LETTERS

Trichostrongylus NC1: 5′-ACGTCTGGTTCAGGGTT to reference levels 3 months later. GTT-3′ (forward) and NC2: 5′-TTAG Specifi c questioning of the 6 colubriformis TTTCTTTTCCTCCGCT-3′ (reverse) persons indicated that the source (3,4), which were used by Hoste et of infection most likely was a meal Infections in al. for spp. typing eaten in April 2009, which included (3). ITS2 rDNA was sequenced, and strawberries picked in the vegetable Humans, France third-stage larvae sequences were garden of the patient’s mother. The To the Editor: In April 2009, a registered in GenBank (accession nos. patient’s father and brother did not eat 47-year-old woman in Saint-Jeannet HQ174256 and HQ174257). any strawberries and did not have any in southern France reported stomach Complete (100%) homology symptoms. The garden was fertilized aches, abdominal bloating, and was obtained with known sequences yearly with dried manure from a local occasional diarrhea. Blood analyses (3,4) for adult Trichostrongylus sheep farm. Lack of dried manure in found an increased eosinophil level colubriformis from sheep 2009 led to use of fresh sheep manure (8,800 cells/mm3), which represented (GenBank accession nos. S69220, from the same farm. Sheep manure 52% of 16,900 leukocytes/mm3. X78063, and EF427624). Parasite from breeding stock on the farm was Parasitologic examinations for sequences also showed 100% examined. Trichostrongylus spp. helminths were conducted with 6 fecal homology with the main haplotype third-stage larvae were found despite specimens obtained during June 9– observed in humans in Laos (2). prophylactic treatment of sheep on the July 2, 2009. Analyses included direct If one considers the absence of farm against helminths. wet mount microscopic examination, intraspecifi c variability within T. T. colubriformis nematodes Merthiolate–iodine–formaldehyde colubriformis nematodes (3,4), the are mainly parasites of herbivorous concentration, formalin–ethyl acetate specimens isolated from the patient mammals and have a worldwide concentration, and Baermann larval and most likely from the other 5 distribution. Human infections are extraction. persons presumed to be affected in found predominantly in warm areas. Results of direct examination and this outbreak belong to this species. They are usually asymptomatic or the Baermann technique were negative The 6 symptomatic patients as described in the present case. for all samples. The formalin–ethyl were treated according to published T. colubriformis adults live in the acetate concentration technique recommendations (5) with intestines of the host (6). The female detected a parasite egg (Figure, panel , 400 mg/day for 10 days. lays eggs, which are excreted in feces. A) and fi rst-stage larvae. Fecal cultures Clinical remission was obtained in <3 Eggs then hatch and mature into grew mature third-stage larvae (length days, and eosinophil counts returned infectious larvae. Humans become 700–800 μm, 16 intestinal cells, length of the sheath <40 μm), belonging to the genus Trichostrongylus (Figure, panel B). Because of the ambiguous morphologic features of this genus, a molecular approach was necessary for specifi c identifi cation (1,2). Identical symptoms developed in 2 children of the patient and in 2 friends. The mother of the patient had additional symptoms (weight loss 5 kg in 1 month and 35,000 eosinophils/ mm3, which represented 85% of 43,200 leukocytes/mm3). However, results of fecal examinations were negative for these 5 persons. DNA was extracted separately from 2 third-stage larvae (Figure, Figure. Trichostrongylus colubriformis nematode isolated from feces of a 47-year-old woman, panel B) by using the DNA Tissue Mini France. A) Egg (length 89 μm) isolated by using direct examination (original magnifi cation Kit (QIAGEN, Hilden, Germany). To ×200). B) Third-stage larvae (length 740 μm, 16 intestinal cells, length of distal part of the amplify internal transcribed spacer 2 sheath <40 μm) isolated by using fecal culture (original magnifi cation ×50). A color version (ITS2) sequences, we used primers of this fi gu re is available online (www.cdc.gov/EID/content/17/7/1301-F.htm).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 7, July 2011 1301 LETTERS infected by ingesting unwashed Vittier); Services Vétérinaires des Alpes- Adult Opisthorchis vegetables contaminated by 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 , 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 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: ). 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