Entamoeba bangladeshi nov. sp., Bangladesh

To the Editor: Diarrheal diseases have a major effect on global health, particularly the health of malnourished children (1). The enteric parasites Entamoeba histolytica and E. moshkovskii are potential causes of diarrheal disease in children (2). For the last 20 years, we have been studying Entamoeba infections in children from the urban slum of Mirpur in Dhaka, Bangladesh (3).

only risk factor identifi ed among the persons reported here was older age. Unlike persons in other reports, persons in our report were all men, and 2 reported GI symptoms. The mechanism for UTI in these cases is unclear but could have included ascending and hematogeneous spread.
We calculated incubation periods for GI symptoms for 6 persons as the time between onset of GI symptoms and the May 14 wedding (5 persons) or last travel date (1 person). The incubation period was 5-7 days (average 5.5 days). The incubation period for UTI, which could be calculated for 2 persons, was an average of 25.5 days. Long incubation periods for Salmonella spp. infections have been reported (6-9); reasons include exposure to a low dose of bacteria, specifi c populations (e.g., young children, child day care attendees), and method of food preparation (6)(7)(8)(9). The age of persons in our investigation did not affect the length of the incubation period. The amount of food eaten was not collected during the interview; however, most persons in our investigation reported eating a wide variety of foods, and 1 reported eating small portions. All food was prepared during the week before the wedding and served cold. This length of time and the potential for temperature abuse could have increased the infectious dose and decreased the incubation period (6). In addition, the 1 person with travel-related infection was not exposed to these food items. We found no literature on the incubation period for S. enterica ser. Agbeni. The reason for the long incubation period in this investigation is unclear and could be due to host-specifi c factors, the implicated serotype, or the food source.
The 3-day time frame for exposures was not suffi cient to identify appropriate exposures. Expanding the period for collecting exposure information about Salmonella spp. infections and the reporting and investigation of persons with Salmonella spp. identifi ed in urine to public health authorities might be needed to help identify and solve outbreaks.

Entamoeba bangladeshi nov. sp., Bangladesh
To the Editor: Diarrheal diseases have a major effect on global health, particularly the health of malnourished children (1). The enteric parasites Entamoeba histolytica and E. moshkovskii are potential causes of diarrheal disease in children (2). For the past 20 years, we have been studying Entamoeba infections in children from the urban slum of Mirpur in Dhaka, Bangladesh (3).
E. histolytica infections can be detected through fecal microscopy, culture, PCR, and antigen detection. Microscopy and culture have limited specifi city because several species of Entamoeba, which vary in their pathogenic potential, have morphologically similar cysts and trophozoites (4). In 2010-2011, during analysis of feces positive for Entamoeba organisms by microscopy or culture but negative for E. histolytica, E. dispar, and E. moshkovskii by PCR, a new species was identifi ed, which we have named Entamoeba bangladeshi nov. sp. in recognition of the support of the Bangladesh community for this research.
Feces from both diarrheal and surveillance specimens were collected from a cohort of children living in Mirpur (3). A total of 2,039 fecal samples were examined microscopically (0.9% saline smear) and/or by fecal culture for amebic trophozoites and cysts (4 ENTAGEN-F and ENTAGEN-R primers, which exhibit a broad specifi city for the small subunit ribosomal RNA (SSU rRNA) gene sequences of Entamoeba, were used in PCR to amplify DNA fragments from 43 of the samples that were negative by PCR for the 3 Entamoeba species; amplifi cation conditions were adapted from Stensvold et al. (10). The amplifi ed DNA was separated by electrophoresis by using a 2% agarose gel. Bands of the size predicted for the Entamoeba spp. SSU rRNA gene amplicon were detected in 15 samples (online Technical Appendix Table, wwwnc.cdc.gov/ EID/pdfs/12-0122-Techapp.pdf). The PCR products were extracted by using the QIAquick Gel Extraction Kit (QIAGEN) and cloned by using the Zero Blunt TOPO Cloning Kit (Invitrogen, Carlsbad, CA, USA). The sequenced clones from 2 different isolates, 1 diarrheal and 1 surveillance specimen, were completely novel when compared with the SSU rRNA gene sequences from other organisms and did not match any previously sequenced Entamoeba species. These isolates represent a new species of Entamoeba (GenBank accession nos. JQ412861 and JQ412862), here named E. bangladeshi (online Technical Appendix) We examined the phylogenetic relationship between E. bangladeshi and other Entamoeba parasites by using maximum-likelihood analysis as implemented in MEGA 5 (online Technical Appendix Figure, panel  A). E. bangladeshi, although distinct, clearly grouped with the clade of Entamoeba infecting humans, including E. histolytica. E. bangladeshi, however, appeared more distantly related than the noninvasive E. dispar, but closer than E. moshkovskii, to E. histolytica.
To further characterize E. bangladeshi, we established it in xenic culture, and it displayed the ability to grow at 37°C and 25°C, a characteristic shared with E. moshkovskii and E. ecuadoriensis but that distinguishes it from E. histolytica and E. dispar. Cultured trophozoites were evaluated through light and transmission electron microscopy (online Technical Appendix Figure, panel B). By light microscopy, we detected no apparent differences between E. bangladeshi and E. histolytica. The physical resemblance between E. histolytica and E. bangladeshi is notable because direct microscopic examination of fecal samples is still used as a diagnostic tool in areas to which these species are endemic to detect E. histolytica parasites.  Our fi ndings add to the diversity of Entamoeba species found in humans. The incidence and effect of infection in infants by the newly recognized species E. bangladeshi await future epidemiologic studies.