ISSN (Print) ISSN (Online) CASE REPORT Korean J Parasitol Vol. 50, No. 4: , December

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1 ISSN (Print) ISSN (Online) CASE REPORT Korean J Parasitol Vol. 50, No. 4: , December An Outbreak of Trichinosis with Molecular Identification of Trichinella sp. in Vietnam Nguyen Van De, *, Nguyen Vu Trung 2, Nguyen Hong Ha 2, Vu Thi Nga 3, Nguyen Minh Ha 2, Pham Thanh Thuy 4, Le Van Duyet 2 and Jong-Yil Chai 5 Department of Parasitology, Hanoi Medical University, Vietnam; 2 National Hospital of Tropical Diseases, Hanoi, Vietnam; 3 National Centre for Veterinary Diagnosis, Hanoi, Vietnam; 4 Bach Mai Hospital, Hanoi, Vietnam; 5 Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul 0-799, Korea Abstract: The 5th outbreak of trichinosis occurred in a mountainous area of North Vietnam in 202, involving 24 patients among 27 people who consumed raw pork together. Six of these patients visited several hospitals in Hanoi for treatment. Similar clinical symptoms appeared in these patients within 5-8 days after eating infected raw pork, which consisted of fever, muscle pain, difficult moving, edema, difficult swallowing, and difficult breathing. ELISA revealed all (6/6) positive reactions against antigen and all cases showed positive biopsy results for Trichinella sp. larvae in the muscle. The larvae detected in the patients were identified as T. spiralis (Vietnamese strain) by the molecular analysis of the mitochondrial cytochrome c oxidase subunit III (cox3) gene. Key words:, muscle pain, pork, fever, ELISA, Vietnam INTRODUCTION Received 9 June 202, revised August 202, accepted 6 August 202. * Corresponding author (ngvdeyhn@gmail.com) 202, Korean Society for Parasitology and Tropical Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. adult worms parasitize the intestine and their larvae encyst in muscles of humans and animals [-4]. In Asia, Trichinella spp. infection has been documented in humans in 8 countries, in domestic animals (mainly pigs) in 9 countries, and in wildlife in 4 countries [5,6]. Molecular identification of Trichinella papuae was reported in Thailand [3] and T. spiralis in Korea [7], but no reports have been available in Vietnam. In Vietnam, the first outbreak of trichinosis was reported in 970 in a mountainous area of Yen Bai province with 26 patients by eating raw pork in a wedding-party and 4 of them died [2]. The second outbreak occurred in 200 in a mountainous commune in Dien Bien province with 22 patients who ate raw pork in a wedding-party also and 2 of them died [2]. The third outbreak was also in Dien Bien province in 2004 with 20 patients by eating raw pork in a funeral party [2]. The fourth outbreak occurred in a mountainous area of Son La province in 2008 [8]. In this outbreak, 22 patients were involved after eating raw pork in a wedding-party from pig together, and 2 of them died. The fifth outbreak occurred in February 202 in a mountainous area of Thanh Hoa province involving 24 patients who ate raw pork in a lunar New Year party (6 of them are reported in this paper) (Fig. ). However, additional reports are needed to provide better understanding of the molecular and genetic characteristics of the Vietnamese Trichinella species. CASE RECORD In February 202, 24 of 27 people, who ate raw pork together in a lunar Year party in Muong Lat district, Thanh Hoa province, Vietnam, were proved as trichinosis patients by ELISA test using T. spiralis antigen. They all revealed similar clinical symptoms, i.e., fever and muscle pain, and were treated with albendazole 800 mg/day for 0 days. Among them, 6 patients visited hospitals in Hanoi, Vietnam, and their clinical histories were obtained as follows. The 6 patients included 3 men and 3 women between 30 and 43 years of age. The clinical symptoms included fever in 6 of 6 patients, muscular pain in 6 of 6 patients, difficult moving in 6 of 6 patients, edema in the leg/hand in 6 of 6 patients, edema around the eyes in 6 of 6 patients, difficult swallowing in 6 of 339

