Minute Intestinal Flukes Research Articles

Gastrointestinal Helminthic Infection among the Population in Northern Thailand.

Gastrointestinal (GI) helminthic infections pose substantial public health threat, particularly in northern Thailand, with a heightened concern in Nan province. This study aims to evaluate the prevalence of GI helminthic infections in Nan province and identify associated risk factors in local population. A cross-sectional survey was conducted in 2022. Fecal specimens were collected and examined using Kato-Katz technique and Formalin Ethyl-Acetate Concentration Technique (FECT). Univariate analysis employed the Chi-square test to explore correlations, while logistic regression was defined risk factors associated with parasite infections. Among 739 individuals surveyed, 14.34% were revealed GI helminthic infections. Notably, infection rates were higher in males (20.40%) compared to females (8.95%), with a notable prevalence among individuals aged ≥ 60 (17.78%) and high infection rate was demonstrated in Kiew Chan village (21.67%). The most commonly detected helminths were minute intestinal flukes (MIFs) (10.01%) and Capillaria spp. (3.11%). Males were significantly associated with GI helminthic infections, while individuals aged 40-49 and aged 50-59 commonly displayed of parasite infections. Additionally, Kiew Chan village exhibited a significant association with GI helminthic infections, followed by Sop Puen village. Furthermore, fecal specimens of 13 rodent specimens were frequency revealed Strongyloides spp. (92.30%) and mostly exposed Ancylostoma caninum (41.93%) among 93 canines. These findings highlight the imperative for immediate public health interventions in Nan province and analogous areas in northern Thailand. Implementing strategies to enhance sanitation infrastructure and promote hygiene education can significantly contribute for reducing the prevalence of GI helminthic infections and improve overall community health.

Unveiling the Transmission Potential of Opisthorchis viverrini and Intestinal Helminths Along the Thailand-Laos Border in Thailand.

In pursuit of enhancing prevention efforts for the notably high endemic Opisthorchis viverrini infection in lower Mekong sub-region countries, particularly Thailand and Laos, this cross-sectional study investigated the transmission potential of O. viverrini and other intestinal helminths along the Thailand-Laos border in seven Thai villages. Human and pet faecal samples, Bithynia snails and cyprinid fish were analysed for helminth infections. Additionally, a questionnaire survey assessed relevant risk factors among the human population. Two groups of helminth infections were detected in humans: foodborne infections (FBIs) including O. viverrini, minute intestinal flukes (MIFs), and Taenia spp., and soil-transmitted infections (STIs) including hookworm and Strongyloides stercoralis, with prevalence rates of 7.4%, 0.5%, 2.5%, 0.5%, 2.5% and 3%, respectively. Smoking was identified as a risk factor for O. viverrini infection [adjusted odds ratios (ORa) = 3.12, 95% confidence intervals (CI): 1.33-7.30, p = 0.009] and FBIs (ORa = 2.47, 95% CI: 1.14-5.33, p = 0.022), while male was a risk factor for FBIs (ORa = 2.62, 95% CI: 1.16-5.94, p = 0.021). In dogs, hookworm, Toxocara spp., Spirometra mansoni, Trichuris vulpis and Hymenolepis diminuta were identified with prevalence rates of 35.6%, 8.1%, 8.1%, 1.2% and 1.2%, respectively. In cats, hookworm, Toxocara spp., S. mansoni, Strongyloides spp., Platynosomum fastosum, MIFs and H. diminuta were identified with prevalence rates of 50%, 17.9%, 10.7%, 7.1%, 3.6%, 3.6% and 3.6%, respectively. Bithynia snails showed 2% virgulate and 0.7% unknown cercariae infections, while among 19 cyprinid fish species, only unknown metacercariae were found. Our findings underscore the necessity of an integrated approach following the One Health concept to effectively combat these parasitic diseases while addressing human, animal and environmental health.

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

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. 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. 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.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Effectiveness of health education in improving knowledge, attitude and practice related to foodborne zoonotic trematodes in Vietnam, with a particular focus on Clonorchis sinensis.

