Fishborne Zoonotic Intestinal Trematodes, Vietnam

These parasites are an unrecognized food safety risk in a population with a tradition of eating raw fish.

The exponential increase in aquaculture is suggested to be the major cause of the emergence of FZTs in east and Southeast Asia (2,3). For example, in the People's Republic of China, the land devoted to aquaculture increased 75% (to 4.9 million hectares) since 1970, accompanied by a tripling of cases of infection with C. sinensis (3). The association of O. viverrini in Thailand and Lao People's Democratic Republic with fi sheries has also been reported (9,10). However, wild fi sh are also frequently infected, but epidemiologic information to compare relative infection risks from eating wild fi sh and farmed fi sh in many FZTendemic loci is insuffi cient (1,4).
A recent review of publications on FZTs in Vietnam indicated infections with only liver fl ukes (C. sinensis and O. viverrini) in humans (11). However, recent Vietnamese surveys for zoonotic parasites in cultured and wild fi sh in northern and southern Vietnam identifi ed metacercarial stages of several zoonotic intestinal trematode species in fi sh (12,13). This fi nding is of concern because fi sh production has increased 9.3-fold (to 400,000 tons) over the past 40 years in Vietnam (3). Furthermore, human intestinal fl ukes are highly prevalent in neighboring countries such as Thailand (14), Lao People's Democratic Republic (15), and the People's Republic of China (16), which further raises the issue of whether human infections might be present in Vietnam but overlooked because of diagnostic diffi culties in differentiating liver and intestinal fl uke eggs in fecal examinations (11,17). A more reliable approach to detect and characterize human FZTs is to treat egg-positive patients and recover and identify the expelled adult worms (7,15).
We conducted a study in April 2005 in Nam Dinh Province, an area of Vietnam in which persons are known for eating raw fi sh, and where previous investigations have shown a high prevalence of liver fl ukes (11,13). Identifi cation of worms expelled from egg-positive persons showed that intestinal FZTs are present in Vietnam and represent a major public health risk for a population with the habit of eating raw fi sh.

Site, Sampling, and Examination Procedure
A cross-sectional survey for fecal eggs was conducted in 2 communes in Nghia Hung District, Nam Dinh Province, Vietnam, southeast of the capital of Hanoi ( Figure 2), a clonorchiasis-endemic area (11). The rural population in these communes is mostly farmers with fi sh ponds that are integrated into their farming systems, e.g., pig farming. In 2005, the Nghia Phu commune had a population of 9,608, including 2,214 families, and the Nghia Lac commune had a population of 9,147, including 2,160 families. Households in these 2 communes were randomly selected from a list provided by community authorities, and from each household 1 man or 1 woman who was head of household was selected. Trained personnel delivered labeled plastic containers to the selected persons and instructed them how to collect a fecal sample and store it until it was retrieved the next day. The label requested the person's name, age, address, and date of stool collection.
Permission to conduct this research was obtained from the National Institute of Malariology, Parasitology and Entomology (NIMPE), Hanoi, and the Faculty of Tropical Medicine, Mahidol University, Bangkok. Each study participant signed a consent form, which is on fi le at NIMPE.
From each stool sample, 2 Kato-Katz smears were prepared and analyzed by using the standard kit provided to NIMPE by the World Health Organization and originally obtained from Vestegaard Frandsen Pvt. Ltd. (New Delhi, India). Fecal slides were examined by light microscopy (×400). Helminth eggs were identifi ed and enumerated, and the number of eggs was multiplied by 23 to obtain the number of eggs per gram (epg) of feces.

Parasite Expulsion
Thirty-three persons who had >1,000 epg on fecal examinations were selected for worm expulsion. Selected patients were asked to eat a light liquid dinner the evening before treatment. The following morning they were given oral praziquantel, 25 mg/kg; 1 hour later, they were given a saturated solution of 30 g of MgSO 4 dissolved in water. Subsequently, 3-4 consecutive posttreatment stools were collected. Worms were recovered by a series of washing steps (15).  All persons who were positive for eggs were provided free drug treatment. Patients with nematode infections were given 1 dose of albendazole (400 mg) or mebendazole (500 mg), and patients with trematode infections were given praziquantel (25 mg/kg, 3× a day for 1 day).

Identifi cation of Adult Worms Recovered
Adult worms were identifi ed by direct light microscopy while still alive; those that needed further examination were fi xed in 10% formalin and stained with Semichon acetocarmine, mounted on a slide, and measured (18). Identifi cations were made by using published taxonomic references (19,20).

Data Analysis
Results of fecal examinations for helminth eggs were analyzed for prevalence and intensity of infection (epg), as measured by enumeration of eggs per gram of feces. Species infection rates (number of expelled worms) were descriptively analyzed by using SPSS version 11.0 software (SPSS Inc., Chicago, IL, USA) and χ 2 and Student t tests.