2 340 Korean J Parasitol Vol. 50, No. 4: , December 202 Table. The clinical symptoms in 6 trichinosis patients Endemic areas of trichinosis:. Yen Bai province 2. Dien Bien province 3. Son La province 4. Thanh Hoa province Symptoms Gender Man Man Woman Woman Man Woman Age Fever Muscular pain Edema in leg/hand Edema around eyes Difficult moving Difficult swallowing Difficult breath Loss of weight Itching Diarrhea Time of symptom appearance (days) Table 2. Clinical laboratory findings in 6 trichinosis patients Findings Fig.. Map of the endemic areas of trichinosis in Vietnam. 6 patients, loss of weight in 5 of 6 patients, difficulty in breathing in 5 of 6 patients, itching in 3 of 6 patients, and diarrhea in of 6 patients (Table ). Laboratory findings in these patients included eosinophilia in 5 of 6 patients from 8.5% to 5.8%; increased transaminase for AST in 6 of 6 patients in the range U/L (normal values 37 U/L in man and 3 U/L in woman) and for ALT in 6 of 6 patients in the range U/L (normal values 40 U/L in man and 3 U/L in woman). Positive ELISA test to T. spiralis antigen was found at 30 days after eating raw pork in 6 of 6 patients, and T. spiralis larvae were detected by muscule biopsy in 6 of 6 patients. Leptospira test and bacterial culture were negative in 6 of 6 patients (Table 2). The 6 patients were treated with albendazole 800 mg/ day for 0 days, and the symptoms disappeared after month. Molecular studies The larvae from the muscle of patients (Fig. 2A, B) were identified by molecular methods using the mitochondrial cytochrome c oxidase subunit III (cox3) gene in comparison with that of the standard strains in GenBank (Table 3). Comparison Erythrocyte/mm Leukocyte/mm Eosinophils (%) AST (U/L) ALT (U/L) Serum ELISA test for Trichinella Biopsy on Trichinella larvae Leptospira test Bacterial culture of 200 nucleotides of a portion of cox3 mitochondrial genome between the Vietnamese (ATGX-VN) and other geographical isolates of T. spiralis, including the Chinese (Tspi, Tspi2, Tspi3, and Tspi4), US (Tspi5), Spanish (Tspi6), Bulgarian (Tspi7), Finnish (Tspi8), and GenBank (Tspi9) showed that the nucleotide homology between the Vietnamese and Chinese isolates were 00% and that between the Vietnamese and other isolates were 99% (Table 4). A phylogenetic tree was constructed for T. spiralis Vietnam and other isolates using cox3 nucleotide sequences as estimated by neighbor-joining (NJ) using MEGA4.0 [9]; Vietnamese T. spiralis was group together with T. spiralis in the world (Fig. 3). DISCUSSION All 5 trichinosis outbreaks were in the mountainous region

De et al.: Trichinosis outbreak and molecular identification of Trichinella sp. in Vietnam 34 A B Fig. 2. A larva in the human muscle (A) and section of a T. spiralis larva in the muscle (B). Table 3.

Bulgaria Finland GenBank Host Human Mephitis mephitis Length Species GenBank no. GU33948. GU33947. GU33946. GU33945. GU33942. GU33939. GU33935. GU33934. GU38634. Author De et al.a Webb et al.

