primer set targeting <i>N</i>. <i>fowleri</i> virulence-related protein used for amplification in the LAMP assay.</p

Development of a Rapid, Simple Method for Detecting <i>Naegleria fowleri</i> Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

<div><p><i>Naegleria fowleri</i> is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of <i>N</i>. <i>fowleri</i> 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 <i>N</i>. <i>fowleri</i>. Time to results is about 90 min and amplification products were easily detected visually using <i>hydroxy naphthol blue</i>. 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. <i>N</i>. <i>fowleri</i> 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 <i>N</i>. <i>fowleri</i>. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing <i>N</i>. <i>fowleri</i> in water and clinical samples, particularly in resource-poor settings.</p></div

Specificities of the LAMP assay for the detection of <i>N</i>. <i>fowleri</i>.

<p>(A) Specificity of LAMP assay using HNB (note the sky-blue color for a positive sample). (B) Confirmation of results of the LAMP products using agarose gel (2%) electrophoresis. In panels A and B: M, 100 bp DNA Ladder (Thermo Scientific); 1, <i>N</i>. <i>fowleri</i>; 2, <i>N</i>. <i>gruberi</i>; 3, <i>Acanthamoeba</i> spp.; 4, <i>G</i>. <i>duodenalis</i>; 5, <i>C</i>. <i>parvum</i>; 6, <i>E</i>. <i>histolytica</i>; 7, <i>Entamoeba coli</i>; 8, <i>T</i>. <i>gondii</i>; 9, <i>N</i>. <i>caninum</i>; 10, <i>Blastocystis</i>; 11, <i>E</i>. <i>bieneusi</i>; 12, <i>Escherichia coli</i>; 13, <i>C</i>. <i>neoformans</i>; 14, <i>M</i>. <i>tuberculosis</i>; 15, CSF from non-PAM patients; 16, blood sample of healthy donor; 17, <i>N</i>. <i>fowleri</i>-free pond water; 18, no template control.</p