Parasite Diversity, Prevalence, and Intensity
A total of 615 persons, 563 men (91.5%) and 52 women (8.5%), were selected and submitted stool for examination. Fecal egg examinations showed that 554 persons (90.1%) were positive for helminth parasites (Table 1). Trichuris trichiura (whipworm) nematode eggs were found in 58.2% of the stool samples. A total of 64.9% were infected with small trematode eggs (<50 μm long), and 39.5% were infected with Ascaris lumbricoides (roundworm). Hookworm eggs (3.1%) and large (>50 μm long) trematode eggs (0.8%) were infrequently seen (Table 1). Multiparasitism was common in this community, with 65.1% of the persons expelling eggs having >2 species or types of eggs. Small trematode eggs, all <50 μm long, were presumed to be either those of C. sinensis or of intestinal trematodes of the family Heterophyidae. However, differentiation was not considered reliable by light microscopy, and selected persons were treated to expel their helminth parasites.
On the basis of egg count data, small trematode infection prevalence differed signifi cantly between men (68.7%) and women (23.1%) (χ 2 43.56, p<0.05). The infection rate for small trematode infection in men also differed signifi -cantly between age groups; it was signifi cantly higher for those >40 years of age (χ 2 7.95, p<0.05). In contrast, women did not show a signifi cant difference in infection rates between age groups (χ 2 0.85, p>0.05).
The prevalence of A. lumbricoides was 39.3% in men and 42.3% in women. There was a signifi cant increase in prevalence with age only in women (χ 2 6.4, p<0.05). Infection with T. trichiura infection did not differ signifi cantly by sex or age (p>0.05).

FZT Species Identifi cation
Trematodes responsible for releasing small eggs were identifi ed by using morphologic characterization of adult stages expelled from patients. A total 15,185 adult worms were collected from 33 patients. The number and prevalence of individual species of expelled trematodes are shown in Table 3. C. sinensis and 4 species of intestinal fi shborne zoonotic fl ukes were identifi ed ( Figure 3) Multiple infections with FZTs were common ( Figure  4): 54.5% of patients were infected with 2 trematode spe- cies, 33.3% with 3 species. A total of 9% were infected with only 1 species. One person (3%) was infected with 4 FZT species and F. buski.

Discussion
Our results demonstrate that zoonotic fi shborne intestinal trematodes are endemic in Vietnam. These trematodes represent, to our knowledge, a new and previously unrecognized public health problem. To our knowledge, in the many publications on human parasites originating in Vietnam since the 19th century colonial era, no reports on these intestinal fi shborne parasites have appeared (11). Whether this zoonosis is newly emerging in Vietnam because of changes in agriculture/aquaculture, demographics, social, or environmental changes or if it has been overlooked because of diagnostic problems is not known. However, snail vectors (e.g., Melanoides tuberculata) and suitable vertebrate intermediate (fi sh) and reservoir hosts (fi sh-eating birds, dogs, cats, pigs) for FZTs are common in this country (1,4,11,19,21). Furthermore, H. taichui, H. pumilio, H. yokogawai, and S. falcatus are endemic in neighboring countries such as Thailand (14), Lao People's Democratic Republic (10,15), and the People's Republic of China (16).
It is puzzling why zoonotic heterophyids have only recently been isolated from fi sh in Vietnam (12,13,22) if they are endemic. These parasites may have been recently introduced into this country and then became a zoonotic risk.
Intensifi cation of aquaculture, use of human and animal manure for pond fertilization, and increased consumption of fi sh because of increasing affl uence by a population with a tradition of eating raw fi sh may be contributing factors for infection. These issues need to be investigated if effective means for prevention of transmission are to be developed. Use of manure and waste water in aquaculture is a well-recognized risk factor for trematode infections in fi sh (2,12,13) and has been the focus of 2 hazard analysis, critical control point-based control projects (4). Major sources of infected fi sh responsible for trematode transmission to humans must be ascertained because FZT metacercariae have been found in both wild and farmed fi sh in Vietnam, as well as elsewhere in Asia (4,10,(12)(13)(14)(15)(16).
Public health and agricultural/fi shery agencies should consider intestinal and liver fl ukes as an FZT complex because they share most biologic features and are risk factors for human infection. Although intestinal fl ukes are less well characterized clinically than liver fl ukes, they are increasingly being recognized as a cause of intestine, heart, brain, and spinal cord abnormalities in humans (1,4,8,23).
The potential economic effect of FZTs on alleviation of poverty is also a concern. Aquaculture in Vietnam is a major economic activity in rural areas. During 2000-2006, Vietnam tripled the value of its export of fi sh, increasing its revenue to >3 billion US dollars. Domestic availability of farmed fi sh is also a way of increasing protein availability  to humans. Therefore, a newly recognized fi sh safety risk associated with aquaculture could have a serious constraint on market access (24), especially because consumer expectations and economic levels are increasing as predicted for Vietnam. These expectations can result in greater demand for safe fi sh by consumers, marketing agencies, and the tourist industry.
Our results showed a difference in infection rates of small trematodes between men (68.7%) and women (23.1%). Differences in liver infections with C. sinensis by sex of the patients are well known (1,4,6). Our results are similar to those of surveys conducted for this parasite in northern Vietnam (25,26). High infection rates for men in Vietnam are often associated with male-oriented social gatherings during which they consume raw or pickled fi sh, although this sex-related difference appears to be narrowing in some countries (4,9).
The relationship between prevalence and intensity of infection and age of the host is also a characteristic of FZT epidemiology (1,4). In our study, infection rates were higher for persons >40 years of age, a pattern believed to be caused by longer exposure and accumulation of parasites (4,9,11). However, the life span of intestinal fl ukes in humans is not well documented. Therefore, accumulation of worms as an explanation for age-related infection patterns is speculative. These behavioral factors in the epidemiology of FZT warrant greater collaboration between epidemiologists and anthropologists in designing approaches for mitigating risk in a population with great resistance to change in eating habits. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13