LETTERS infected by ingesting unwashed Vittier); Services Vétérinaires des Alpes- Adult Opisthorchis vegetables contaminated by animal Maritimes, Sophia-Antipolis, France (E. feces containing strongyloid larvae. Coulibaly); and Université de Nice–Sophia viverrini Flukes in Larvae mature into adults in the Antipolis–Inserm 0895, Nice (P. Marty) Humans, Takeo, intestines. DOI: 10.3201/eid1707.101519 Cambodia Sporadic cases of this infection in humans have been reported in References To the Editor: Opisthorchis many countries (7). In France, viverrini and Clonorchis sinensis, the several autochthonous cases were 1. Yong TS, Lee JH, Sim S, Lee J, Min DY, 2 major species of small liver fl ukes suspected, but because of their rarity Chai JY, et al. Differential diagnosis of (family Opisthorchiidae), cause and diffi culty in identifi cation, they Trichostrongylus and hookworm eggs chronic infl ammation in the bile duct, via PCR using ITS-1 sequence. Korean are not commonly reported (8). Eggs J Parasitol. 2007;45:69–74. doi:10.3347/ which leads to cholangitis and cirrhosis of Trichostrongylus spp. can be kjp.2007.45.1.69 of the liver, and are a predisposing differentiated from those of Necator 2. Sato M, Sanguankiat S, Yoonuan T, factor for cholangiocarcinoma (1). and Ancylostoma spp. because they are Pongvongsa T, Keomoungkhoun M, Human infections with O. viverrini Phimmayoi I, et al. Copro-molecular longer, narrower, and elongated. After identifi cation of infections with hook- fl ukes are found along riverside areas 6 days of culture, T. colubriformis worm eggs in rural Lao PDR. Trans R of Indochina (Thailand, Lao People’s nematodes can be distinguished from Soc Trop Med Hyg. 2010;104:617–22. Democratic Republic [PDR], and similar stages in Strongyloides and doi:10.1016/j.trstmh.2010.06.006 Vietnam) (2). 3. Hoste H, Chilton NB, Gasser RB, Bev- Ancylostoma spp. by the bead-like eridge I. Differences in the second inter- Small trematode eggs (length swelling at the tip of the tail. Except nal transcribed spacer (ribosomal DNA) 20–32 μm) have been found in human for isolation of adult worms, which between fi ve species of Trichostrongylus fecal samples in Cambodia (1,3,4). are rarely found in feces, sequencing (Nematoda: Trichostrongylidae). Int J Par- During 1981–1982, two of 102 asitol. 1995;25:75–80. doi:10.1016/0020- of the ITS2 region is the most accurate 7519(94)00085-3 Cambodian refugees in the United method for specifi c identifi cation of 4. Hoste H, Gasser RB, Chilton NB, Mal- States were found to be positive for C. Trichostrongylus spp. isolated from let S, Beveridge I. Lack of intraspecifi c sinensis (likely O. viverrini) eggs (3). humans. variation in the second internal transcribed Egg-positive cases were later detected spacer (ITS-2) of Trichostrongylus colu- This familial outbreak highlights briformis ribosomal DNA. Int J Parasitol. in several provinces of Cambodia increased risk for animal parasitosis in 1993;23:1069–71. doi:10.1016/0020- (4,5). Presence of O. viverrini fl ukes humans in an industrialized country, 7519(93)90128-L in Cambodia was verifi ed by detection which may have been caused by an 5. Ghadirian E. Human infection with of metacercariae in freshwater fi sh in a Trichostrongylus lerouxi (Biocca, Cha- increasing trend of persons using baud, and Ghadirian, 1974) in Iran. Am J lake on the border between Takeo and ecologic and organic farming methods. Trop Med Hyg. 1977;26:1212–3. Kandal Provinces and by isolation of These cases confi rm that hygienic 6. Boreham RE, McCowan MJ, Ryan adult fl ukes in experimentally infected recommendations for use of organic AE, Allworth AM, Robson JM. Human hamsters (6). trichostrongyliasis in Queensland. Pa- fertilizer must be disseminated on a thology. 