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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 specificity 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 identified, 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). One hundred forty-nine (7%) of the samples were positive by microscopy and/or culture for an Entamoeba parasite with both cysts and trophozoites that closely resembled those of E. histolytica, E. moshkovskii, and E. dispar.

DNA was extracted directly from fecal samples by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. DNA from positive fecal cultures was isolated by using the cetyl-trimethylammonium bromide extraction method (5). PCR was conducted to detect E. histolytica, E. dispar, and E. moshkovskii, all of which are morphologically indistinguishable by microscopy and are endemic to Bangladesh (Table) (69). An antigen detection test (TechLab Inc., Blacksburg, VA, USA) was also used to identify fecal samples positive for E. histolytica.

Fecal samples (129) and cultures derived from fecal material (20) were tested by PCR. Forty-four fecal samples were positive for E. histolytica, 42 for E. dispar, and 7 for E. moshkovskii. PCR results for 48 samples were negative for all 3 parasites (mixed infections account for the total being >129); 5 cultures also were negative for all 3 parasites.

ENTAGEN-F and ENTAGEN-R primers, which exhibit a broad specificity 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; amplification conditions were adapted from Stensvold et al. (10). The amplified 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, 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[degrees]C and 25[degrees]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 findings 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.


We thank B. Mann for careful reading of this manuscript.

This investigation was supported by grant R01AI043596 from the National Institute of Allergy and Infectious Diseases to W.A.P.

Tricia L. Royer, [1] Carol Gilchrist, [1] Mamun Kabir, [1] Tuhinur Arju, Katherine S. Ralston, Rashidul Haque, C. Graham Clark, and William A. Petri, Jr.

[1] These authors contributed equally to this article.

Author affiliations: University of Virginia School of Medicine, Charlottesville, Virginia, USA (T.L. Royer, C. Gilchrist, K.S. Ralston, W.A. Petri, Jr.); International Centre for Diarrheal Disease Research, Dhaka, Bangladesh (M. Kabir, T. Arju, R. Haque); and London School of Hygiene and Tropical Medicine, London, UK (C.G. Clark)



(1.) Mondal D, Haque R, Sack RB, Kirkpatrick BD, Petri WA Jr. Attribution of malnutrition to cause-specific diarrheal illness: evidence from a prospective study of preschool children in Mirpur, Dhaka, Bangladesh. Am J Trop Med Hyg. 2009;80:824-6.

(2.) Shimokawa C, Kabir M, Taniuchi M, Mondal M, Kobayashi S, Ali IK, et al. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice. J Infect Dis. 2012 Jun 21; [Epub ahead of print]. http://dx.doi. org/10.1093/infdis/jis414

(3.) Haque R, Mondal D, Karim A, Molla IH, Rahim A, Faruque ASG, et al. Prospective case-control study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clin Infect Dis. 2009;48:1191-7. http://dx.doi. org/10.1086/597580

(4.) Petri WA Jr, Haque R. Entamoeba species, including Amebiasis. In: Mandell GL, JE Bennett, R Dolin, eds. Mandell, Douglas, and Bennett's principles and practice of infectious diseases, 7th ed. Philadelphia: Churchill Livingstone; 2010. p. 3411-25.

(5.) Ali IKM, Zaki M, Clark CG. Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica. J Clin Micro biol. 2005;43:5842-7. http://dx.doi. org/10.1128/JCM.43.12.5842-5847.2005

(6.) Stensvold CR, Lebbad M, Victory EL, Verweij JJ, Tannich E, Alfellani M, et al. Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist. 2011;162:525-41. http://dx.doi. org/10.1016/j.protis.2010.11.002

(7.) Ali IKM, Hossain MB, Roy S, AyehKumi PF, Petri WA, Haque R, et al. Entamoeba moshkovskii infections in children, Bangladesh. Emerg Infect Dis. 2003;9:580-4. eid0905.020548

(8.) Haque R, Kabir M, Noor Z, Rahman SMM, Mondal D, Alam F, et al. Diagnosis of amebic liver abscess and amebic colitis by detection of Entamoeba histolytica DNA in blood, urine, and saliva by a real-time PCR assay. J Clin Microbiol. 2010;48:2798-801. http://dx.doi. org/10.1128/JCM.00152-10

(9.) Haque R, Ali IK, Akther S, Petri WA Jr. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. J Clin Microbiol. 1998;36:449-52.

(10.) Haque R, Faruque AS, Hahn P, Lyerly DM, Petri WA. Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh. J Infect Dis. 1997;175:734-6.

Address for correspondence: William A. Petri, Jr., Division of Infectious Diseases and International Health, University of Virginia Health System, Carter Harrison Bldg, Rm 1709A, 345 Crispell Dr, PO Box 801340, Charlottesville, VA 22908-1340, USA; email:
Table. Oligonucleotide primers used for screening and sequencing
of Entamoeba bangladeshi nov. sp., Bangladesh *

Target organism       Primer name      Primer sequence,     Reference
                                     5' [right arrow] 3'

Broad specificity      Entagen-F     ACT TCA GGG GGA GTA       (6)
                                          TGG TCA C
Entamoeba sp.          Entagen-R       CAA GAT GTC TAA         (6)
                                        GGG CAT CAC AG
E. histolytica           Eh-F        AAC AGT AAT AGT TTC       (8)
                                      TTT GGT TAG TAA AA
                         Eh-R        CTT AGA ATG TCA TTT       (8)
                                        CTC AAT TCA T
                     Eh-YYT Probe     YYT *-ATT AGT ACA        (8)
                                       AAA TGG CCA ATT
                                         CAT TCA-Dark
                                          Quencher #
E. moshkovskii           Em-1          CTC TTC ACG GGG         (7)
                                           AGT GCG
                         Em-2          TCG TTA GTT TCA         (7)
                                           TTA CCT
                         nEm-1         GAA TAA GGA TGG         (7)
                                           TAT GAC
                         nEm-2         AAG TGG AGT TAA         (7)
                                           CCA CCT
E. dispar                 E-1          TTT GTA TTA GTA         (9)
                                            CAA A
                          E-2          GTA [A/G]TA TTG         (9)
                                          ATA TAC T
                         Ed-1          AGT GGC CAA TTT         (9)
                                          ATG TAA GT
                         Ed-2          TTT AGA AAC AAT         (9)
                                          GTT TCT TC

* Boldface indicates the probe fluorophore and quencher.

Note: The probe fluorophore and quencher indicated with #.
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Title Annotation:LETTERS
Author:Royer, Tricia L.; Gilchrist, Carol; Kabir, Mamun; Arju, Tuhinur; Ralston, Katherine S.; Haque, Rashi
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Geographic Code:9BANG
Date:Sep 1, 2012
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