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.

Seroprevalence of Tissue and Luminal Helminths among Patients in Hanoi Medical University Hospital, Vietnam, 2018.

A serological survey was performed using ELISA to estimate the prevalence of tissue and luminal helminthic infections among hospital patients in Hanoi region, Vietnam. An overall seroprevalence of tissue and luminal helminthiases was 64.0% (95% CI 61.2-66.8) among 1,120 patients who visited Hanoi Medical University Hospital, Vietnam in 2018. The highest seroprevalence was observed against Toxocara spp. (59.0%), followed by Strongyloides stercoralis (46.3%), Gnathostoma spp. (25.5%), cysticercus (12.8%), Angiostrongylus cantonensis (10.5%), Fasciola spp. (11.1%), and Clonorchis sinensis (8.7%). Mono-infection by one species (11.1%) was lower than multiple infections (53.0%) (P<0.05). The seroprevalence in males (59.3%) was lower than in females (66.2%) but not statistically significant (P>0.05). Children (<15 years) revealed lower seroprevalence (34.0%) than adults (68.4%), and the age group 51-70 years revealed the highest seroprevalence (76.0%). Among the seropositive patients, eosinophilia (≥8.0%) was noted in 80.2%. The present results suggested active transmission of various tissue and luminal helminths among people in Hanoi, Vietnam.

Two Human Cases of Echinococcus ortleppi Infection in the Lung and Heart in Vietnam.

This is a report of 2 cases of human hydatidosis caused by Echinococcus ortleppi in Vietnam. The patients were a 12-year-old male (case 1) having a cyst of 10.0×9.0 cm size in the lung and a 50-year-old female with a 3.0×3.3 cm-sized cyst in the heart. Eosinophilia was 33.7% in the male and 45.8% in the female patient. C-reactive protein was increased to 16.5 mg/L in the male and 18.2 mg/L in the female. Both patients were positive for ELISA at OD=2.5 and 3.1, respectively. Echinococcus protoscolices were collected from the cysts by amniocentesis and surgery. The protoscolices were identified as E. ortleppi by morphology and analysis of mitochondrial NADH dehydrogenase 1 (nad1) gene sequence. Both patients were cured by surgical resection of the hydatid cyst combined with albendazole medication. The E. ortleppi infection in lung is the second report, and the other in the heart is the first in Vietnam.

Early Postnatal and Preschool-Age Infection by Fasciola spp.: Report of Five Cases from Vietnam and Worldwide Review.

Fascioliasis is reported in five Vietnamese children aged 4 years or younger. A 10-month-old girl child and a 12-month-old boy child are the youngest patients ever diagnosed. Eggs in stools suggested an infection occurred at 5-6 months and 7-8 months of age, respectively. DNA sequencing and egg size indicated this to be the first report of a verified Fasciola gigantica infection in so small children. No specific diagnosis could be obtained in two 3-year-old children detected in the acute phase. A big and gravid ectopic F. gigantica-like worm was surgically found in a 4-year-old boy presenting with peritonitis. A worldwide review showed only 38 past cases in preschool children. They included 3, 7, 12, and 16 cases of 1, 2, 3, and 4 years, respectively, with a faster infection increase in males from 2 years onward. Reports were from all continents, except Oceania, including severe complications and death. The causal agent, when specifically diagnosed, was always Fasciola hepatica. Analyses include detection in hospital, surveys, and family outbreaks; infection sources; disease phases; parasite burden; ectopic cases; symptom onset; eosinophilia; biochemical markers; and clinical complications. C-reactive protein, creatinine, and γ-glutamyl transferase are the most useful biomarkers. A serological test and a coprological analysis are recommended for so small children, in which typical symptoms may be overlooked. Treatment problems were described with many drugs, except triclabendazole. Triclabendazole should be considered the drug of choice for such small children. The possibility of a very early infection by Fasciola spp. should be henceforth considered.

Detection of four patients who were infected by Schistosoma haematobium in Vietnam.

INTRODUCTION: Schistosoma lives as a parasite in the portal vein causing intestinal lesions. It also lives in the liver, spleen, and the vein of the urinary bladder causing lesions in the urinary system. Angola is an endemic area of Schistosoma haematobium, which causes lesions in the urinary system, including the urinary bladder. In this study, we aimed to identify and classify the parasites that were collected from four patients from Angola, who currently live in Vietnam, by morphological and molecular methods. PATIENTS AND METHODS: The main clinical symptoms of the patients were collected, and Schistosoma eggs were taken from urine by a centrifugal method from the four patients in 2016. Identification of the species by morphological method was taken using a microscope. The DNA of the Schistosoma was also isolated and was identified by cytochrome C oxidase subunit 1 (Cox1) sequence. RESULTS: The four Vietnamese patients infected with S. haematobium in Angola returned to Vietnam. All the patients felt strange and had cystalgia and hematuria (blood urine), and one of them was diagnosed with urinary bladder cancer, where surgery was necessary for that patient. Schistosoma eggs, which were collected from the urine of the four patients, were identified as S. haematobium by morphological and molecular methods. These patients were the first reports of Schistosoma in Vietnam. CONCLUSION: Four Vietnamese schistosomiasis patients returned from Angola: three were diagnosed with schistosomiasis and one was diagnosed with urinary bladder cancer. They had similar symptoms including a strange feeling, cystalgia, hematuria, and eosinophilia and were detected with Schistosoma eggs in urine.

Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam.

The aim of this study is to delineate 'admixed hybrid' and 'introgressive' Fasciola genotypes present in the Fasciola population in Vietnam. Adult liver flukes collected from ruminants in 18 Provinces were morphologically sorted out by naked eyes for small (S), medium (M) and large (L) body shapes; and human samples (n=14) from patients. Nuclear ribosomal (rDNA) ITS1 and ITS2, and mitochondrial (mtDNA) nad1 markers were used for determination of their genetic status. Total 4,725 worm samples of ruminants were tentatively classified by their size: 6% (n=284) small (S)-, 13% (n=614) medium (M)-, and 81% (n=3,827) large (L)-forms. All the representative (n=120, as 40 each group) and 14 human specimens, possessed maternal mtDNA of only F. gigantica and none of F. hepatica. Paternally, all (100%) of the L-(n=40) and 77.5% (n=31) of the M-flukes had single F. gigantica rDNA indicating 'pure' F. gigantica. A majority (90%, n=36) of the S- and 15% (n=6) of the M-worms had single F. hepatica rDNA, indicating their introgressive; the rest (10%, n=4) of the S- and 7.5% (n=3) of the M-flukes had mixture of both F. gigantica and F. hepatica rDNAs, confirming their admixed hybrid genetic status. Fourteen human samples revealed 9 (64%) of pure F. gigantica, 3 (22%) of introgressive and 2 (14%) of admixed hybrid Fasciola spp. By the present study, it was confirmed that the small worms, which are morphologically identical with F. hepatica, are admixed and/or introgressive hybrids of Fasciola spp., and able to be the pathogens of human fascioliasis.

Prevalence of Soil-Transmitted Helminths and Molecular Clarification of Hookworm Species in Ethnic Ede Primary Schoolchildren in Dak Lak Province, Southern Vietnam.

To know the infection status of helminths in primary schoolchildren of southern parts of Vietnam, we performed an epidemiological study in Krong Pac district, Dak Lak Province, Vietnam. A total of 1,206 stool specimens were collected from ethnic Ede schoolchildren in 4 primary schools in 2015 and examined by the Kato-Katz technique. In addition, stool cultures were done by the Harada-Mori method to obtain hookworm larvae and then to clarify the species of hookworms infected. The results showed that the helminth infection rate was 25.0%, including 2.0% Ascaris lumbricoides, 0.33% Trichuris trichiura, and 22.8% hookworm infections. The average intensity of infection was 102.0 eggs per gram of feces (EPG) for Ascaris, 36.0 EPG for Trichuris, and 218.0 EPG for hookworms. ITS1 gene sequences of the hookworm larvae were identical with those of Necator americanus (100% homology) reported in GenBank. It has been confirmed in this study that the hookworm, N. americanus, is a dominant helminth species infected in primary schoolchildren of a southern part of Vietnam. Public health attention is needed for control of hookworm infections among schoolchildren in surveyed areas of Vietnam.

A Case of Ocular Angiostrongyliasis with Molecular Identification of the Species in Vietnam.

A 23-year-old female residing in a village of Cao Bang Province, North Vietnam, visited the Hospital of Hanoi Medical University in July 2013. She felt dim eyes and a bulge-sticking pain in her left eye for some days before visiting the hospital. In the hospital, a clinical examination, an eye endoscopy, and an operation were carried out. A nematode specimen was collected from the eye of this patient. The body of this worm was thin and long and measured 22.0×0.3 mm. It was morphologically suggested as an immature female worm of Angiostrongylus cantonensis. By a molecular method using 18S rRNA gene, this nematode was confirmed as A. cantonensis. This is the first molecular study for identification of A. cantonensis in Vietnam.

Foodborne trematode metacercariae in fish from northern Vietnam and their adults recovered from experimental hamsters.