1995;27:182–5. doi:10.1080/ In May 2010, we analyzed fecal large scale. It is also mandatory that 00313029500169842 samples from 1,993 persons in 3 fresh vegetables be washed carefully 7. Gutierrez Y, Guerrant RL, Walker DH, villages (Ang Svay Chek, Kaw Poang, and thoroughly before ingestion, and Weller PF. Other tissue nematode infec- and Trartang Ang) in the Prey Kabas tions. In: Guerrant RL, Walker DH, Weller only dried manure should be used as PF, editors. Tropical infectious diseases, District, Takeo Province, Cambodia, an organic fertilizer. principles, pathogens and practice. 2nd ≈45 km south of Phnom Penh, to ed. Oxford: Churchill Livingstone; 2006. confi rm the presence of O. viverrini Stéphanie Lattès, Hubert Ferté, p. 1231–47. fl ukes among humans. We found an Pascal Delaunay, 8. Thibert J-B, Guiguen C, Gangneux J-P. Human trichostrongyloidosis: case report egg-positive rate of 32.4% for small Jérôme Depaquit, and microscopic diffi culties to identify an- trematode eggs. Because these eggs Matteo Vassallo, Mélanie Vittier, kylostomidae eggs. Ann Biol Clin (Paris). may be those of Haplorchis spp. Sahare Kokcha, Eric Coulibaly, 2006;64:281–5. fl ukes (H. taichui, H. pumilio, and and Pierre Marty H. yokogawai) and lecithodendriid Address for correspondence: Pascal Delaunay, Author affi liations: Centre Hospitalier fl ukes (Prosthodendrium molenkampi Parasitologie–Mycologie, Centre Hospitalier Universitaire de Nice, Nice, France (S. and Phaneropsolus bonnei) (1), we Universitaire l’Archet, BP 3079, 06202 Nice Lattès, P. Delaunay, M. Vassallo, S. Kochka); attempted to detect adult fl ukes that Cedex 3, France; email: delaunay.p@chu-nice. Université de Reims Champagne–Ardenne, are responsible for these eggs. fr Reims, France (H. Ferté, J. Depaquit, M.

1302 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 7, July 2011 LETTERS

Six of the small trematode Five worms were lanceolate and had a and Stellantchasmus falcatus), and egg–positive villagers, 1 man and 5 mean length of 9.5 mm (range 6.5–12.0 lecithodendriids have been found in women (age range 16–72 years), who mm), a mean width of 1.5 mm (range humans (1,7,9). In several provinces had occasional epigastric discomfort 1.2–1.7 mm), and 2 characteristic in Lao PDR, mixed infections with were selected for anthelmintic 4–5-lobulated testes (Figure, panel O. viverrini and heterophyids or treatment, purgation, and recovery of A). Ten eggs in uteri were 27 μm long lecithodendriids were common (7,9), adult worms. Fecal examination and (range 25–29 μm) and 15 μm wide and the relative prevalence of each anthelmintic treatment of villagers were (range 13–16 μm). fl uke species varied by locality. In approved by the Ministry of Health, To detect the source of Vientiane, Lao PDR, O. viverrini was Cambodia, under the agreement with infection, 2 freshwater fi sh species, the predominant species, whereas the Korea–Cambodia International Puntioplites proctozysron (n = 5) in Saravane Province, H. taichui Collaboration on Intestinal Parasite and Cyclocheilichthys apagon (n = predominated (7). In a mountainous Control in Cambodia (2006–2011). 10), were caught in nearby Ang Svay area of Phongsaly Province, H. After obtaining informed consent, the Chek village and examined for O. taichui and H. yokogawai worms were villagers were treated with a single viverrini metacercariae by using a obtained from 10 villagers; however, oral dose of praziquantel, 40 mg/kg digestion technique (8). A total of 50 no O. viverrini worms were detected (Shinpoong Pharmaceutical Co., Seoul, metacercariae (Figure, panel B) were (10). Thus, in Cambodia, the presence South Korea), and given a purgament obtained from 5 P. proctozysron fi sh of human infections with intestinal

