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

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

Molecular identification of

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

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

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

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

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

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

Lower limits of detection of LAMP and PCR assays.

<p>^<i>a</i> +, triplicated assay showed all positive; ±, triplicated assay showed both positive and negative (positive number/test number);-, triplicated assay showed all negative.</p><p>Lower limits of detection of LAMP and PCR assays.</p