Parasitology International 62 (2013) 268–271
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Case report
First identification of eggs of the Asian fish tapeworm Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) in human stool Hélène Yera a,⁎, Roman Kuchta b, Jan Brabec b, François Peyron c, Jean Dupouy-Camet a a b c
Université Paris Descartes, Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Service de Parasitologie-Mycologie, 27 rue du Fbrg St Jacques, 75014, Paris, France Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Department of Helminthology, Branišovská 31, 370 05 České Budějovice, Czech Republic Hospices Civils de Lyon, Hôpital de la Croix Rousse, Service de Parasitologie et Pathologie Exotique, F-69317, Lyon, France
a r t i c l e
i n f o
Article history: Received 16 November 2012 Received in revised form 10 February 2013 Accepted 11 February 2013 Available online 17 February 2013 Keywords: COI Diphyllobothrium French Guiana Man Molecular identification rDNA
a b s t r a c t We report the first case of egg isolation of the Asian fish tapeworm Bothriocephalus acheilognathi (Bothriocephalidea) from human stool. A male patient from Saint Laurent du Maroni (French Guiana) presenting abdominal pain was examined in France for the diagnosis of intestinal parasites. Diphyllobothrium-like eggs were observed in his stool. However, molecular phylogenetic analyses based on sequences of rDNA and COI genes showed that the eggs observed belong to a bothriocephalidean cestode B. acheilognathi. The adult life stages of B. acheilognathi cestodes are known as invasive parasites of a wide spectrum of fish; however, they have not been described to parasitize any mammals. This human infection seems to be accidental and represents a parasite passage through human intestine after the consumption of an infected fish host. © 2013 Published by Elsevier Ireland Ltd.
1. Introduction
2. Material and methods
Tapeworms have been recognized as human parasites since antiquity and remain a cause for concern in many endemic world regions [1]. Only two of the 17 recognized tapeworm orders involve parasites that have an impact on human health [2,3]. Cyclophyllidea, the most evolutionary derived order, accommodate around 30 species of several genera known to ordinarily infect humans (e.g. Taenia, Echinococcus, Hymenolepis, Dipylidium). The order Diphyllobothriidea includes up to 23 human infecting species of either so-called broad fish tapeworms (Diphyllobothrium, Diplogonoporus) or sparganum (Spirometra). From the two orders, only the following four species are globally distributed and responsible for the absolute majority of human intestinal infections: beef tapeworm Taenia saginata, pork tapeworm Taenia solium, dwarf tapeworm Hymenolepis nana, and broad fish tapeworm Diphyllobothrium latum. Tapeworms from the remaining orders have never been reported from man, with the exception of three cases of “pseudo-parasitic” infections apparently caused by a passage of plerocercoid larvae of cestodes of the order Trypanorhyncha [4,5]. In this paper, we report the first evidence of a bothriocephalidean cestode egg presence in human stool and also describe its identification based on coprological (morphological) and molecular phylogenetic techniques.
2.1. Patient and parasite isolation A 32 year old male, who lived in Saint Laurent du Maroni (French Guiana), was on holiday in Lyons (France) for two weeks in April 2009. He visited the International Travel Vaccination Centre — tropical medicine department to receive a yellow fever vaccination. As he complained of abdominal pain persisting several weeks, his stool was examined for the presence of parasites. Eggs resembling either diphyllobothriidean or bothriocephalidean cestode eggs were found (Fig. 1) and Diphyllobothrium was identified based on these morphological criteria as the causative agent. The patient was treated with a dose of niclosamide after which the abdominal pain disappeared and the stool control was negative. No other recorded sign of parasitic infection was found, except for a blood hypereosinophilia (2000/mm3) in 2007. Blood cell counts performed in April and June 2009 were normal. The patient described that he regularly ate raw freshwater and brackish fish such as aymara (Hoplias aimara), tiger fish (Pseudoplatystoma sp.), acoupa (Plagioscion squamosissimus), or various machoiron catfish (e.g. Sciades proops) in Saint Laurent du Maroni. 2.2. Parasite molecular identification
⁎ Corresponding author. E-mail address:
[email protected] (H. Yera). 1383-5769/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.parint.2013.02.001
The patient stool was sent in the Parasitology Department of Cochin Hospital in Paris in order to exactly identify the Diphyllobothrium
H. Yera et al. / Parasitology International 62 (2013) 268–271
Fig. 1. Eggs of bothriocephalidean cestodes from human stools (×400).
