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Volume 18, Number 9—September 2012

Letter

Entamoeba bangladeshi nov. sp., Bangladesh

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Technical Appendicies

Suggested citation for this article

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).

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) (710). 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. (6). The amplified DNA was separated by electrophoresis by using 2% agarose gel. Bands of the size predicted for the Entamoeba spp. SSU rRNA gene amplicon were detected in 15 samples (Technical Appendix Table [PDF - 305 KB - 3 pages]). 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 (Technical Appendix [PDF - 305 KB - 3 pages])

We examined the phylogenetic relationship between E. bangladeshi and other Entamoeba parasites by using maximum-likelihood analysis as implemented in MEGA 5 (Technical Appendix Figure, panel A [PDF - 305 KB - 3 pages]). 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 (Technical Appendix Figure, panel B [PDF - 305 KB - 3 pages]). 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.

Tricia L. Royer1, Carol Gilchrist1, Mamun Kabir1, Tuhinur Arju, Katherine S. Ralston, Rashidul Haque, C. Graham Clark, and William A. PetriComments to Author 
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)

Acknowledgments

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.

References

  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;80:8246.PubMed
  2. Shimokawa C, Kabir M, Taniuchi M, Mondal M, Kobayashi S, Ali IK, 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].
  3. Haque R, Mondal D, Karim A, Molla IH, Rahim A, Faruque ASG, Prospective case-control study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clin Infect Dis. 2009;48:11917. DOIPubMed
  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 Microbiol. 2005;43:58427. DOIPubMed
  6. Stensvold CR, Lebbad M, Victory EL, Verweij JJ, Tannich E, Alfellani M, Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist. 2011;162:52541. DOIPubMed
  7. Ali IKM, Hossain MB, Roy S, Ayeh-Kumi PF, Petri WA, Haque R, Entamoeba moshkovskii infections in children, Bangladesh. Emerg Infect Dis. 2003;9:5804. DOIPubMed
  8. Haque R, Kabir M, Noor Z, Rahman SMM, Mondal D, Alam F, 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;48:2798801. DOIPubMed
  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;36:44952.PubMed
  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;175:7346. DOIPubMed

Table

Technical Appendix

Suggested citation for this article: Royer TL, Gilchrist C, Kabir M, Arju T, Ralston KS, Haque R, et al. Entamoeba bangladeshi nov. sp., Bangladesh [letter]. Emerg Infect Dis [serial on the Internet]. 2012 Sep [date cited]. http://dx.doi.org/10.3201/eid1809.120122

DOI: 10.3201/eid1809.120122

1These authors contributed equally to this article.

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Table of Contents – Volume 18, Number 9—September 2012

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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


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