Foodborne zoonotic trematodes (FZT), which infect the liver, lungs and intestines of humans, are an emerging public health concern in tropical countries including Vietnam. In northern Vietnam, Clonorchis sinensis is recognised as the most important species of the FZT. Up to now, small-scale studies conducted in the country have indicated that health education could improve participants' knowledge and practices related to clonorchiasis, however strong evidence is still lacking. We conducted an intervention trial between 2020 and 2021 in four communes in Yen Bai and Thanh Hoa province, aiming to evaluate the impact of an extended educational package on knowledge, attitude and practices related to clonorchiasis, as well as on knowledge on the large liver fluke and minute intestinal flukes. To this end, baseline and post-intervention questionnaires were conducted. Generalised estimating equations models were run to analyse the impact of the intervention on knowledge, attitudes and practices over time in the intervention versus control group. Finally, 172 people in the intervention group and 162 in the control group were included for analysis. Results demonstrated that the intervention significantly improved clonorchiasis knowledge, attitudes and practices, with higher odds for a correct knowledge response (odds ratio (OR) = 2.80, 95% confidence interval (CI) = 1.84-4.27, p-value < 0.001), increased average mean attitude score (mean = 0.363, 95%CI = 0.182-0.544, p-value < 0.001), and a reduced odds of consuming raw fish (OR = 0.15, 95%CI = 0.06-0.40, p = 0.002) in the intervention group compared to the control group post-intervention versus baseline. Additionally, participants in the intervention group exhibited enhanced basic knowledge of the large liver fluke and minute intestinal flukes post-intervention. These findings demonstrate the potential of health education programs in preventing clonorchiasis in endemic areas, emphasising the importance of continued health education as a critical component of integrated control programs for clonorchiasis.

An Approach for Egg Parasite Classification Based on Ensemble Deep Learning

Opisthorchis viverrini and minute intestinal fluke (MIF) infections are heavily epidemic in northeastern Thailand. Their primary cause is eating raw or undercooked cyprinid fishes, and they cause health problems in the human digestive system. In cases of liver fluke, these parasites can go through the bile duct system, which may cause cholangiocarcinoma (bile duct cancer). When a medical doctor suspects that a patient is infected with parasites, they typically request a stool analysis to determine the type of egg parasites using microscopy. Both parasites have similar characteristics, thus, it is necessary for a specialist to identify the specific type of egg parasites present. Many automatic systems have been developed using deep learning to assist doctors in diagnosing the type of egg parasite. In this study, we proposed three models of deep learning architectures and created voting ensembles to analyze egg parasite images. Images of similar liver fluke eggs and MIF eggs were taken from the Parasitology Laboratory, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. Image data augmentation is used to expand images from different perspectives and assist the system in acquiring a greater variety of images. Three models performed effectively, by employing the hard voting ensemble, the accuracy increased to 86.67%, while for the second group, the accuracies reached 68.00%, 76.00%, and 77.33%, respectively. Using the soft voting ensemble, the accuracy improved to 79.33%. These outcomes highlight the potential of ensemble deep learning in image classification. Furthermore, these results align closely with those achieved by several experts in image classification. Hence, a promising ensemble approach can aid doctors in accurately classifying images of egg parasites.

High endemicity of Opisthorchis viverrini infection among people in northern Cambodia confirmed by adult worm expulsion