(solution containing 30–40 g MgSO4). and fed to a hamster. Six weeks later, fl ukes, including Haplorchis spp. and Feces was obtained 3 or 4 times in 42 young O. viverrini fl ukes (Figure, lecithodendriids, cannot be ruled out. a 2–3-hour period after purgation, panel C) were isolated from the biliary pooled individually, and processed as tract of the hamster. Acknowledgment described (7). Worms obtained were Our study identifi ed only O. We thank the staff of the Centre for fi xed with 10% formalin, stained viverrini infections in humans in Parasitology, Entomology, and Malaria with acetocarmine, and identifi ed by Cambodia. However, eggs of other Control, Phnom Penh, Cambodia, and morphologic features. hepatic and intestinal fl ukes also Dong-Min Lee, Yoon-Hee Lee, Ji-Hae A total of 34 O. viverrini adult can be found in humans (1). In Kang, Hyun-Ju Woo, and Jun-Yong worms were obtained from the 6 Thailand, Vietnam, and Lao PDR, Hong for assistance with Kato-Katz fecal villagers (14, 9, 5, 3, 2, and 1 from opisthorchiids (O. viverrini and C. examinations. each villager, respectively). No other sinensis), heterophyids (Haplorchis species of trematodes were obtained. spp., Centrocestus formosanus, Woon-Mok Sohn,1 Eun-Hee Shin,1 Tai-Soon Yong, Keeseon S. Eom, Hoo-Gn Jeong, Muth Sinuon, Duong Socheat, and Jong-Yil Chai Author affi liations: Gyeongsang National University School of Medicine, Jinju, South Korea (W.-M. Sohn); Seoul National University College of Medicine, Seoul, South Korea (E.-H. Shin, J.-Y. Chai); Seoul National University Bundang Hospital, Sungnam, South Korea (E.-H. Shin); Yonsei University College of Medicine, Seoul (T.- S. Yong); Chungbuk National University College of Medicine, Chongju, South Korea (K. S. Eom); Korea Association of Figure. A) Adult Opisthorchis viverrini liver fl uke (length 12.0 mm) isolated from a human after chemotherapy and purgation in Takeo Province, Cambodia, showing the Health Promotion, Seoul (H.-G. Jeong, characteristic morphology of the two 4–5-lobulated testes. B) Metacercaria of O. viverrini J.-Y. Chai); and Centre for Parasitology, fl uke (diameter 0.22 mm) detected in a freshwater fi sh (Puntioplites proctozysron). C) Entomology, and Malaria Control, Phnom Young adult O. viverrini fl uke (length 5.5 mm) isolated 6 weeks after experimental infection Penh, Cambodia (M. Sinuon, D. Socheat) of a hamster with metacercariae from P. proctozysron fi sh. Original magn ifi cation levels ×8 1 (A), ×120 (B), ×9 (C). A color version of this fi gure is available online (www.cdc.gov/EID/ These authors contributed equally to this content/17/7/1302-F.htm). article.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 7, July 2011 1303 LETTERS

DOI: 10.3201/eid1707.102071 Address for correspondence: Jong-Yil Chai, patients from India, 20 PKDL patients Department of Parasitology and Tropical from India, and 40 VL patients from References Medicine, Seoul National University College of Brazil. VL was confi rmed by fi nding Medicine, Seoul 110-799, South Korea; email: parasites in aspirates. Serum samples 1. Chai JY, Murrell KD, Lymbery AJ. Fish- [email protected] borne parasitic zoonoses: status and is- were collected before chemotherapy sues. Int J Parasitol. 2005;35:1233–54. was given. PKDL was diagnosed as doi:10.1016/j.ijpara.2005.07.013 described (3). Control samples were 2. Andrews RH, Sithithaworn P, Petney TN. collected from 24 healthy persons Opisthorchis viverrini: an underestimated parasite in world health. Trends Para- from non–disease-endemic areas sitol. 2008;24:497–501. doi:10.1016/j. in India; 15 healthy persons from pt.2008.08.011 disease-endemic areas in India; 20 3. Parish RA. Intestinal parasites in South- healthy persons from disease-endemic east Asian refugee children. West J Med. Easy Test for 1985;143:47–9. areas in Brazil; and 21 persons with 4. Stich AH, Biays S, Odermatt P, Men C, Visceral Hansen disease, 7 with fi lariasis, 4 Saem C, Sokha K, et al. Foci of Schis- Leishmaniasis with tuberculosis, 1 with lymphoma, tosoma mekongi, northern Cambodia: 1 with leukemia, 2 with virus-induced II. Distribution of infection and morbid- and Post–Kala-azar ity. Trop Med Int Health. 1999;4:674–85. fever, and 5 with malaria. Consent doi:10.1046/j.1365-3156.1999.00474.x Dermal was obtained from all human donors. 