Misclassification of Opisthorchis viverrini and Minute Intestinal Fluke Eggs by Routine Laboratory Staff Using Images from the Kato-Katz Method.

BACKGROUND: The Kato-Katz method is a commonly used diagnostic tool for helminth infections, particularly in field studies. This method can yield inaccurate results when samples contain eggs that are similar in appearance, such as Minute Intestinal Fluke (MIF) and Opisthorchis viverrini (OV) eggs. The close resemblance of eggs can be problematic and raises the possibility of false diagnoses. The objectives were to compare the diagnostic performance of the Kato-Katz method for accurately identifying MIF and OV and to provide evidence of possible misclassification.  Methods: Based on questionnaire responses from 15 (young parasitologists and public health staff), the test comprised 50 MIF egg images and 50 OV egg images, for a total of 100 Google Form questionnaires. RESULTS: The morphology of MIF and OV eggs found size and shape similarity and found that the shoulder rims were small, while the OV egg found the knobs had disappeared. The opercular conjunction was apparent, the shoulder rims and miricidium were prominent. The average percentage of correctly classified infections was 61.6 ± 12.1%. The accuracy percentages for both public health staff and young parasitologists in identifying were found to be 59.0 ± 14.8 and 66.8 ± 2.8, respectively. There was no significant difference observed in both groups. CONCLUSION: These findings highlight the need for improving the accuracy of parasite identification. Preserving stool samples before the Kato-Katz method can help mitigate the potential degradation or distortion of parasite eggs. The incorrect classification of both eggs had an impact on treatment plans and the policy of parasite control programs.

Formalin-fixed stool improves the performance of the Kato-Katz method.

Background and Aim: Parasitic infections are a public health problem worldwide, including in Thailand. An epidemiological survey for helminthiasis based on stool examination uses the Kato-Katz method as recommended by the World Health Organization. Limitations of this method include the need for fresh stool, time requirement, and lack of quality control. The aim of this study was to enhance the efficiency of the Kato-Katz technique using formalin and glycerol solutions and to implement specimen preparation in fieldwork. Materials and Methods: For the Kato-Katz method, stool samples were divided into formalin-fixed and unfixed groups at various time points and processes. Fresh echinostome eggs were added to each stool group. Incubation with glycerol increased the clearing process. Each group was observed and photographed using a light microscope. Parasite eggs were imaged and compared using the standard Kato-Katz method. Results: Visualization of echinostome eggs from formalin-fixed stool slides was significantly better than that from unfixed stool slides (p < 0.01). Stool samples fixed for 7 days retained normal echinostome eggs morphology. Incubation with glycerol for 1 h resulted in increased Kato-Katz performance by digesting the stool content and enhancing egg observation. Moreover, the results of the Kato-Katz method using fixed and fixed stool plus glycerol for natural helminth infection showed good quality of Opisthorchis viverrini and Taenia egg visualization and normal morphology with a clear background of slides. Conclusion: Formalin-fixed stool could be more suitable than fresh stool for the Kato-Katz method.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Comparison of 3 DNA extraction methods for extracting DNA from an adult Culex quinquefasciatus (Diptera: Culicidae).

The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ±â€…3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ±â€…4.57 ng/µl) and a microwave-based method (119.26 ±â€…3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ±â€…0.02, 1.79 ±â€…0.01, and 1.87 ±â€…0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.

Reduction of carcinogens in fermented fish (pla-ra and pla-som) by heating.