Opisthorchis viverrini infection is an emerging disease in Cambodia, especially in central and southeastern areas. However, its status in northern areas bordering Lao PDR has been relatively unknown. The present study was performed to investigate the status of O. viverrini infection among people in Preah Vihear and Stung Treng provinces through fecal examination to detect eggs and recovery of adult flukes from some of the egg-positive cases. Fecal examinations were performed on a total of 1101 people from 10 villages in the 2 provinces using the Kato-Katz thick smear technique. For recovery of adult flukes and other helminth parasites 10 volunteers positive for eggs of Opisthorchis viverrini and/or minute intestinal flukes (Ov/MIF), in Kampong Sangkae village, Preah Vihear province, were administered a single oral dose of 40 mg/kg praziquantel plus 5–10 mg/kg of pyrantel pamoate and purged with 40–50 g magnesium salts. Adult trematodes, together with nematodes and cestodes expelled in diarrheic stools were collected under a stereomicroscope or with the naked eye. The proportion of egg-positive cases for overall liver and intestinal helminths was high but not notably different between the 2 provinces, 65.5% in Preah Vihear versus 64.7% in Stung Treng. The average proportion of Ov/MIF egg-positive cases was 59.8%. A total of 315 adult specimens of O. viverrini were recovered from the 10 volunteers (4–98 specimens per individual; mean, 32). A smaller number of Haplorchis taichui adults, an intestinal fluke, were found mixed-infected in 7 (103 specimens in total; 1–31 per individual; mean, 15) of the 10 volunteers. Adult specimens of hookworms, Enterobius vermicularis, Trichostrongylus sp., and a Taenia tapeworm strobila were recovered in some cases. Based on the results, it has been confirmed that the surveyed areas in Preah Vihear and Stung Treng provinces, Cambodia, are highly endemic areas of O. viverrini infection with a low-grade mixed infection with H. taichui.

Performance of Mini Parasep® SF stool concentrator kit, Kato-Katz, and formalin-ethyl acetate concentration methods for diagnosis of opisthorchiasis in Northeast Thailand

BackgroundControl and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections.MethodsA cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases.ResultsThe initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05).ConclusionsAs the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis.Graphical abstract

DETEKSI KEBERADAAN TELUR Soil Transmitted Helminth (STH) PADA KUKU PETANI

Soil-Transmitted Helminth (STH) is an intestinal nematode that requires soil for the process of reproduction and maturation from non-infective to infective. The nematodes included in this species include Ascaris lumbricoides, Trichuris trichiura, and two species of hookworm, Necator americanus, and Ancylostoma duodenale. Personal hygiene can affect the incidence of helminth infections in humans. The purpose of this study was to determine the relationship between personal hygiene and the presence of Soil-Transmitted Helminth (STH) eggs on farmers' nails. This research was conducted by analytical observation with a cross-sectional design in April-May 2021. The research method was filling out questionnaires and identifying eggs using the 10% KOH sedimentation method. Data were analyzed by univariate and Pearson Chi-Square tests. The results of this study showed that 4 fingernail samples (47.05%) were positive for Ascaris lumbricoides eggs and 2 fingernail samples (11.76%) were positive for Minute Intestinal Fluke, as many as 8 toenail samples (82.35%) were positive for Ascaris lumbricoides eggs. and 1 sample of toenails (5.88%) positive for Minute Intestinal Fluke eggs. There was no relationship between personal hygiene and the presence of STH eggs on farmers' nails (p = 0.05). STH eggs were found in the fingernails and toenails of farmers in Kaligondang District, Purbalingga Regency. Farmers need to improve personal hygiene to avoid helminth infections.

Comparative analysis of the characteristics of Haplorchis taichui mitochondrial genome with Metagonimus yokogawai and its ribosomal transcription unit with H. pumilio (family Heterophyidae)