5. Lee KJ, Bae YT, Kim DH, Deung YK, Leishmaniasis This study was approved by Ethical Ryang YS, Kim HJ, et al. Status of intes- Committee on Human Subjects at tinal parasites infection among primary To the Editor: Diagnosis of school children in Kampongcham, Cam- Indian Institute of Chemical Biology bodia. Korean J Parasitol. 2002;40:153–5. visceral leishmaniasis (VL), fatal if and the Ethical Board for Human doi:10.3347/kjp.2002.40.3.153 untreated, is complex because the Subjects and Animal Experimentation 6. Touch S, Komalamisra C, Radomyos symptoms are the same for many of the Federal University of Piauí. P, Waikagul J. Discovery of Opisthor- fever-associated ailments. Despite chis viverrini metacercariae in fresh- We developed a diagnostic water fi sh in southern Cambodia. Acta limitations, diagnosis remains based ELISA with modifi cations of our Trop. 2009;111:108–13. doi:10.1016/j. on fi nding Leishmania amastigotes in previous method (6). Microtiter actatropica.2009.03.002 spleen and/or bone marrow aspirates plates were coated with 2.5 μg 7. Chai JY, Park JH, Han ET, Guk SM, (1). Sophisticated laboratory Shin EH, Lin A, et al. Mixed infections LAg at pH 7.5 (100 μL/well) and with Opisthorchis viverrini and intestinal methods, although sensitive, are kept at 4°C overnight, after which fl ukes in residents of Vientiane Munici- costly. The immunochromatographic they were blocked with 1% bovine pality and Saravane Province in Laos. J strip test that uses recombinant K39 serum albumin, dried, and stored at Helminthol. 2005;79:283–9. doi:10.1079/ antigen (rK39), although satisfactory JOH2005302 4°C as precoated plates. The assay 8. Rim HJ, Sohn WM, Yong TS, Eom KS, in India, is less sensitive in Africa, performed at room temperature Chai JY, Min DY, et al. Fishborne trema- Latin America, and Mediterranean took ≈2.5 h. Test and control serum tode metacercariae detected in freshwater regions (2). Post–kala-azar dermal samples (1:1,000 dilution, 100 μL/ fi sh from Vientiane Municipality and Sa- leishmaniasis (PKDL), a sequel vannakhet Province, Lao PDR. Korean J well) were applied to the plates for Parasitol. 2008;46:253–60. doi:10.3347/ to VL in India and Africa, is often 45 min and shaken occasionally. kjp.2008.46.4.253 confused with other skin diseases Horseradish peroxidase–conjugated 9. Chai JY, Han ET, Shin EH, Sohn WM, (3,4). Diagnosis of VL in dogs in goat anti-human immunoglobulin Yong TS, Eom KS, et al. High preva- Latin America and Mediterranean lence of Haplorchis taichui, Prosthoden- (Ig) G (Genei, Bangalore, India) was drium molenkampi, and other helminth countries remains confusing because applied at 1:5,000 (100 μL/well) for 45 infections among people in Kham- of rampant asymptomatic infections min. Color development with ortho- mouane Province, Lao PDR. Korean J and elevated antibodies against phenylenediamine (Sigma-Aldrich, Parasitol. 2009;47:243–7. doi:10.3347/ Leishmania spp. (5). kjp.2009.47.3.243 St. Louis, MO, USA) was allowed 10. Chai JY, Yong TS, Eom KS, Min DY, Shin Earlier we reported the diagnostic for 5–10 min. Positive results were EH, Banouvong V, et al. Prevalence of the potential of L. donovani (MHOM/ determined by comparing colors with intestinal fl ukes Haplorchis taichui and IN/83/AG83) promastigote membrane those on a card previously prepared H. yokogawai in a mountainous area of antigens (LAg) (3,6). Here we report Phongsaly Province, Lao PDR. Korean J for positive and negative wells. Parasitol. 2010;48:339–42. doi:10.3347/ applicability of LAg-based ELISA and ELISA, performed for the VL and kjp.2010.48.4.339 dipstick systems even at primary health PKDL patients from India, was 100% centers. Using randomized sampling, sensitive (percentage of patients we tested samples from 122 kala-azar with confi rmed disease and positive

1304 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 7, July 2011