Forensic Study of Schistosoma Japonicum Eggs Found in an Autopsy Case

Parasitology International 65 (2016) 285–287

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Case report Forensic study of Schistosoma japonicum eggs found in an autopsy case

Ayumi Motomura a,⁎, Kazumi Norose b, Yumi Hoshioka a, Suguru Torimitsu c, Fumiko Chiba c, Yohsuke Makino c, Go Inokuchi a, Daisuke Yajima a, Nobuo Ohta d, Takashi Kumagai d,HirotaroIwasea,c a Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan b Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan c Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan d Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan article info the central Boso peninsula in Chiba, which is part of a different river system than the Tone River. Kojima et al. found S. japonicum eggs in path- Article history: ological tissues, such as the liver and rectum, in 15 cases around these Received 13 October 2015 Received in revised form 12 January 2016 areas [3]. However, a study in 1985 on Oncomelania snails in the Obitsu Accepted 15 February 2016 River did not detect any cercaria [3], and no eggs were found in the Available online 16 February 2016 stools of patients who tested positive with a schistosomiasis skin test and/or an enzyme-linked immunosorbent assay (ELISA) with Keywords: S. japonicum antigen [4]. It is also worth noting that in Japan the last re- Schistosoma japonicum Forensic autopsy port of infected snails was in 1977 [5,6], and no new human infections Identification have been reported since 1978 [2]. Liver Here, we report an autopsy case with the forensic consideration of S. japonicum eggs unexpectedly detected in the liver tissue. We discuss the significance of the S. japonicum eggs in the deceased body from the point of view of forensic medicine. An 80-year-old man with over 100 stab and incision wounds on his head, face, chest, upper limbs, and all over his body was found dying in Schistosomiasis is caused by infection with one of the three main his bedroom. A forensic autopsy revealed that he died from a hypovole- schistosomes: Schistosoma japonicum, Schistosoma mansoni,and mic shock with a blunt ascending aortic rupture due to a knife-thrust to Schistosoma haematobium. This disease affects more than 240 million the center of his chest. people worldwide and more than 700 million people live in endemic Postmortem computed tomography (PMCT) was performed using a areas [1]. S. japonicum was discovered in 1904 by Dr. Katsurada, who 16-row multidetector CT scanner (ECROS, Hitachi Medical Corporation, found a male worm in a dead cat in Kofu, and 4 days later, Professor Tokyo, Japan) prior to the autopsy. The following conditions were used Fujinami found a female S. japonicum worm body during the forensic for scanning from the superior margin of the sternum to the toes: tube autopsy of a homicide victim in the Katayama district, Hiroshima, voltage, 120 kV; tube current, 200 mA; collimation, 1.25 mm; and rota- Japan [2]. Japan previously had some endemic areas of S. japonicum, tion time, 1 r/s. Acquired images were reconstructed using an interval of but this parasite has been eradicated. Before its eradication, the major 1.25-mm slice thickness with a 300-mm field of view and soft tissue and most consistently S. japonicum-endemic areas were the Kofu basin kernels. The data were sent to a workstation (SYNAPSE VINCENT, in Yamanashi, the Katayama district in Hiroshima, and the Chikugo Fujifilm Medical Co., Ltd., Tokyo, Japan) for analyses. No specific findings River basin in Fukuoka and Saga. There were also other minor, unstably indicating septal calcification, such as a network or a turtle back pattern, infected areas, such as the Numazu district in Shizuoka, the Tone River scattered calcified nodules, or band-like linear calcification of the liver, basin, Nakatomi-cho in the Fuji River basin in Yamanashi, and the were obtained by PMCT (Fig. 1a) [7,8]. Obitsu River bank in Chiba. In these areas, especially the major areas, Overall, the patient's liver showed a slightly atrophic change the number of schistosomiasis patients increased continuously until (weight: 1090 g; size: 25 × 15 × 1.5 cm; body length ratio (liver the intermediate hosts of S. japonicum, Oncomelania snails, were drasti- weight/body length): 6.07, which is within the normal range for an cally eliminated by the coordinated effort of governmental workers and 80-year-old Japanese man (mean ± standard deviation (SD) of body local people. length ratio: 6.88 ± 1.30)), and white linear lesions extended over the In addition to areas within the Tone River basin, where human infec- surface (Fig. 1b). We did not observe any gross anatomical changes in tions of S. japonicum were detected along riverbeds in the early 1970s, the vascular system or lobular structure on the transverse section of Oncomelania snails have also been found in the Obitsu River bank in the liver (Fig. 1c). We also did not find any adult schistosomes macro- scopically around his liver during autopsy. The liver slices were pre- ⁎ Corresponding author. pared and stained with a hematoxylin and eosin (HE) stain by E-mail address: [email protected] (A. Motomura).

http://dx.doi.org/10.1016/j.parint.2016.02.008 1383-5769/© 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 286 A. Motomura et al. / Parasitology International 65 (2016) 285–287

Fig. 2. Microscopic ﬁndings in the patient's liver. (a) A representative image of a HE- stained section of the liver containing portal veins in Glisson's capsule. Arrows mark oval bodies (×40). (b) A representative magniﬁed HE-stained image of the oval bodies (×400). (c) Immunohistostaining of the liver was performed with polyclonal rabbit anti-S. japonicum antibody. A representative image of the resulting staining is shown here (×100). (d) Liver sections were stained using the von Kossa method. A representative image of the resulting staining is shown here (×100).