3 De et al.: Trichinosis outbreak and molecular identification of Trichinella sp. in Vietnam 34 A B Fig. 2. A larva in the human muscle (A) and section of a T. spiralis larva in the muscle (B). Table 3. Sequencing of a portion of cox3 of different Trichinella isolates from GenBank compared with in Vietnam Notation Origin ATGX-VN Tspi Tspi2 Tspi3 Tspi4 Tspi5 Tspi6 Tspi7 Tspi8 Tspi9 Vietnama USA Spain Bulgaria Finland GenBank Host Human Mephitis mephitis Length Species GenBank no. GU GU GU GU GU GU GU GU GU Author De et al.a Webb et al., 200 Results of this study. a of North Vietnam (Fig. ). The local people have the habit of feeding pigs outside (free-roaming) and eating raw pork, particularly at parties. This habit in Vietnam is similar in Thailand, Laos, India, and other parts of Asia and the South Pacific [4]. The second outbreak occurred 3 year after the first outbreak in Vietnam [2]. It is questioned why there were no other cases of trichinosis for such a long duration. Sometimes, parasitic diseases such as trichinosis may be neglected or misdiagnosed as other diseases by local health authorities due to lack of knowledge. Actually, all the trichinosis patients in 5 outbreaks of Viet nam were misdiagnosed as leptospirosis at first (because this disease caused clinical symptoms, such as fever, illness, and muscle pain), and treated with antibiotics but not anthelmintic drugs. Only after some patients died, they made contact with the national health level and diagnosed as trichinosis. Moreover, trichinosis can be clinically mild and can resemble other diseases, because it does not have pathognomonic signs or symptoms, and in endemic areas, if people frequently eat Trichinella-infected meat, they can develop an asymptomatic form of the disease [5]. Hence, the first step for reasonable diagnosis of diseases is thinking every reasons, including parasites. Therefore, the diagnostic clue to differentiate trichinosis from leptospirosis for future outbreaks will become better, especially in the mountainous areas. The main clinical symptoms in our 6 patients were fever, muscle pain, difficult moving, edema, difficult swallowing, and others which were similar in each patient (Table ). Comparing with review of 98 trichinosis patients in 3 outbreaks in Viet nam [2], the clinical symptoms were similar in all patients and the symptoms developed within -30 days after eating infected raw pork (7.9 days in average). The symptoms included fever (00% of patients), muscle pain (00%), difficult moving (90.9-

4 342 Korean J Parasitol Vol. 50, No. 4: , December 202 Table 4. Percentage identity in nucleotide sequences of cox3 of a Vietnamese isolate in comparison with other T. spiralis in GenBank ATGX-VN Tspi Tspi2 Tspi3 Tspi4 Tspi5 Tspi6 Tspi7 Tspi8 Tspi9 ATGX-VN Tspi Tspi Tspi Tspi Tspi Tspi Tspi Tspi Tspi ATGX-VN is Vietnamese ; Tspi, Tspi2, Tspi3, and Tspi4 are Chinese isolates (GenBank no. GU33948., GU33947., GU33946., GU relatively); Tspi5, Tspi6, Tpsi7, Tspi8, and Tspi9 are isolates from USA, Spain, Bulgaria, Finland, and GenBank (GeneBank no. GU33942., GU33939., GU33935., GU33934., and GU38634.), respectively. cox3 Asuu (X54253) Tnin (DQ58085) Tspi8 (GU33934) Tspi9 (GU38634) Tspi7 (GU33935) Tspi5 (GU33942) Tspi6 (GU33939) ATGX-VN Tspi (GU33948) Tspi2 (GU33947) Tspi3 (GU33946) Tspi4 (GU33945) Name (AJ4779) Tpap (AY85286) Teju (EU887092) T. spiralis Drep- (AJ2764) Drep-2 (DQ35884) Fig. 3. Phylogenetic tree of Vietnam and other strains from a part of cox3 nucleotide sequences estimated by Neighbor-Joining (NJ) method using MEGA4.0 [6]. Note: ATGX- VN=Vietnamese; Tspi, Tspi2, Tspi3, Tpsi4=Chinese; Tspi5= US; Tspi6 =Spanish; Tspi7 =Bulgarian; Tspi8 =Finnish; Tspi9 = GenBank; Name=Necator americanus (GenBank no. AJ4779); Tpap=Trichinella papuae (GenBank no. AY85286); Teju=Troglosiro cf. juberthiei (GenBank no. EU887092); Tnin= Troglosiro ninqua (GenBank no. DQ58085); Asuu=Ascaris suum (GenBank no. X54253); Drep- and Drep-2= Italian Dirofilaria repens (Gen- Bank no. AJ2764 and DQ35884). 00%), edema ( %), difficult swallowing ( %), weight loss ( %), itching ( %), difficulty in breathing ( %), diarrhea (50.0%), lisping ( %), abdominal pain ( %), and stool with blood (0-9.%), and 6 patients died. In an outbreak, serum ELISA test was performed in 2 patients using T. spiralis antigen, which showed 00% positivity [2]. In our report, the biopsy for detection of larvae in the muscles of patients showed 00% positive results for Trichinella larvae (Fig. 2A, B). The results of ELISA test with T. spiralis antigen were positive in 6 of 6 patients and 8 other patients in this outbreak. The number of leukocytes, especially eosinophils, increased remarkably. Transaminases increased also in 6 of 6 patients. However, the results of Leptospira test and bacterial culture were negative. In every outbreak, patients ate raw pork from domestic pigs, and the pigs were infected with T. spiralis larvae. For example, in the first outbreak, a pig used for eating was a female pig 8 years old and weighed 50 kg and was infected with 879 larvae/g muscle [2]. Another male pig in this area was 7 years old, which was infected with 70 larvae/g muscle. In the second outbreak, the patients ate raw pork from a female pig, which was 5 years old and weighed 70 kg and was infected with encysted larvae of T. spiralis. In the third outbreak, the patients consumed raw pork from a female pig, which was 3 years old and weighed 60 kg. In the fourth outbreak, the patients ate raw pork from a female pig, which was 5 years old and weighed 70 kg and was infected by -3 larvae/g muscle [2,8]. In the fourth outbreak, 206 (9.9%) of,035 domestic pigs (free-roaming) were positive for T. spiralis ELISA. Muscle samples from 76 serologically positive pigs were tested by artificial digestion, and Trichinella larvae were detected in (4.5%) of them [8]. Appearance of clinical symptoms in our cases was not longer (6.0 days in average) than in cases of previous outbreaks (7.9 days in average). In our study, the larvae from humans were identified by molecular methods as T. spiralis. This is the first time when T. spiralis from human infections was identified by the molecular method in Vietnam.