species of the eggs with molecular methods. The eggs were concentrated from the stool using Para-Selles/Kop-Color kit, Fumouze (France), then washed three times in physiological solution. After 3 cycles of freezing at −80 °C and defreezing at 90 °C, the DNA was extracted and used for PCR amplification followed by sequencing nuclear ribosomal small and large subunit rDNA (ssrDNA and lsrDNA), internal transcribed spacers 1 and 2 (ITS1 and ITS2), and mitochondrial cytochrome oxidase subunit 1 gene (COI), as previously described [6,7]. Sequences were submitted to GenBank (accession numbers HM367066, HM367067 and HM439384). BLAST analyses (http://www.ncbi.nlm.nih.gov/Blast.cgi) were performed. Phylogenetic analyses were carried out in the Institute of Parasitology in České Budějovice, Czech Republic under maximum likelihood criteria [8] using all genes characterized. First, ssrDNA data have been analyzed together with representatives of each of the known 17 tapeworm groups. Next, more detailed analyses were run on lsrDNA data to reliably pinpoint the phylogenetic affiliation of the eggs. 3. Results The eggs obtained from the patient stools (Fig. 1), were oval, 47–52 μm long, 33–36 μm wide, unembryonated, bearing an operculum on one end. As such, they appeared to be smaller than those of Diphyllobothrium human parasites such as D. dendriticum (52–76 by 38–52 μm), D. latum (54–76 by 35–57 μm), D. nihonkaiense (53–59 by 35–40 μm), or D. pacificum (49–63 by 33–45 μm) [9,10]. According to its size, these eggs would most closely resemble the eggs of either the marine D. pacificum reported from South America [11] or the recently resurrected D. arctocephalinum (41–56 by 37–44 μm) [12]. Each PCR amplification of the eggs' DNA produced a single amplicon and no multiple signals were observed from the sequences' chromatograms. BLAST searches based on ssrDNA, lsrDNA and ITS1 + ITS2 found bothriocephalidean cestodes of the genus Bothriocephalus instead of Diphyllobothrium as the closest match. Phylogenetic analysis of the ssrDNA data set confirmed the affiliation of the eggs to a derived lineage of bothriocephalidean cestodes with its closest relative being Bothriocephalus claviceps (data not shown). However, complete ssrDNA data of bothriocephalidean cestodes are still relatively scarce and do not suit lower level phylogenetic studies as some other markers. For this reason, D1–D3 region of lsrDNA was phylogenetically analyzed allowing a greater diversity of bothriocephalidean cestode sequences from GenBank to be included. Here our sample clearly formed a monophyletic and strongly statistically supported lineage together with other Bothriocephalus acheilognathi tapeworms within a group of bothriocephalidean cestodes from Africa [13,14] (Fig. 2).
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Also the results of other analyses support the identification of the eggs as B. acheilognathi. BLAST search of the entire ITS1 + ITS2 sequence revealed a high sequence similarity of our sample (~95%) with a group of B. acheilognathi cestodes (GenBank accession nos. AF362419–24) compared to the pairwise sequence similarity to a distinct closest species of Bothriocephalus (~74%, AF362434). However, running a phylogenetic analyses on the ITS data was not feasible thanks to the enormous variability of both ITS1 and ITS2 sequences and the impossibility to align them unambiguously. Alignment of the only available B. acheilognathi ssrDNA sequence (AY340106) with our ssrDNA then found only 2 base pairs (bp) difference out of 459 bp long sequence of this V4 variable ssrDNA region. The partial sequence of the COI gene of our B. acheilognathi specimen (HM439384) represents the first B. acheilognathi COI sequence characterized to be available for future studies and a rare case of bothriocephalidean COI sequence (the only other bothriocephalidean COI sequences deposited in GenBank to this date are the two from Anchistrocephalus and Abothrium, JQ268539 and JQ280884). If phylogenetic analysis based on COI is carried out with at least one representative of each of the 17 recognized clades of tapeworms, our sequence would create a sister lineage of the bothriocephalidean taxa Anchistrocephalus and Abothrium. This molecular identification was in concordance with the morphology of the eggs as contrary to Diphyllobothrium, the eggs of B. acheilognathi are ovoid in shape and measure approximately 42–62 by 22–40 μm [15]. Variation in the egg size has been shown to change depending on the time of the year as also on the host size and species [16]. 4. Discussion The eggs observed in the patient stool were initially identified as those of Diphyllobothrium sp. based on morphological criteria. They could be identified as the eggs of the marine D. pacificum reported from South America, usually from the Pacific coast of Chile, Ecuador and Peru with few cases from Argentina [11], but never from tropical part such as French Guiana. French Guiana is close to Brazil, where D. latum and Diphyllobothrium sp. cases have been described in humans after consumption of raw fish in sushi and sashimi or freshwater fish [11] and Diphyllobothrium sp. has been identified in cetaceans off the northeastern coast [17]. However, species identification has never been confirmed by molecular methods [11]. Moreover, our patient regularly ate raw freshwater and brackish fish such as aymara, tiger fish, acoupa, or various machoiron catfish in Saint Laurent du Maroni, that have not been described as intermediate