(normal: 0–38 IU/L), ALT (alanine aminotransferase) was 13 IU/L (normal: 0–44 IU/L), and γ-GTP (gamma-glutamyltransferase) was 14 IU/L (normal: 5–80 IU/L). The patient also previously had a gastric ulcer, gastric polyps, and an esophageal hiatal hernia. From a pathological viewpoint, it was primarily important to determine if these eggs were alive or not in order to prevent the potential spread of infection. Our findings that the serum was negative for anti- S. japonicum antibody, the inner structures of the eggs were decomposed, Fig. 1. Macroscopic findings of the patient's liver. (a) A representative PMCT image of the the egg shells were highly calcified and stained black with von Kossa liver. (b) A representative image of the liver surface. Arrows mark white linear lesions. method, and the inflammatory responses around the eggs were minimal (c) A representative image of a horizontal sectional view of the liver. clearly show that the infection of S. japonicum in this case was old and no longer active. conventional techniques, which incidentally revealed a number of oval In general, S. japonicum cercaria grow up in Oncomelania snails and bodies with ambiguous inner structures and thick calcified rims around then invade human bodies percutaneously. Secondary immature flukes the portal veins in the Glisson's capsule (Fig. 2a, b). Immunohistostaining travel through the blood vessels to portal veins, and adult fluke parasites using a polyclonal rabbit anti-S. japonicum antibody provided pathologic live mainly in the portal veins and lay eggs in the walls of the digestive confirmation of S. japonicum eggs (Fig. 2c). Although mild fibroses were tract [2]. In this case, an extensive pathological examination of the di- found around these eggs, no active inflammatory responses were seen. gestive tract was not performed because the patient's history of The egg shells stained black with the von Kossa method, reflecting S. japonicum infection had not yet been identified when the autopsy their high levels of calcification (Fig. 2d) and indicating that these eggs was performed; however, gross anatomical changes in the digestive are very old. The patient's spleen showed mild splenomegaly (weight: tract were not recognized during the autopsy. S. japonicum eggs were 145 g; body length ratio (spleen weight/body length): 0.81, which is not detected in any other pathological samples, including the brain, within the normal range for an 80-year-old Japanese man (mean ± SD lungs, cardiac muscle, spleen, pancreas, kidneys, adrenal grand, or of body length ratio: 0.62 ± 0.56)). No eggs were found in any other test- thyroid. ed organs, such as the brain or lungs. ELISAs using soluble egg antigen or In schistosomiasis, the eggs generally cause thromboses in various or- soluble adult worm antigen preparations to test for the presence of gans, such as the liver and brain. Notably, obstruction of the intrahepatic serum anti-S. japonicum antibodies were negative. port system leads to inflammation of the surrounding tissues and subse- According to the information provided by his family, the patient was quently causes periportal fibrosis. Finally, these schistosomiasis patients born and grew up in Kisarazu, Chiba, and moved around Chiba and develop cirrhosis, which causes the development of ascites. Thus, in en- Tokyo from his late teens through his twenties. After that, he returned demic areas, S. japonicum infection is suspected in cases with unex- to Kisarazu and made a living cultivating seaweed and clamming. To plained pooling ascites. their knowledge, the patient had never cultivated rice in a paddy nor The pathological liver lesions caused by S. japonicum infection are had he ever been abroad, although he had traveled within Japan a few typically linear, high echoic/density lesions whose appearance in ultra- times. He took insulin for diabetes and had cut down on his drinking sonography/CT is referred to as having a “Network pattern” or “Fish- after developing diabetes. To their knowledge, the patient had never scale pattern” that reflects the resulting calcification or fibrosis [8,9]. had liver dysfunction. An abdominal ultrasonography did not detect In this case, these characteristic patterns were not detected by PMCT, any abnormalities. The results of his last blood test were as follows: presumably because the patient's S. japonicum infection was mild. The for diabetes markers, HbA1c was 5.8% (normal: 4.6–6.2%) and fasting levels of AST/ALT and γ-GTP provided by the patient's primary care doc- blood glucose was 89 mg/dL (normal: 70–110 mg/dL), and for liver tor suggest that he did not have ante-mortem hepatic dysfunction. Our function markers, AST (aspartate aminotransferase) was 20 IU/L microscopic examination of the patient's liver revealed the presence of A. Motomura et al. / Parasitology International 65 (2016) 285–287 287 many calcified S. japonicum eggs in the Glisson's capsule and moderate distribution of Oncomelania snails in Japan is so restricted that finding fibrosis of the portal area; however, neither obvious inflammatory cell S. japonicum eggs during an autopsy may make it possible to estimate infiltration nor the formation of a pseudolobule were detected. These an unidentified cadaver's origin. Additionally, a DNA analysis of the findings indicate that this patient was previously infected with S. japonicum in each endemic area may provide more focused informa- S. japonicum without any symptoms and all that remains of this infection about their living places [19]. tion are the calcified eggs in his portal vein. The patient's route of infection was suspected to be via the freshwa- Conflict of interest ter around his residence because he lived in a rural area around Kisarazu, Chiba, collected seashells and harvested seaweed for a living, The authors have no conflicts of interest to declare in association and had presumably never left Japan. The Obitsu River runs through with this study. the Central Boso Peninsula in Chiba toward Kisarazu city, meeting the Tokyo bay. 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