Background and Aim: The risk factors for cholangiocarcinoma (CCA) are opisthorchiasis and the intake of a combination of nitroso compounds through the consumption of traditionally fermented fish, which is very popular in areas where liver flukes are endemic. The incidence of CCA remains high because this cultural habit of rural people has been altered. Therefore, decreasing nitrate and nitrite concentrations in fermented fish are an alternative approach to reducing the risk of CCA. Thus, this study aimed to reduce nitrate and nitrite concentrations in fermented foods by heating and investigated its effect on CCA development in a hamster model. Materials and Methods: We used Association of Official Analytical Chemists method 973.31 to measure the nitrate and nitrite concentrations in both fermented fish (pla-ra [PR]) and pickled fish (pla-som [PS]) before and after boiling for 5 and 30 min, respectively. The same samples were fed to Opisthorchis viverrini (OV)-infected or -uninfected hamsters for 3 months. Thereafter, the hamsters' liver and blood were collected for analysis. Results: The levels of nitrates and nitrites in PS and PR significantly decreased following boiling for 5 and 30 min. The OV-PR and OV-PS groups showed dramatically increased numbers of inflammatory cells, fibrosis surrounding the bile duct, and focal fibrotic areas. However, after boiling the fermented dishes for 5 and 30 min, the extent of inflammatory cell infiltration and intensity of fibrosis in these groups were decreased. Conclusion: Our findings suggest that boiling reduces nitrate and nitrite toxicity in fermented dishes, as evidenced by reduced hepatic inflammation. However, regardless of heating, kidney tissues are adversely affected when fermented meals are consumed daily.

Endoplasmic Reticulum Stress-Mediated Apoptosis Induced by VR12684 Isolated from Mallotus spodocarpus in Cholangiocarcinoma Cell Line.

INTRODUCTION: Cholangiocarcinoma (CCA) is a poor prognosis of a malignant tumor that has been unresponsive to conventional chemotherapeutic agents. Effective and novel therapeutic agents are urgently needed. VR12684 (isolated from Mallotus spodocarpus) has been reported to exhibit growth inhibitory activities in cancer cell lines. The present study investigated the growth inhibitory mechanisms of this compound in a human CCA cell line (KKU-M156). METHODS: The effects of VR12684 on anti­proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by SRB assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) staining and western blot analysis. RESULTS: Treatment with VR12684 decreased cell proliferation in a dose- and time-dependent manner in the KKU-M156 cell line. VR12684 induced cell cycle arrest in the G2 phase in KKU-M156 through down-regulation of cyclin B1 and Cdk1 and up-regulation of p21, p27 and p53 levels. VR12684 induced mitochondria-mediated apoptosis by increasing DNA fragmentation, the Bax/BCL-2 ratio and AIF, and decreasing survivin with subsequent activation of caspase-9 and -3. This compound could induce apoptosis through the endoplasmic reticulum (ER) stress-mediated pathway by up-regulation of GRP78, IRE1α and GADD153 levels leading to down-regulation of Bcl-2 and activation of calpain-1, caspase-7 and -12. CONCLUSION: These results suggested that VR12684 inhibited KKU-M-156 cell growth by way of cell cycle arrest and induction of apoptosis, at least in part, through the mitochondria- and ER-associated intrinsic pathways. Such compounds warrant evaluation as a candidate for the treatment of human CCA.

Correction: Perakanya et al. Prevalence and Risk Factors of Opisthorchis viverrini Infection in Sakon Nakhon Province, Thailand. Trop. Med. Infect. Dis. 2022, 7, 313.

The authors wish to make the following corrections to this paper [...].

Monks: A Population at Risk for Liver Fluke and Skin-Penetrating Helminths.

Monks cannot cook received raw meat dishes and should walk barefoot while working. This population lacks both a survey of parasitic infection and a proper prevention and control policy. Five hundred and fourteen monks from the Ubolratana, Ban Haet, and Ban Phai Districts of Kh on Kaen Province were enrolled in this study. A stool container and questionnaire were collected from each study participant. Stool samples were processed by formalin ethyl acetate concentration and agar plate culture techniques. We then analyzed the results and risk factors to demonstrate associations. The prevalence of overall parasites, liver flukes, and skin-penetrating helminths were 28.8%, 11.1%, and 19.3%, respectively. Raw fish dish offerings were associated with opisthorchiasis (ORcrude 3.32; 95% CI 1.53-7.20). The risk factors for skin-penetrating helminths were older age (ORcrude 5.02; 95% CI 2.2-11.17), being a long-term ordinate (ORcrude 3.28; 95% CI 1.15-9.34), smoking (ORcrude 2.03; 95% CI 1.23-3.36), and chronic kidney disease with other underlying disease (ORcrude 20.7; 95% CI 2.54-190.1). The protective factors for skin-penetrating helminths were secular education above primary education (ORcrude 0.41; 95% CI 0.25-0.65) and having received health education about parasitic infection (ORcrude 0.47; 95% CI 0.28-0.80). Wearing shoes at times other than alms work does not show a protective effect against skin-penetrating helminths (ORcrude 0.86; 95% CI 0.51-1.46). These findings support the recommendation for a strict Rule of Discipline regarding raw meat ingestion and allowing shoes to be worn for protection against skin-penetrating helminths in high-risk situations.