The prevalence of foodborne trematode (FBT) metacercariae was investigated in fish from 2 localities of northern Vietnam in 2004-2005. Freshwater fish (9 species) were collected from local markets in Hanoi City (n=76) and Nam Dinh Province (n=79), and were examined for FBT metacercariae using the artificial digestion technique. Adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post-infection. Three (Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium) and 6 (Haplorchis taichui, H. pumilio, C. formosanus, P. varium, Stellantchasmus falcatus, and Heterophyopsis continua) species of FBT metacercariae were detected in the 2 regions, respectively. Overall, among the positive fish species, H. pumilio metacercariae were detected in 104 (80.0%) of 130 fish examined (metacercarial density per infected fish; 64.2). C. formosanus metacercariae were found in 37 (40.2%) of 92 fish (metacercarial density; 14.7). P. varium metacercariae were detected in 19 (63.3%) of 30 fish (Anabas testudineus and Mugil cephalus) (metacercarial density; 247.7). S. falcatus metacercariae were found in all 10 M. cephalus examined (metacercarial density; 84.4). H. continua metacercariae (2 in number) were detected in 1 fish of Coilia lindmani. Morphologic characteristics of the FBT metacercariae and their experimentally obtained adults were described. The results have demonstrated that various FBT species are prevalent in northen parts of Vietnam.

Dirofilaria repens in Vietnam: detection of 10 eye and subcutaneous tissue infection cases identified by morphology and molecular methods.

From 2006 to 2010, hospitals in Hanoi treated 10 human patients for dirofilariasis. The worms were collected from parasitic places, and identification of the species was completed by morphology and molecular methods. Ten parasites were recovered either from the conjunctiva (n=9) or subcutaneous tissue (n=1). The parasites were 4.0-12.5 cm in length and 0.5-0.6 mm in width. Morphological observations suggested all parasites as Dirofilaria repens. Three of the 10 parasites (1 from subcutaneous tissue and 2 from eyes) were used for molecular confirmation of the species identification. A portion of the mitochondrial cox1 (461 bp) was amplified and sequenced. Nucleotide and amino acid homologies were 95% and 99-100%, respectively, when compared with D. repens (Italian origin, GenBank AJ271614; DQ358814). This is the first report of eye dirofilariasis and the second report of subcutaneous tissue dirofilariasis due to D. repens in Vietnam.

Development and evaluation of a single-step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gigantica (family Fasciolidae, class Trematoda, phylum Platyhelminthes).

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis.

Human infections of fish-borne trematodes in Vietnam: prevalence and molecular specific identification at an endemic commune in Nam Dinh province.

The prevalence of fish-borne trematodes in humans and their molecular identification was investigated in the Rang Dong commune of Nam Dinh province, Vietnam, between January 2009 and December 2010. A total of 405 people in this commune were interviewed on the habit of eating raw fish and all of their stool samples were collected using the Kato-Katz technique for examination of the presence of fish-borne trematodes. The worms (and eggs) were first morphologically examined, counted, described and identified, then the representative isolates were subjected for molecular species confirmation. A total of 385 adult flukes collected from 10 patients were morphologically identified to species and defined as Clonorchis sinensis (14.58%) in Opisthorchiidae family, Haplorchis taichui (32.29%), Haplorchis pumilio (52.08%) and Centrocestus formosanus (1.04%) in Heterophyidae family. A high rate (77.8%) of the interviewees was found to have the habit of eating raw fish. This habit was attributed to the high infection rate of fish-borne trematode in humans (22.72%; OR=2.486). The infection rate of fish-borne trematodes in males was higher (29.3%) than that in females (16.0%) and increased by age, reaching the highest in the patients aged 40-59 years (28.2-28.7%). The infection intensity of fish-borne trematode was found light (336 EPG). Adult flukes were collected from a group of the patients with the highest intensity of infection and subjected to molecular and phylogenetic analysis using a portion (326 bp) of mitochondrial cox1. Phylogenetic tree inferred from cox1 sequences using sequence data for 34 isolates of opisthorchid, heterophyid, fasciolid, paragonimid, schistosomid trematodes and taeniid cestodes revealed that they are distinct groups. The newly collected with the known clonorchid and heterophyid isolates form the well defined taxonomic groups, respectively, confirming that C. sinensis and Haplorchis spp. (H. pumilio and H. taichui) were among the collected samples.

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam.

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.

Molecular confirmation that Fasciola gigantica can undertake aberrant migrations in human hosts.

Two cases of aberrant migration by the liver fluke Fasciola gigantica in humans are reported. In both cases, subadult worms emerged through the skin. The identity of the worms was confirmed from their DNA sequences. This uncommon human pathogen might be more likely than F. hepatica to undertake aberrant migrations in humans.

Cross-sectional parasitological survey for helminth infections among fish farmers in Nghe An province, Vietnam.