5 De et al.: Trichinosis outbreak and molecular identification of Trichinella sp. in Vietnam 343 ACKNOWLEDGMENTS We acknowledge the funds supported from the National Foundation for Science and Technology Development (NAF- OSTED) in Vietnam (No to Nguyen Van De), National Hospital of Tropical Diseases, Bach Mai Hospital and National Centre for Veterinary Diagnosis, Hanoi, Vietnam. REFERENCES. Cook GC. Trichinosis (). Manson s Tropical Diseases. London, UK. W.B. Saunder Co. Ltd p De NV, Dorny P, Waikagul J. Trichinelliasis in Vietnam. Seminar on Food- and Water-borne Parasitic Zoonoses (5th FBPZ), November Intapan PM, Chotmongkol V, Tantrawatpan C, Sanpool O, Morakote N, Maleewong W. Molecular identification of Trichinella papuae from a Thai patient with imported trichinellosis. Am J Trop Med Hyg 20; 84: Miyazaki I. Trichinelliasis. Helminthic Zoonoses. Tokyo, Japan. Southeast Asian Medical Information Center. 99. p Owen IL, Morales G, Pezzotti MA, Pozio E. Trichinella infection in a hunting population of Papua New Guinea suggests an ancient relationship of Trichinella with human beings. Trans R Trop Med Hyg 2005; 99: Pozio E. World distribution of Trichinella spp. infections in animals and humans. Vet Parasitol 2007; 49: Sohn WM, Huh S, Chung DI, Pozio E. Molecular identification of Korean Trichinella isolates. Korean J Parasitol 2003; 4: Vu Thi N, Dorny P, La Rosa G, To Long T, Nguyen Van C, Pozio E. High prevalence of anti-trichinella IgG in domestic pigs of the Son La province, Vietnam. Vet Parasitol 200; 68: Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2007; 0:

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