hosts of Diphyllobothrium [11,18]. Together, parasite morphology, geographical origin and the mode of contamination were uncharacteristic for D. pacificum, thus molecular methods were used to confirm the taxonomic identification. The members of the genus Bothriocephalus are parasites of freshwater and marine fish, with about 100 nominal species [19]. One of the most common species is the Asian fish tapeworm, B. acheilognathi Yamaguti, 1934, a widely distributed parasite of freshwater fish. B. acheilognathi is not only unique in its extraordinary wide geographical distribution, but also in the extremely wide host spectrum that includes more than 200 species of freshwater fish. This tapeworm is an important pathogen of cultured and feral fish, especially carp fry [13]. It is believed to have disseminated around the world due to an uncontrolled movement of carp, guppies and other fish from Asia to six continents, including isolated oceanic islands and subterranean sinkholes (cenotes) during the last six decades [13]. Nevertheless, B. acheilognathi has been rarely described in South America. There are just two reports in Brazil both from cultured hosts including the common carp (Cyprinus carpio). Adult worms were occasionally reported also from amphibians, reptiles and birds, but these infections seem to be accidental (i.e. parasite passage after the consumption of an infected fish host) [13]. B. acheilognathi has a two-host life cycle. Fish are the final host to the parasite and get infected by eating a parasitized copepod, the intermediate host. Depending on water temperature, the life cycle may be completed between about one
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H. Yera et al. / Parasitology International 62 (2013) 268–271
Bothriocephalus acheilognathi JQ811834 Bothriocephalus acheilognathi JQ811839 D1–D3 lsrDNA 55 HUMAN SAMPLE HM367067 Ichthybothrium ichthybori JQ811837 100 Kirstenella gordoni JQ811838 48 85 76 Polyonchobothium polypteri JQ811836 Tetracampos ciliotheca JQ811835 96 Senga sp. JQ811840 45 Bothriocephalus claviceps DQ925323 31 Anantrum tortum AF286941 Bothriocephalus scorpii AF286942 100 Ptychobothrium belones DQ925333 18 Parabothriocephaloides segmentatus DQ925330 88 46 44 Paraechinophallus japonicus DQ925331 0.02 Echinophallus wageneri DQ925329 100 Anchistrocephalus microcephalus AF286946 64 Anonchocephalus chilensis DQ925320 88 Milanella familiaris DQ925319 99 Bathycestus brayi DQ925322 Eubothrium crassum AF286947 100 Philobythoides stunkardi DQ925332 Triaenophorus crassus DQ925334 (OUTGROUP) 49 Marsipometra hastata AY584867 (OUTGROUP) 67 Bathybothrium rectangulum DQ925321 (OUTGROUP) Abothrium gadi AF286945 (OUTGROUP) 76 100
Fig. 2. Maximum likelihood phylogenetic tree based on the analysis of D1–D3 lsrDNA sequences with the Bothriocephalus acheilognathi eggs isolated from human stool marked in uppercase bold letters. GenBank accession nos. are given behind the terminal node names. The tree was constructed using the GTR+Γ model of nucleotide evolution in the program RAxML v.7.2.8, nodal labels show bootstrap nodal support calculated from 100 repetitions in RAxML.
and three months. The adult worm lives in the intestine of fish and it releases eggs into the water with the fish's feces. A larva called coracidium hatches from the egg, leaving through an operculum. The larva is eaten by the copepod, and penetrates the gut where it develops into a procercoid stage. The life cycle is completed when fish ingest infected copepods. The larva attaches to the intestinal wall of a suitable fish and transforms into an adult parasite in 23 days [13]. In consequence, our finding of eggs of B. acheilognathi in human stool allows several explanations. The most probable explanation would be that the eggs were transmitted through the digestive tract after consumption of a raw fish with parasitized internal organs. However, the patient denied consumption of any raw fish, such as carp — a possible B. acheilognathi host in metropolitan France, during the last several days prior to the stool examination. Another unlikely possibility could be that adults of B. acheilognathi survived for several weeks in the patient's intestine after being ingested in South America. Indeed, the patient reported the consumption of raw fish in the weeks preceding his arrival to metropolitan France and complained of abdominal pain persisting several weeks. The last hypothesis of an unusual development of a procercoid larva into an adult in a human intestine after the consumption of an infected fish could not be eliminated, regarding the presence of blood hypereosinophilia in the patient two years before the identification of B. acheilognathi in his stool. Indeed, B. acheilognathi is notorious for an exceptionally low definitive-host specificity, enabling it to infect a wide range of new hosts in its expanded range and it could have been reported from non-fish species [13]. We have been the first to describe an unusual case of D. nihonkaiense acquired in France after the consumption of imported raw Pacific salmon [20]. We and others have then reported human infections with exotic species of Diphyllobothrium in Europe, in travelers or after consumption of various imported raw salmonids [10,21]. Without the use of molecular diagnostic, all these infections would have been diagnosed as D. latum cases. Here, we showed that our patient case would have been diagnosed as yet another Diphyllobothrium
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