Human Taeniasis and Cysticercosis and Related Factors in Phu Tho Province, Northern Vietnam.

Several factors presumed to facilitate the transmission of Taenia spp. were reported in Vietnam. We conducted a cross-sectional study taking questionnaires from 1,185 participants, and collecting 1,151 sera and 1,036 stool samples in northern Vietnam. Sera were examined for circulating antigens of Taenia solium cysticerci using ELISA, stools for Taenia eggs by Kato-Katz smear, and copro-antigens by ELISA. Ag-ELISA revealed 4.6% antigen positivity, indicating infection with viable cysticerci. Taenia eggs were detected in 1.5% of participants. Copro-antigens were found in 2.8% of participants. Eating raw meat and/or vegetables was significantly associated with the presence of copro-antigen (OR=8.6, 95% CI: 1.16-63.9, P=0.01). Considering the high taeniasis prevalence and the associated threat, public health attention should be given to treat the tapeworm carriers in the projected areas.

A multidisciplinary analysis of over 53,000 fascioliasis patients along the 1995-2019 countrywide spread in Vietnam defines a new epidemiological baseline for One Health approaches.

Fascioliasis, only foodborne trematodiasis of worldwide distribution, is caused by Fasciola hepatica and F. gigantica, liver flukes transmitted by freshwater snails. Southern and southeastern Asia is an emerging hot spot of F. gigantica, despite its hitherto less involvement in human infection. In Vietnam, increasing cases have been reported since 1995, whereas only sixteen throughout 1800-1994. A database was created to include epidemiological data of fascioliasis patients from the 63 Vietnam provinces throughout 1995-2019. Case profiles were based on serology, symptoms, eosinophilia, imaging techniques, stool egg finding, and post-specific-treatment recovery. Radio broadcasting about symptoms and costless diagnosis/treatment led patients to hospitals after symptom onset. Yearly case numbers were modelled and spatio-temporally analyzed. Missing data and confounders were assessed. The countrywide spread has no precedent. It started in the central coast, including 53,109 patients, mostly adults and females. Seasonality, linked to vegetable consumption, peaks in June, although the intensity of this peak differs according to relief/climatic zones. Incidence data and logistic regression curves are obtained for the first time in human fascioliasis. Fasciolid hybrids accompanying the spreading F. gigantica flukes, and climate change assessed by risk index correlations, are both ruled out as outbreak causes. Human-guided movements of livestock from an original area prove to be the way used by fasciolids and lymnaeid vectors to expand geographically. Radix viridis, a highly efficient transmitting and colonizing vector, played a decisive role in the spread. The use of irrigated crop fields, widely inhabited by R. viridis, for livestock grazing facilitated the transmission and spread of the disease. General physician awareness and diagnostic capacity improvement proved the successful impact of such knowledge transfer in facilitating and increasing patient infection detection. Information, education and communication to the public by radio broadcasting demonstrated to be very helpful. Fasciola gigantica is able to cause epidemic and endemic situations similar to F. hepatica. The magnitude of the human outbreak in Vietnam is a health wake-up call for southern and southeastern countries of Asia which present the highest human population densities with increasing food demands, uncontrolled livestock inter-country exchange, foreign import practices, and monsoon's increasing climate change impact.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis.

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

Evaluation of a facility-based inspection tool to assess lymphedema management services in Vietnam.

Assuring availability of services for patients with lymphedema is required for countries to be validated as having achieved elimination of lymphatic filariasis (LF). A direct inspection protocol (DIP) tool, designed to measure the readiness to provide quality lymphedema management services, has recently been developed. The DIP tool includes 14 indicators across six quality themes: trained staff, case management and education materials, water infrastructure, medicines and commodities, patient tracking system, and staff knowledge. We evaluated the use of the tool in Vietnam, where data were needed to inform validation efforts. To apply the tool in Vietnam, we compiled a list of 219 commune health stations (CHS) with known lymphedema patients and conducted a cross-sectional survey in 32 CHS; including 24 in Red River Delta region, 2 in the North Central region, and 6 in the South Central Coast region. The mean facility score, calculated by assigning 1 point per indicator, was 8.8 of 14 points (63%, range 4[29%]-13[93%]). Percentage of surveyed facilities with staff trained in last two years was 0%; availability of lymphedema management guidelines (56%); availability of information, education, and communication materials (16%); reliable improved water infrastructure (94%); availability of antiseptics (81%), antifungals (44%), analgesics or anti-inflammatories (97%), antibiotics (94%); supplies for lymphedema and acute attack management (100%); lymphedema patients recorded in last 12 months (9%); staff knowledge about lymphedema signs/symptoms (63%), lymphedema management strategies (72%), signs/symptoms of acute attacks (81%), and acute attack management strategies (75%). The tool allowed standardized assessment of readiness to provide quality services. Lack of trained health staff, limited patient tracking, and depletion of education materials were identified as challenges and addressed by the national program. Survey data were included in the validation dossier, providing evidence necessary for WHO to validate Vietnam as having eliminated lymphatic filariasis in 2018.

Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker.

This study aimed to discriminate infections of two common fish-borne trematodes in Thailand, Opisthorchis viverrini from Haplorchis taichui, based on mitochondrial cytochrome c oxidase subunit I (COI) gene. Designed primers (COI-OV-Hap F&R primers) amplified partial COI fragments of O. viverrini and H. taichui with high sensitivity in different developmental stages (adult, metacercaria, and egg). Polymerase chain reaction (PCR) amplicons were generated with low genomic DNA concentration ( approximately 10(-4)ng) of O. viverrini and H. taichui at 50 and 56 degrees C annealing temperatures, respectively. At 50 degrees C, COI fragments of Clonorchis sinensis and H. taichui were also obtained, but this was less sensitive than O. viverrini. At 56 degrees C, only H. taichui could be amplified and discriminated from H. pumilio, H. yogokawai, O. viverrini, and C. sinensis. Between 50 and 56 degrees C, the PCR amplicons of H. pumilio and H. yogokawai were amplified with low specificity and low sensitivity. The genetic characters among O. viverrini, C. sinensis, and H. taichui were distinguished by PCR-RFLP method. The PCR-RFLP profiles might be useful for diagnosing mixed O. viverrini and H. taichui infections in endemic areas, and for detecting metacercariae of O. viverrini, C. sinensis and H. taichui in epidemiological surveys of infections in fish hosts.

The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam.

BACKGROUND: Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis. RESULTS: The near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies. CONCLUSIONS: The 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon (Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.

Risk factors for Clonorchis sinensis infection transmission in humans in northern Vietnam: A descriptive and social network analysis study.

Clonorchis sinensis is major fish-borne trematode, endemic in North Vietnam. Risk factors described so far include individual eating behaviors and environmental factors. Here, additional to conventional risk factors, we report on socially influenced liver fluke transmission in endemic communities. A cross-sectional study on risk factors and fish sharing networks was conducted in 4 villages of Gia Thinh Commune, Ninh Binh Province. A total of 510 residents in 272 households were recruited for risk factor analysis while 220 households, 28 fishermen and 10 fish-sellers were enrolled for social network study. Fecal examination for C. sinensis eggs was performed. Average C. sinensis infection rate at Gia Thinh commune was 16.5% (range 2% to 34.4%). Higher infection rates were significantly associated with males, lower educational levels, eating raw fish, and location of the villages. Social network analysis (SNA) showed a strong positive correlation between ego network size (number of households in fish sharing network) and quantity of raw fish consumed (r=0.603, P<0.05). The infection rate in people who ate raw-fish caught from a nearby river was significantly higher than those who consumed fish taken from farmed ponds (P<0.05). The amount of raw-fish meal consumed per resident/year was significantly higher in villages that had a strong network of sharing raw-fish food (P<0.001). This study reports for the first time on fish-food sharing among neighbors, proximity to water bodies, frequency of eating raw fish from natural water bodies and low education were key risk factors in C. sinensis infection transmission in northern Vietnam.

A complete mitochondrial genome from Echinochasmus japonicus supports the elevation of Echinochasminae Odhner, 1910 to family rank (Trematoda: Platyhelminthes).

The complete mitochondrial (mt) genome of the trematode Echinochasmus japonicus Tanabe, 1926 was fully determined and annotated. The circular mt molecule of this species is 15,865bp in length, containing 12 protein-coding genes (arranged in the following order: cox3-cob-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-cox2-nad6-nad5), two ribosomal RNA genes (rrnL and rrnS) and 22 transfer RNA genes (trnH; trnQ; trnF; trnM; trnV; trnA; trnD; trnN; trnP; trnI; trnK; trnS1(AGN); trnW; trnT; trnC; trnL1(CUN); trnS2(UCN); trnL2(UUN); trnG; and trnE). The atp8 gene is absent. The 3' end of nad4L overlaps the 5' end of nad4 by 40bp. An array of eight identical tandem repeats (240bp each) was found between trnG and trnE in the long non-coding region of the individual worm sequenced. Numbers of these repeats varied among E. japonicus samples. Phylogenetic analysis of concatenated mt protein sequences of 40 trematode species/strains supports the elevation of Echinochasminae Odhner, 1910 to family rank, close to the families Echinostomatidae and Fasciolidae. As echinochasmid and echinostomatid species can parasitize humans, the future characterization of additional mt genomes is needed for development of mt markers for identification and phylogenetic, population, epidemiological and hybridization studies.

Microbial contamination along the main open wastewater and storm water channel of Hanoi, Vietnam, and potential health risks for urban farmers.