Prevalence of gastrointestinal parasites in captive mammals at Khon Kaen Zoo, Thailand.

Background and Aim: Captive animals are susceptible to parasitic diseases due to the stress and confinement they experience. In addition, they can serve as reservoirs of zoonotic parasites that have the potential to infect humans. To investigate this possibility, we estimated the prevalence of gastrointestinal (GI) parasites in captive mammals at Khon Kaen Zoo, Thailand. Materials and Methods: One hundred and forty-seven individual mammals (37 primates, 43 carnivores, 62 herbivores, and 5 rodents) were examined for parasitic infections by fecal examination daily for 3 consecutive days using the formalin-ethyl acetate concentration technique (FECT) and the agar plate culture method. Results: According to FECT, the overall prevalence of GI parasites was 62.6% (92/147). Within animal groups, the numbers were as follows: 67.6% (25/37) in primates, 23.3% (10/43) in carnivores, 85.5% (53/62) in herbivores, and 80.0% (4/5) in rodents. Using the agar plate culture method, 21.43% (27/126) were positive for Strongyloides spp. and hookworm infections. The GI parasites identified belonged to three categories: protozoa (including Entamoeba histolytica species complex, Entamoeba coli, Giardia spp., coccidia, and ciliated protozoa), trematodes (minute intestinal flukes and rumen flukes), and nematodes (strongyle/hookworm, Strongyloides spp., Ascarididae, and Trichuris spp.). Conclusion: The findings of this study indicate the prevalence of several GI parasites in zoo animals with the potential for transmission to humans, given the animals' close proximity to both visitors and animal caretakers.

Green-synthesized silver nanoparticle coating on paper for antibacterial and antiviral applications.

The use of active packaging has attracted considerable attention over recent years to prevent and decrease the risk of bacterial and viral infection. Thus, this work aims to develop active packaging using a paper coated with green-synthesized silver nanoparticles (AgNPs). Effects of different silver nitrate (AgNO3) concentrations, viz. 50, 100, 150, and 200 mM (AgNPs-50, AgNPs-100, AgNPs-150, and AgNPs-200, respectively), on green synthesis of AgNPs and coated paper properties were investigated. A bio-reducing agent from mangosteen peel extract (ex-Garcinia mangostana (GM)) and citric acid as a crosslinking agent for a starch/polyvinyl alcohol matrix were also used in the synthetic process. The presence of AgNPs, ex-GM, and citric acid indicated the required synergistic antibacterial activities for gram-positive and gram-negative bacteria. The paper coated with AgNPs-150 showed complete inactivation of virus within 1 min. Water resistance and tensile strength of paper improved when being coated with AgNPs-150. The tensile strength of the coated paper was found to be in the same range as that of a common packaging paper. Result revealed that the obtained paper coated with AgNPs was proven to be effective in antibacterial and antiviral activities; hence, it could be used as an active packaging material for items that require manual handling by a number of people.

Prevalence of Ehrlichia-, Babesia-, and Hepatozoon-infected brown dog ticks in Khon Kaen Province, Northeast Thailand.

