1. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice Shinjiro Hamano Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Japan The 3 rd International Conference, Sept 1, 2012 at ICDDR, B.

2. ICDDR, B.: Mr. Kabir, Ms. Sobuz, Dr. Mondal, Dr. Haque University of Virginia: Dr. Taniuchi, Dr. Ali Prof. Houpt, Prof. Petri 約 1km 2 Bihari ethnic origin Settled there after 1971 Poor sanitary and hygienic conditions Use pit latrines The mean monthly family income was around $65 U.S 1

3. Life cycle of Entamoeba histolytica (an extra-cellular protozoan parasite) 5-20  m 10-60  m Haque R et al. N Engl J Med 2003;348:1565-1573 2

4. 1.E. histolytica is pathogenic, although it was recently shown that all of genotypes of E. histolytica are not equally capable of causing disease. 2.E. dispar is non-pathogenic. Backgrounds Then, the question is if E. moshkovskii is……? 3

5. Entamoeba moshkovskii Development of molecular biological method Development of PCR method  was first identified in 1941 and was thought to be a free- living common protozoan species.  The first human isolate was obtained from a resident of Laredo, Texas, who suffered from diarrhea, weight loss, and epigastric pain in 1961.  This isolate was named E. histolytica Laredo and shared biological features with E. moshkovskii.  E. histolytica Laredo is identical with E. moshkovskii. Thus, E. moshkovkii is able to at least parasitize in human. 4

6. An unrooted phylogenetic reconstruction among Entamoeba species. Silberman J.D. et al. Mol. Biol. Evol. 16(12):1740–1751. 1999 E. moshkovskii E. histolytica E. dispar E. hartmanni E. invadens 4 nuclei per cyst E. ranarum E. sp. NIH:1091:1 E. insolita E. gingivalis E. polecki E. coli strain HU-1:CDC E. coli strain IH:96/135 1 nucleus per cyst 8 nuclei per cyst non-cyst former E. chattoni E. terrapinae 16S-like rDNA Within the group of four nucleated-cyst-producing species, Entamoeba moshkovskii joins Entamoeba histolytica and Entamoeba dispar in a well-supported clade. 5

7. E. moshkovskii  is morphologically similar to E. histolytica and E. dispar.  is microscopically indistinguishable from them.  is biochemically and genetically different from them.  High incidence of E. moshkovskii has been reported in developing countries by PCR in human fecal samples. However, it remains unclear whether E. moshkovskii is pathogenic or non-pathogenic. 6

8. we tested the ability of E. moshkovskii to cause colitis and diarrhea in a murine model system. at first, In order to clarify the pathogenicity of E. moshkovskii, 7

9. 1.In the murine model of intestinal amebiasis, E. moshkovskii caused diarrhea, weight loss and colitis. 2.In this way, E. moshkovskii shared the ability to cause disease with E. histolytica, but not with non-pathogenic E. dispar. Summary 8 Shimokawa, C., et al. J. Infect. Dis. 2012; 206: 744-51

10. Then, the association between diarrheal episodes and infection with each Entamoeba spp. was examined in children in Mirpur, Dhaka, Bangladesh. These studies were part of a prospective cohort study on diarrheal diseases in newborns, which prospectively followed for diarrheal illness by every other day home visits. 9

11. ICDDR, B.: Mr. Kabir, Ms. Sobuz, Dr. Mondal, Dr. Haque University of Virginia: Dr. Taniuchi, Dr. Ali Prof. Houpt, Prof. Petri 約 1km 2 Bihari ethnic origin Settled there after 1971 Poor sanitary and hygienic conditions Use pit latrines The mean monthly family income was around $65 U.S 10

12. Field research assistants (FRAs) visited each study house every other day and collected information related to child morbidity, especially for diarrheal illness. FRAs collected non-diarrheal monthly stool specimens as well as diarrheal stool specimens from the home or in the study field clinic. All stool specimens were transported from the field to the clinic, then to ICDDR, B. using a cold box. 11