A total of 964 adult fish farmers in five eastern districts in Nghe An province, Vietnam were investigated in late 2004 for food-borne trematodes and other helminth infections using duplicate Kato-Katz thick smears prepared from single stool samples. Eggs of fish-borne trematodes and of Fasciolopsis buski were found in 0.6 and 0.7% of farmers, respectively. Infection prevalences with soil-transmitted helminths (STHs), namely Ascaris lumbricoides, Trichuris trichiura and hookworm, were 34.8, 50.7 and 51.3%, respectively, and 81.8% were infected with at least one of the three STHs. While A. lumbricoides and T. trichiura were more prevalent in the eastern districts compared to the more western districts, the opposite was true for hookworm infections. The widespread prevalence of STH infections in fish farmers suggests that control of these infections in school-age children only may be inadequate. Identification of the human behavioural factors and environmental features responsible for the distribution and frequency of STHs among adults is needed as well as a sensitive diagnostic test of fish-borne trematodes at species level.

Current status of soil-transmitted helminths in Vietnam.

This paper provides a review of surveys on soil-transmitted helminths that were done in Vietnam between 1990 and 2001. Prevalence estimates could be obtained for 29 of the 61 provinces. Extrapolating from this, it is estimated that 33.9 million people in Vietnam are infected with Ascaris (prevalence 44.4%), 17.6 million with Trichuris (prevalence 23.1%), and 21.8 million with hookworm (prevalence 28.6%). Prevalence of Ascaris and Trichuris showed a declining trend from the north to the south of the country. This is probably related to differences in climatic conditions. Hookworm prevalence is more evenly distributed throughout the country, but is concentrated in peri-urban and rural agricultural areas. Vegetable cultivation in which nightsoil is used as fertilizer is a risk factor for hookworm infection, especially among adult women. Helminth control programs should be targeted at school-age children in the northern provinces. Specific interventions are needed throughout the country for women of agricultural communities that are at risk for hookworm infection. There is clearly a need for more detailed analysis of risk factors to quantify the relative contribution of climatic, environmental, and human behavioral factors in the transmission of intestinal nematode infections in Vietnam.

The food-borne trematode zoonoses of Vietnam.

During the past couple of decades, numerous surveys for the occurrence and distribution of food-borne trematodes in Vietnam have been carried out. However, the majority of the data obtained have not been published in international journals, and therefore, the seriousness of these trematode parasitic zoonoses in the country has not been fully appreciated. To correct this, over 40 Vietnamese language reports and local publications were translated and organized as a status review for an international audience. The results show that such serious trematode zoonoses as clonorchiasis, opisthorchiasis, paragonimiasis and fascioliasis are common in many regions of Vietnam, and, in the case of fascioliasis and paragonimiasis, are increasing. Data on the species of intermediate hosts involved in the transmission of these zoonoses, and the effect of host sex and age on infection frequencies are presented, along with findings on food preference/behavior investigations. Finally, the authors present recommendations for further research to provide a more comprehensive picture of the status of these zoonoses, and to obtain the risk assessment information needed to design prevention and control programs.

Current status of cysticercosis in Vietnam.

This English review concerning the current status of cysticercosis in Vietnam has been compiled from various reports of studies conducted over the past 15 years, which have appeared in national publications in Vietnamese, in order to make the information available to the international community. Hospital surveys indicate that cysticercosis is emerging as a serious health problem in the country though most of the information comes from the Hanoi area. Many more men than women are being treated for cysticercosis with most patients being young to middle-aged adults though several juvenile cases have been seen in the south. Clinical manifestations of the disease in humans include subcutaneous nodules, epileptic seizures, severe headache, impaired vision and memory loss. Albendazole has been found to be the best drug for treating cysticercosis though it does not appear to be totally effective for curing cerebral cysts. Information concerning porcine and bovine cysticercosis is very limited and based mostly on passive surveillance at Hanoi slaughterhouses. Surveys for human taeniasis in central and northern provinces indicate a prevalence of 0.2-7.2%. However, techniques of low sensitivity were used and the results are inconclusive since it is unknown with which species of tapeworm the people were infected. In addition to Taenia solium which causes human cysticercosis, T. saginata and T. asiatica are also known to be present in Vietnam. Risk factors investigated thus far with regard to transmission of T. solium suggest that consumption of raw pork, inadequate or absent meat inspection and control, poor sanitation in some areas, and the use of untreated human waste as fertilizer for crops may play important roles in Vietnam but this remains to be validated. The evidence thus far collected suggests that a national surveillance program for cysticercosis is a great need for Vietnam. The authors recommend further research on the epidemiology and impact of cysticercosis in both human and pig hosts in order to determine whether a prevention and control program in Vietnam would be merited and cost effective.