Background and Aim: The brown dog tick, Rhipicephalus sanguineus sensu lato, is the most common tick found on domestic dogs in Southeast Asia, including Thailand. Canine tick-borne pathogens are a public health concern worldwide. Tick-borne diseases are diagnosed by identifying pathogens based on the morphological or molecular analyses of dog blood samples. However, the collection of ticks, a non-invasive procedure, is easier than drawing blood. This study aimed to demonstrate the usefulness of collecting brown dog ticks for the diagnosis of tick-borne diseases and for estimating the prevalence of tick-borne pathogens among companion dogs in Khon Kaen, Northeast Thailand. Materials and Methods: Seventy brown dog ticks from 70 companion dogs in Khon Kaen Province, Thailand, were evaluated for molecular evidence of tick-borne pathogens, including Babesia spp., Ehrlichia canis, and Hepatozoon canis. Ticks were collected from dogs at a private animal hospital based on the presence of at least one of the three inclusion criteria: fever, anorexia, or lethargy. Molecular diagnosis was performed using conventional polymerase chain reaction for the detection of pathogens. Results: Of the 70 ticks collected from 70 sick dogs, 55 (78.57%) were positive for tick-borne pathogens. The most common infection was a single infection with H. canis (65.71%) followed by Babesia spp. (31.43%) and E. canis (30.00%). Coinfection was observed in 14 ticks (20.00%), and coinfection with Babesia spp. and E. canis was the most prevalent double infection (n = 6). The prevalence of coinfection was identical for H. canis mixed with Babesia spp. and H. canis mixed with E. canis (n = 4). Conclusion: The present study showed that tick-borne pathogens are highly prevalent among companion dogs in Khon Kaen Province. Therefore, we encourage an increase in tick control or the reduction and prevention of tick-borne diseases in this region. Furthermore, this study revealed that ticks are valuable samples for the molecular detection of tick-borne pathogens.

Prevalence and Risk Factors of Opisthorchis viverrini Infection in Sakon Nakhon Province, Thailand.

Opisthorchiasis is a parasitic infection caused by the liver fluke Opisthorchis viverrini. This parasite is widely distributed and well documented in Thailand, Lao PDR, Southern Vietnam, Cambodia, and Myanmar. However, its prevalence is a major problem in these countries. Thus, the aim of this study was to determine the prevalence and risk factors of O. viverrini infection from 2017 to 2020 in Sakon Nakhon province, Thailand. Questionnaires were used to interview 320 participants (160 cases and 160 controls) in a random selection of 18 districts across Sakon Nakhon province. Univariate logistic regression was used to identify the factors associated with O. viverrini infection. The overall prevalence levels of O. viverrini infection in Sakon Nakhon province for 2018, 2019, and 2020 were 3.60%, 5.21%, and 7.01%, respectively. Raw fish consumption was a positive risk factor for its infection in endemic areas. Factors associated with O. viverrini infection were the habit of consuming unsafely prepared fish (OR = 6.33, 95%CI = 0.32-0.59), the medical history of O. viverrini examination (OR = 8.93, 95%CI = 5.15-15.47), a history of O. viverrini infection (OR = 3.64, 95%CI = 1.17-1.44), and a history of taking praziquantel (OR = 3.64, 95%CI = 1.17-1.44). These results identified gaps in the epidemiological knowledge of O. viverrini in this region that need addressing to identify and develop innovative methods for prevention, control, and support efforts to permanently overcome O. viverrini infection in endemic regions.

Myiasis in Ulcerated Breast Carcinoma: First Case Record in Myanmar.

Myiasis is an infestation of maggot, which is frequently associated with poor personal hygiene and environmental sanitation. A 78-year-old female breast cancer patient visited clinic complaining of irritation, itching, and pain within the ulcerous cancer lesion for 3 weeks. Many maggots were found in the lesion. A total of 30 maggots were removed and identified to be 3rd stage of larvae of metallic fly. This is the first case of wound myiasis in advanced breast carcinoma as a complication of untreated or drug-induced ulcer.

Disinfection efficiency test for contaminated surgical mask by using Ozone generator.