13.  Diarrhea was defined as having three or more unformed or abnormal stools (as per the mother’s perception) in a 24 hour period.  A diarrheal episode was defined as being separated from another episode by at least three diarrhea-free days. A total of 1426 diarrheal episodes were recorded during the first 12 months of life in 385 children. 12

14. 39 were positive for E. dispar (35.8%), 23 were positive for E. moshkovskii (21.1%), 17 were positive for E. histolytica (15.6%). Ali, I.K. et al.: Entamoeba moshkovskii infections in children, Bangladesh. Emerg Infect Dis. 2003; 9(5): 580-584. Among 109 stool specimens that were positive for Entamoeba spp. from Bangladeshi preschool children 13

15. The frequency of E. moshkovskii in diarrheal stools is comparable to that of E. histolytica in Mirpur children. E. moshkovskii (2.95%)E. histolytica (4.63%) 4064 2 E. dispar (0.35%) 5 The 1426 episodes of diarrhea were observed in 385 children during the first 12 months of life. 0 0 0 2 14

16. Both diarrheal severity score and duration are comparable between episodes positive for E. histolytica and E. moshkovskii. Pathogen Severity score mean ±SE Duration in days mean ± SE E. histolytica E. moshkovskii Others 4.89 ± 0.224.44 ± 0.44 4.71 ± 0.244.74 ± 0.49 4.84 ± 0.054.84 ±0.10 The mean age of the onset of diarrheal episodes positive for E.h., E.m. and E.d. are observed at 235, 277 and 510 days old, respectively. * * *No significant difference between means. 15

17. 16 As there are many microbes that can potentially induce diarrhea, the presence of other diarrheagenic microbes was examined in the 42 diarrheal samples that were associated with E. moshkovskii.  standard bacterial culture  fecal antigen detection  multiplex PCR combined with probe-based detection with Luminex beads

18. Many enteropathogens were detected in 42 of Em+ diarrheal stools Name of organismNumber of samples Encephalitozoon intestinalis1 Cyclospora cayetanensis1 Cystoisospora belli1 Enterocytozoon bieneusi6 Adenovirus1 Astrovirus4 Sapovirus2 Norovirus G10 Norovirus G24 Rotavirus1 E. histolytica2 Giardia intestinalis10 Cryptosporidium spp.1 Name of organismNumber of samples Vibrio cholera / parahaemolyticus 3 EAEC15 ETEC3 EPEC5 EHEC0 EIEC / Shigella spp23 Salmonella (pan)2 Aeromonas (pathogenic)14 Yersinia (pan)0 Campylobacter jejuni / coli23 17

19. The diarrheal episodes associated with E. moshkovskii were commonly co-infected. No. of other infection No. of samples Percent of infection (%) 049.52 11535.71 21638.09 3511.90 4+24.76 18 The application of these state-of-the-art diagnostic techniques in this cohort has on average identified a minimum of two different enteropathogens in every diarrheal stool sample (Houpt E and Taniuchi M personal communication).

20. Prevalence of E. moshkovskii asymptomatic infection preceding E. moshkovskii-associated diarrhea in 42 children Category Preceding 1 month surveillance stool Preceding 2 month surveillance stool E. moshkovskii (+)36 E. moshkovskii (-)3936 Total42 19 The new acquisition of E. moshkovskii infection was associated with diarrhea.

21. Conclusions 1.E. moshkovskii caused diarrhea, colitis and weight loss in mice. 2.In children in Bangladesh, the new acquisition of E. moshkovskii infection was associated with diarrhea. 3.These data are consistent with E. moshkovskii causing diarrhea, and indicate that it is important to re-examine its pathogenicity. 20 Shimokawa, C., et al. J. Infect. Dis. 2012; 206: 744-51

22. Dhaka Charlottesville, Virginia Nagasaki Chikako Dinesh Rashidul Bill Eric Mamun Mami Masataka Seiki (Keio) Ibne Shihab 21 FRAs