The use of wastewater in agriculture and aquaculture has a long tradition throughout Asia. For example, in Hanoi, it creates important livelihood opportunities for >500,000 farmers in peri-urban communities. Discharge of domestic effluents pollute the water streams with potential pathogenic organisms posing a public health threat to farmers and consumers of wastewater-fed foodstuff. We determined the effectiveness of Hanoi's wastewater conveyance system, placing particular emphasis on the quality of wastewater used in agriculture and aquaculture. Between April and June 2014, a total of 216 water samples were obtained from 24 sampling points and the concentrations of total coliforms (TC), Escherichia coli, Salmonella spp. and helminth eggs determined. Despite applied wastewater treatment, agricultural field irrigation water was heavily contaminated with TC (1.3×10(7)colony forming unit (CFU)/100mL), E. coli (1.1×10(6)CFU/100mL) and Salmonella spp. (108 most probable number (MPN)/100mL). These values are 110-fold above Vietnamese discharge limits for restricted agriculture and 260-fold above the World Health Organization (WHO)'s tolerable safety limits for unrestricted agriculture. Mean helminth egg concentrations were below WHO tolerable levels in all study systems (<1egg/L). Hence, elevated levels of bacterial contamination, but not helminth infections, pose a major health risk for farmers and consumers of wastewater fed-products. We propose a set of control measures that might protect the health of exposed population groups without compromising current urban farming activities. This study presents an important example for sanitation safety planning in a rapidly expanding Asian city and can guide public and private entities working towards Sustainable Development Goal target 6.3, that is to improve water quality by reducing pollution, halving the proportion of untreated wastewater and increasing recycling and safe reuse globally.

Current status of fish-borne zoonotic trematode infections in Gia Vien district, Ninh Binh province, Vietnam.

BACKGROUND: Ninh Binh province is known as an endemic area of fish-borne zoonotic trematode (FZT) transmission in Vietnam. A cross-sectional study was conducted in Gia Minh and Gia Thinh communes of Gia Vien district, Ninh Binh province to investigate the infections with different stages of FZT in various host species. METHODS: Faecal samples from 1,857 humans were examined for trematode eggs using the Kato-Katz method, while faecal samples from 104 dogs, 100 cats, and 100 pigs were examined using the Formalin-ethyl acetate concentration technique (FECT). A total of 483 specimens of freshwater fish, representing 9 species, were examined for metacercariae using the artificial digestion method. Three methods of cercarial detection (shedding, crushing and cutting) were applied for examination of 3,972 specimens of freshwater snails, representing 7 species. All relevant data e.g. location, sex, age group, animal species, and habitat were recorded for statistical analyses. RESULTS: Helminth eggs were found in 65.5% of the human faecal samples, including 20.5% of faecal samples containing small trematode eggs. Infection with small trematodes differed among communes, age groups and sexes. Eggs of small trematodes were found in 32.7% of faecal samples from dogs, 49.0% from cats and 13.0% from pigs. The difference in prevalences and intensities were significant among species of animals but did not differ between the two communes. All fish species were infected with FZT, with an average prevalence of 56.1% and a mean intensity of 33.245 metacercariae per gram. Prevalence and intensity in fish differed significantly among cummunes and fish groups. Six species of zoonotic trematodes were identified. Metacercariae of the small liver fluke, Clonorchis sinensis, was only found in Hemiculter leucisculus. A total of 9 specimens from two snail species, Melanoides tuberculata and Bithynia fuchsiana, were infected with trematodes and four cercarial types were detected in the study sites. CONCLUSIONS: We conclude that Gia Minh and Gia Thinh communes are continuing to be hot-spot endemic areas of FZT and other helminths infections where the habit of eating raw fish by the local people is still present.

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP.

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.

Human paragonimiasis in Viet Nam: epidemiological survey and identification of the responsible species by DNA sequencing of eggs in patients' sputum.

Parasitological and sero-epidemiological surveys for human paragonimiasis were conducted in three provinces of Viet Nam. A total of 590 participants from two known endemic areas of human paragonimiasis (Sinho district of Laichau province and Lucyen district of Yenbai province) and from Dakrong district of Quangtri province where we recently found crab hosts heavily infected with Paragonimus westermani metacercariae. By multiple dot-ELISA screening, 28 (12.7%) out of 220 participants in Sinho district of Laichau province and 4 (3.3%) out of 120 participants in Lucyen district of Yenbai province were proven to be antibody-positive against the Paragonimus antigen. None of the 250 sera of the residents in Dakrong, Quangtri province, gave sero-positivity. Among a total of 32 sero-positive patients Paragonimus eggs were found in 6 cases. ITS2 sequences were successfully determined from a single Paragonimus egg from each patient. The results of homology search by BLAST and alignment clearly confirmed that Paragonimus eggs collected from 6 patients were all of Paragonimus heterotremus. The pathogenicity of P. westermani for human paragonimiasis in Viet Nam is still questionable and needs to be explored in the future.

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region.

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.