BACKGROUND: Ozone (O3) is an effective disinfectant agent that leaves no harmful residues. Due to the global health crisis caused by the COVID-19 pandemic, surgical masks are in high demand, with some needing to be reused in certain regions. This study aims to evaluate the effects of O3 for pathogen disinfection on reused surgical masks in various conditions. METHODS: O3 generators, a modified PZ 2-4 for Air (2000 mg O3/L) and a modified PZ 7 -2HO for Air (500 mg O3/L), were used together with 1.063 m3 (0.68 × 0.68 × 2.3 m) and 0.456 m3 (0.68 × 0.68 × 1.15 m) acrylic boxes as well as a room-sized 56 m3 (4 × 4 × 3.5 m) box to provide 3 conditions for the disinfection of masks contaminated with enveloped RNA virus (105 FFU/mL), bacteria (103 CFU/mL) and fungi (102 spores/mL). RESULTS: The virucidal effects were 82.99% and 81.70% after 15 min of treatment with 2000 mg/L O3 at 1.063 m3 and 500 mg/L O3 at 0.456 m3, respectively. The viral killing effect was increased over time and reached more than 95% after 2 h of incubation in both conditions. By using 2000 mg/L O3 in a 1.063 m3 box, the growth of bacteria and fungi was found to be completely inhibited on surgical masks after 30 min and 2 h of treatment, respectively. Using a lower-dose O3 generator at 500 mg O3/L in 0.456 m3 provided lower efficiency, although the difference was not significant. Using O3 at 2000 mg O3/L or 500 mg O3/L in a 56 m3 room is efficient for the disinfection of all pathogens on the surface of reused surgical masks. CONCLUSIONS: This study provided the conditions for using O3 (500-2000 mg/L) to reduce pathogens and disinfect contaminated surgical masks, which might be applied to reduce the inappropriate usage of reused surgical masks.

Prevalence and Associated Risk Factors of Intestinal Parasitic Infections: A Population-Based Study in Phra Lap Sub-District, Mueang Khon Kaen District, Khon Kaen Province, Northeastern Thailand.

Intestinal parasitic infections are still a crucial problem among communities in Northeast Thailand. Misuse of antiparasitic drugs and unhealthy food behaviors are known. This study aimed to explore the prevalence, behavioral health factors, and motivation for self-treatment of anti-parasitic drugs in this area. A community-based cross-sectional study was conducted in Phra Lap sub-district, Mu Khon Kaen district, Khon Kaen province, Northeast Thailand, in 2016. A total of 419 participants were recruited to complete a self-administered questionnaire and stool examination. Binary logistic regression was used to assess the association between the risk factor and parasitic infection. Forty-two participants (10%; 95%CI 7.5-13.3) were positive for at least one parasite species. In this community, the most detected intestinal parasite was Opisthorchis viverrini (5.3%), followed by Strongyloides stercoralis (3.1%). A total of 67.5% of the participants had the experience of anti-parasitic drug treatment within previous 1 year, and "Often eat raw food" was the most common reason for the use of anti-parasitic drugs. On multivariate analysis, parasitic infections were significantly associated with male gender (ORadj. 2.42; 95%CI 1.00-5.85), age ≥ 60 years (ORadj. 7.55; 95%CI 1.60-35.76), and often consuming raw food of at least one type (ORadj. 2.37; 95%CI 1.03-5.44). Given these findings, correction of the dietary habit of eating raw fish/meat, which is the most important measure, and limitation of the use of anthelmintic treatment for individuals with stools positive for ova as well as emphasis on sanitary toilets will be implemented for the prevention and control of parasitic infection in endemic communities.

Acetaminophen Overdose Enhances Early Cholangiocarcinoma in Opisthorchiasis Hamsters.