Minute intestinal flukes, Haplorchis taichui and H. pumilio, belong to the family Heterophyidae (Trematoda: Platyhelminthes), which have been studied very limited, especially the molecular markers of the mitochondrial genomes (mtDNA) and the ribosome transcription units (rTU or rDNA). We have obtained the complete mitochondrial genome of H. taichui and the coding part of ribosome transcription unit of H. taichui and H. pumilio of Vietnam. Nucleotide and amino acid data were compared between H. taichui and Metagonimus yokogawai for genomic/gene composition, codon usage (skew/skewness), and tandem repeat units (TRU). The complete mtDNA of H. taichui (strain Htai-QT3-VN) with the length of 15,120 bp and M. yokogawai (15,258 bp; Korea; KC330755) contains 36 genes, including 12 protein-coding genes (cox1, cox2, cox3, nad1, nad2, nad3, nad4L, nad4, nad5, nad6, atp6 and cob), 2 ribosomal RNA genes (rRNA); 22 transfer RNA (tRNA or trn) and a noncoding region (NCR) between trnE and trnG, divided into 2 sub-regions containing 5 TRUs (182–183 bp/TRU). H. taichui (Vietnam and Laos) uses A = 19.56%, T = 39.71%, G = 28.34%, C = 12.39% (A + T is 59.27% ​​and G + C is 40.73%) for mtDNA construction, whose skew/skewness value at A+T is negative (–0,340) and G+C is positive (0.392); for 12 protein-coding genes (PCGs) is similar; but for the mito-ribosomal genes (MRGs, of 16S/rrnL and 12S/rrnS) it is less for A+T (57.22%) and more for G+C (42.78%). M. yokogawai had lower A+T (mtDNA/55.68%; PCGs/55.96%; MRGs/54.15%) and higher G+C usage rate than H. taichui. H. taichui of Vietnam and Laos has 10,164 bp encoding for 3,376 amino acids to construct 12 PCGs with the mostly used codons as Phenylalanine (Phe-TTT) and Leucine (Leu-TTG), and the leastly used codonsas Glutamine (Gln-CAA), Arginine (Arg-CGC). Additional condon, Thr-ACA/ACC can be added as the least used in M. yokogawai. The rTU (from 5 '18S to 3' 28S) of H. taichui (7,268 bp) and H. pumilio (7,416 bp) were identified with 5 genomic regions including 18S rDNA, ITS1, 5.8S rDNA, ITS2 and 28S rDNA. The 18S and 5.8S genes of both species were of the same length (1,992 bp for 18S, 160 bp for 5.8S), but different for 28S genes (3,875 bp for H. taichui and 3,870 bp for H. pumilio). ITS1 in H. taichui (797 bp) and ITS2 in H. pumilio (280 bp) do not contain TRUs, whilst ITS1 in H. pumilio (1,106 bp) contains 5 TRUs(136 bp for 3 TRU and 116 bp for 2 TRUs); and ITS2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each).

Phylogenetic analyses of ribosomal transcription units from Haplorchis taichui and H. pumilio species of Heterophyidae (Platyhelminthes: Opisthorchiata)

Heterophyidiasis caused by minute intestinal flukes becomes of public concern in many countries worldwide. Haplorchis taichui and H. pumilio, belonging to the family Heterophyidae (Platyhelminthes: Trematoda) are two of many infecting humans and commonly found in Vietnam. Sequence study of these two small intestinal flukes is still very limited, hence we need more prospective markers for taxonomic identification and classification. This study provides complete coding sequence of the ribosomal transcription units (rTU) from H. taichui and H. pumilio (Vietnamese samples) and demonstrates the use of complete 28S rDNA sequences for phylogenetic analysis.The complete coding sequence of the rTU (from 5' 18S to 3' 28S), consisting of complete 18S, ITS-1, 5.8S, ITS2 and complete 28S rRNA genes and spacers, from H. taichui (7,268 bp) and H. pumilio (7,416 bp) from human hosts in Vietnam, were determined and annotated. The 18S and 5.8S genes of both species were of the same length (1,992 bp/18S, 160 bp/5.8S), but 28S genes differed (3,875 bp/H. taichui and 3,870 bp/H. pumilio). ITS-1 in H. taichui (797 bp) and ITS-2 in H. pumilio (280 bp) do not contain tandem repeat units (TRUs), while ITS-1 in H. pumilio (1,106 bp) contains 3 TRUs of 136 bp/each and 2 TRUs of 116 bp/each and ITS-2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each). A phylogenetic tree inferred from the alignment of complete 28S rDNA sequences of 32 trematode strains/species, including 2 Vietnamese Haplorchis spp. and 24 species of 8 families in the suborders Xiphidiata (families Nanophyetidae, Paragonimidae, Collyriclidae), Opisthorchiata (Heterophyidae, Opisthorchiidae), and Echinostomata (Echinostomatidae, Fasciolidae). and Schistosoma japonicum of the family Schistosomatidae is used as an outgroup. The topology of the phylogenetic tree clearly confirmed the status of the Vietnamese H. taichui and H. pumilio species. These species gathered in a group (in the family Heterophyidae) clearly identified in the position of "sister” group to those in the family Opisthorchiidae (suborder Opisthorchiata, superfamily Opisthorchioidea).

Prevalence and intensity of helminths among inhabitants of the Chi River and Lahanna water reservoir areas of Northeastern Thailand.

Helminth infections (HIs) are an important public health problem in tropical countries, and the associated problems have been neglected in rural areas of Thailand. Therefore, this study reports the prevalence and intensity of HIs among inhabitants of the Khon Sawan district, Chaiyaphum province, and Kaeng Samnam Nang district, Nakhon Ratchasima province, which are located near the Chi River and Lahanna water reservoir, northeastern Thailand. A cross-sectional descriptive study was conducted between July 31, 2018, and June 30, 2019, among rural villagers from 40 rural villages in 4 subdistricts. The participants were selected from the village enrolment list after proportional allocation of the total sample size. Faecal samples from 691 inhabitants were prepared using solvent-free faecal parasite concentrator, and helminths were then detected using a light microscope. Statistical analysis included the Chi-square test with Yates correction, and multivariable logistic regression was performed. A P-value of <0.05 was considered statistically significant. The prevalence of HIs was 2.03%. The most prevalent helminths were Opisthorchis viverrini (1.31%), followed by Strongyloides stercoralis (0.44%), Ascaris lumbricoides (0.29%), hookworm (0.15%), Teania spp. (0.15%) and one minute intestinal fluke (0.15%). Coinfections were identified in 2 cases for S. stercoralis and hookworm and 1 case for O. viverrini and S. stercoralis infection. All infected participants had a light intensity of HI. There was no significant difference between general characteristics for all HIs. The prevalence of HIs was not significantly associated with general characteristics. This study indicates that the infections result mainly from foodborne helminths and skin-penetrating nematodes. Therefore, interventions should concentrate on the personal hygiene of the population and improving sanitation to reduce HIs in this area.

Evaluation of Rhophilin Associated Tail Protein (ROPN1L) in the Human Liver Fluke Opisthorchis viverrini for Diagnostic Approach.

Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power.

Genetic Differentiation of Opisthorchis-Like Eggs in Northern Thailand Using Stool Specimens Under National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma.

Liver fluke infection caused by Opisthorchis viverrini is recognized as a potential risk factor for cholangiocarcinoma (CCA). The National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma has implemented microscopic-based stool examination screening. However, eggs of O. viverrini and minute intestinal flukes (MIFs) are nearly morphologically similar and could result in inaccurate O. viverrini diagnosis. Stool specimens were collected from eight districts of Chiang Mai Province in northern Thailand. Opisthorchis-like eggs were identified with the Kato-Katz technique and differentiated for O. viverrini and MIFs using molecular study by PCR and PCR-restriction fragment length polymorphism targeting the internal transcribed spacer 2 (ITS2) gene. Prevalence of Opisthorchis-like eggs was 5.9% from a total of 9,570 specimens. From PCR assays, all liver flukes were O. viverrini and all MIFs were Haplorchis taichui. The distribution of species was H. taichui (38.2%), O. viverrini (10.5%), coinfection of H. taichui and O. viverrini (37.2%), and 14.1% were negative from PCR. Totally, H. taichui was found in 75.4% of infections from Opisthorchis-like specimens. ITS2 nucleotide sequencing analysis showed a single variant of O. viverrini with no variation and two variants of H. taichui. This study first revealed the genetic background of Opisthorchis-like eggs in northern Thailand. Minute intestinal flukes are occasionally misdiagnosed as O. viverrini leading to misinterpretation and overestimation of the burden of O. viverrini infection. Molecular diagnosis such as PCR could effectively discriminate species of Opisthorchis-like eggs and help shape the robustness of epidemiological data to control liver fluke infection and raise awareness of other risk factors for CCA.

Discovery of Carcinogenic Liver Fluke Metacercariae in Second Intermediate Hosts and Surveillance on Fish-Borne Trematode Metacercariae Infections in Mekong Region of Myanmar.

Countries of lower Mekong regions are highly alarmed by the spread of fish-borne trematode infections, i.e., small liver flukes and minute intestinal flukes especially in Thailand, Lao People’s Democratic Republic (Lao PDR), Vietnam, Cambodia and Myanmar. Moreover; the incidence of cholangiocarcinoma has also been increasing in the endemic area of liver fluke infections. Only a few reports have been published concerning the fish-borne trematodes infections in the central region of Myanmar. However; there is still a lack of information regarding the status of trematodes infections in second intermediate hosts in the Mekong region of Myanmar. Therefore, we conducted surveillance on the distribution of trematode metacercariae in small cyprinoid fishes collected from the Mekong region of Myanmar. A total of 689 fishes (12 different species of cyprinoid fishes) have been collected and examined by pepsin digestion methods. We discovered four species of fish-borne trematode metacercariae infections, i.e., carcinogenic liver fluke, Opisthorchis viverrini; minute intestinal flukes, Haplorchis taichui; Haplorchis pumilio and Haplorchoides sp. in Tachileik, the Mekong Region of Myanmar. The outcome of this study could be a useful index for the fish-borne zoonotic trematode epidemiology in the Mekong area. Besides, the results of our study contribute to filling the gap of information necessary for the control and prevention of fish-borne trematode zoonotic infections in the Mekong region.

Intestinal parasites in rural communities in Nan Province, Thailand: changes in bacterial gut microbiota associated with minute intestinal fluke infection.

Gastrointestinal helminth infection likely affects the gut microbiome, in turn affecting host health. To investigate the effect of intestinal parasite status on the gut microbiome, parasitic infection surveys were conducted in communities in Nan Province, Thailand. In total, 1047 participants submitted stool samples for intestinal parasite examination, and 391 parasite-positive cases were identified, equating to an infection prevalence of 37.3%. Intestinal protozoan species were less prevalent (4.6%) than helminth species. The most prevalent parasite was the minute intestinal fluke Haplorchis taichui (35.9%). Amplicon sequencing of 16S rRNA was conducted to investigate the gut microbiome profiles of H. taichui-infected participants compared with those of parasite-free participants. Prevotella copri was the dominant bacterial operational taxonomic unit (OTU) in the study population. The relative abundance of three bacterial taxa, Ruminococcus, Roseburia faecis and Veillonella parvula, was significantly increased in the H. taichui-infected group. Parasite-negative group had higher bacterial diversity (α diversity) than the H. taichui-positive group. In addition, a significant difference in bacterial community composition (β diversity) was found between the two groups. The results suggest that H. taichui infection impacts the gut microbiome profile by reducing bacterial diversity and altering bacterial community structure in the gastrointestinal tract.

Current status of Opisthorchis viverrini, minute intestinal fluke and Taenia spp. infections among inhabitants in the Kenethao district of northern Lao PDR

This study reports the prevalence of Ov/minute intestinal fluke (MIF) and Taenia infections among inhabitants of the Kenethao district, northern Lao PDR. Fecal samples from 580 inhabitants were examined using the formalin-ethyl acetate concentration technique. The prevalence of Ov/MIF, Taenia spp. and coinfection was 45.3, 11.9 and 6.1%, respectively. There was no significant difference between males and females for Ov/MIF (p=0.813) and Taenia infection (p=0.759). The prevalence of Ov/MIF was significantly associated with age (p=0.005), but not for Taenia infection (p=0.836). Consumption of raw fish (p=0.001) and raw meat (p=0.046) was significantly associated with parasitic infections. The results suggest that Ov/MIF and Taenia spp. are highly endemic in this area and there is a need for projects to eliminate these parasites.

Echinostoma aegyptica (Trematoda: Echinostomatidae) Infection in Five Riparian People in Savannakhet Province, Lao PDR

Human infection with Echinostoma aegyptica Khalil and Abaza, 1924 (Trematoda: Echinostomatidae) is extremely rare. In this study, we confirmed E. aegyptica infection in 5 riparian residents living along the Mekong River in Savannakhet Province, Lao PDR. The patients revealed eggs of Opisthorchis viverrini/minute intestinal flukes, echinostomes, and other parasites in fecal examinations using the Kato-Katz technique. Following treatment with praziquantel 30–40 mg/kg and pyrantel pamoate 10–15 mg/kg in a single dose and purging with magnesium salts, adult specimens of various helminth species were collected. Among the trematodes, echinostome flukes of 4.5–7.6 mm in length (n = 134; av. 22.3 specimens per case) were of taxonomic interest and subjected in this study. The flukes were morphologically characterized by having total 43–45 collar spines arranged in 2 alternating rows (corner spines usually 5 on each side) and compatible with previous descriptions of E. aegyptica. The patients were mixed-infected with other helminths, so specific clinical manifestations due to this echinostome fluke were difficult to determine. The present paper describes for the first time human E. aegyptica infections in Lao PDR. This is the second report of human infection (2nd–6th cases) with E. aegyptica in the world following the first one from China.

Rapid label-free analysis of Opisthorchis viverrinieggs in fecal specimens using confocal Raman spectroscopy.

Opisthorchis viverrini, a human liver fluke, is highly prevalent in Southeast Asia. Definitive diagnosis of infection is usually achieved parasitologically through the discovery of fluke eggs in feces. However, the eggs of O. viverrini are difficult to differentiate morphologically from those of other minute intestinal flukes in fecal samples, even for experienced technicians. The present study developed a label-free optical methodology for analysis of O. viverrini eggs using Raman spectroscopy. Raman features of O. viverrini eggs were reported that can be used as marker bands for the efficient analysis of O. viverrini eggs in fecal specimens. The methodology presented here allows for the rapid detection of O. viverrini egg infection and can be readily and practicably applied in any clinical setting, even those in which a trained parasitologist is not available.

Prevalence, Species Distribution, and Related Factors of Fish-Borne Trematode Infection in Ninh Binh Province, Vietnam.

Background. Clonorchis sinensis/Opisthorchis viverrini and minute intestinal flukes (MIF) such as Haplorchis pumilio and H. taichui are fish-borne trematodes (FBT) that may coexist in regions where local people have a habit of eating raw fish like Vietnam. Responses to FBT should be verified according to the data on the distribution of these flukes. This study aims to explore the prevalence of different species of FBT and related factors among local people in a northern province of Vietnam. Methods. A cross-sectional study was conducted in Kim Son and Yen Khanh districts, Ninh Binh province, between March 2016 and March 2017. Four hundred people aged 15 years or older were interviewed and gave stool samples. The FBT eggs in faecal samples were enumerated by modified formalin-ether technique and identified by sequencing of the second internal transcribed spacer (ITS2) region. Result. Among the 400 persons, 19.5% were infected with FBT. On univariate analysis, eating raw fish was the main risk factor (odds ratios (OR)) of 6.769 (95% confidence interval (CI) of 2.655–17.259) followed by being of male gender (3.994 (CI95% 2.117–7.536)) and drinking alcohol (2.680 (CI95% 1.440–4.986)), respectively. There was no risk of increased infection among those living at home without hygienic latrines, those living close to rivers or having ponds, or those raising cats or dogs. By multivariate analysis, FBT infection was only related to the consumption of raw fish and gender. Seventy stool samples with a sufficient amount of faecal matter were subjected to DNA extraction, 42.85% of them yielded DNA production, and all were of Clonorchis sinensis. Conclusion. Results of the study showed the high prevalence of infection of fish-borne trematode, mostly C. sinensis among humans in Ninh Binh province. The prevention of FBT should be strengthened with programs detailed according to the distribution of FBT in different endemic areas.