Opisthorchiasis which exerted by infection of Opisthorchis viverrini is strongly related to the incident of cholangiocarcinoma (CCA) in many Southeast Asian countries northeastern of Thailand. The O. viverrini infection is primarily caused by raw fish consumption, and repeated exposure to liver fluke. Meanwhile, acetaminophen is usually medicated to relieve pain in particularly people in northeast Thailand. OBJECTIVE: This study therefore aimed at investigating effects of acetaminophen on pathogenesis in hamsters for opisthorchiasis. METHODS: There were 4 groups of hamsters: i) uninfected hamster (N); ii) sole acetaminophen administration (N-Ac); iii) sole O. viverrini infection (OV); and iv) combination of O. viverrini infection and acetaminophen (OV-Ac) on pathology of hamsters for 1 month post infection. For analysis of histopathological changes through hematoxylin and eosin, Sirius red and immunohistostaining for Cytokeratin 19 (CK-19), Proliferating cell nuclear antigen (PCNA) and CA 19-9, serum's hamsters were used detected for liver function tests and tumor-related genes expression. RESULTS: After 1 month under these treatments, the OV-Ac showed significantly higher CCA risk, including inflammatory cells were aggregations around bile duct, new bile duct and fibrosis in subcapsular hepatic tissues, than other treatments. These pathological parameters were positively correlated with immunohistochemical staining derived from CK-19, PCNA and CA 19-9. In addition, OV-Ac had significantly higher liver function tests (ALT). CONCLUSION: Combined intake of liver fluke-contaminated raw fishes and acetaminophen rendered more severity of CCA than sole consumption of the contaminated raw fishes.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

Genetic diversity and population structure of Haemonchus contortus in goats from Thailand.

Haemonchus contortus is one of the most economically important parasitic nematodes affecting small ruminant livestock worldwide. This study was conducted to elucidate the genetic diversity and population structure of this nematode in Thailand based on mitochondrial DNA markers, the nicotinamide dehydrogenase subunit 4 (nad4) and the cytochrome c oxidase subunit 1 (cox1) genes. One hundred and thirty-six adult worms were obtained from 86 abomasa of slaughtered goats from 13 different localities in 5 regions of Thailand. Identification to the genus Haemonchus was done using morphology. DNA sequences of the nuclear ribosomal second internal transcribed spacer (ITS2) identified each specimen to species: three fixed nucleotide (SNP) differences distinguished H. contortus from H. placei. Genetic analysis defined 118 and 122 unique haplotypes in partial sequences of nad4 (alignment length 723 bp) and cox1 (645 bp) genes, respectively. Nucleotide diversities were 0.031 and 0.043 for nad4 and cox1 genes, respectively. Low genetic differentiation was observed among H. contortus samples from various provinces in Thailand. This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. This study has provided insights into the transmission dynamics of this parasitic nematode, information which is essential for farm management and parasite control.

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand.

BACKGROUND AND AIM: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene. MATERIALS AND METHODS: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. RESULTS: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). CONCLUSION: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the ß-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

A fluorescence AuNPs-LISA: A new approach for Opisthorchis viverrini (Ov) antigen detection with a simple fluorescent enhancement strategy by surfactant micelle in urine samples.

The colorimetric AuNPs-LISA is a new, powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, the diagnostic sensitivity of the colorimetric AuNPs-LISA is powerless to screen ultralow concentrations of OvAg in urine samples in cases of early stage liver fluke infection. This work, we aimed to improve the diagnostic sensitivity of the colorimetric AuNPs-LISA by developing a new fluorescence AuNPs-LISA. O-phenylenediamine (OPD) was used as the chromogenic substrate instead of the tetramethylbenzidine (TMB) of the colorimetric AuNPs-LISA. Interestingly, the fluorescence of the OPD oxidation product by the peroxidase-like activity of labelled AuNPs can be extremely enhanced by a non-ionic surfactant, especially the Triton X-100. The proposed assay exhibited a dynamic linear detection of OvAg concentration in the range of 34.18 ng mL-1 to 273.44 ng mL-1 with the limit of detection at 36.97 ng mL-1 which the detection sensitivity enhancement around 1200-fold when comparing with the colorimetric AuNPs-LISA. This model exhibits high diagnosis sensitivity, specificity and accuracy, 91.28%, 91.75%, and 91.59%, respectively, compared to the traditional ELISA. The fluorescence AuNPs-LISA showed excellent potential for the diagnosis of OvAg in urine samples from endemic areas. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis.