Mutation detection analysis of a region of 16S-like ribosomal RNA gene of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii
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Address: Department of Microbiology, Jawaharlal Institute of Postgraduate, Medical Education and Research
,
Puducherry, 605006
,
India
Background: The level of intra-species genetic variation in Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii populations in a localized geographic area, like Puducherry, India, remains unknown. Methods: In the present study the existence of genetic variation in the nested multiplex polymerase chain reaction (NM-PCR) amplified region of the 16S-like ribosomal RNA genes of E. histolytica, E. dispar and E. moshkovskii was investigated by riboprinting and single strand conformation polymorphism (SSCP) analysis. Results: We found that 70 stool specimens were positive for E. histolytica, 171 stool specimens were positive for E. dispar, and 37 stool specimens were positive for E. moshkovskii by NM-PCR. Ninety liver abscess pus specimens, 21 urine specimens, and 8 saliva specimens were positive for E. histolytica by NM-PCR. Riboprinting analysis detected a mutation in the PCR product of only one E. histolytica isolate from a stool specimen. However, SSCP analysis detected mutations in the PCR products of five E. histolytica isolates and three E. moshkovskii isolates from stool specimens, and one E. histolytica isolate from a saliva specimen. The mutations detected by riboprinting and SSCP analysis were confirmed by sequencing. All the nucleotide sequences showing mutations in this study have already been deposited into the NCBI GenBank database under accession numbers [GenBank: EF682200 to GenBank: EF682208]. Conclusion: The present study has revealed the subsistence of mutations in the ribosomal RNA genes of E. histolytica and E. moshkovskii, which points towards the existence of intra-species genetic variation in E. histolytica and E. moshkovskii isolates infecting humans.
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Background
The protozoan parasite Entamoeba histolytica is estimated
to infect 50 million people and cause 40,000 to 100,000
deaths annually, making it the second largest cause of
mortality from infection with parasitic protozoa after
malaria [1].
Although the first description of amoebiasis was more
than a century ago [2], there is still uncertainty as to why
symptoms of the disease appear only in 10% of those
infected with E. histolytica while majority remains
asymptomatic [3]. There have been several suggestions regarding
the factors that may contribute to the outcome of amoebic
infection in a susceptible host, which include a range of
virulence levels among the E. histolytica strains and
variability in host immunity against amoebic invasion. While
the variability of human immunity against amoebic
infection is not well understood, the existence of genetic
variation in E. histolytica has been studied in depth recently
[417]. These studies have identified genetic variation in
protein-coding sequences of E. histolytica, such as those for
the serine-rich E. histolytica protein [10-15] and chitinase
[8,11,12], as well as non-protein-coding regions such as
the ribosomal RNA (rRNA) genes [4,5,7] and loci 12 and
56 [11,12,16,17]. In addition, the existence of genetic
variation in non-protein-coding loci 12 and 56 [18], as
well as protein-coding chitinase gene of E. dispar has been
reported recently [19].
These genetic variation studies appear to be promising in
investigating the molecular epidemiology of amoebiasis.
The existence of significant genetic variation among E.
histolytica isolates collected from a wide geographical range,
including Mexico, Bangladesh, India, Venezuela, South
Africa, the Philippines, and Georgia, has already been
demonstrated [8,9,13,14]. However, whether
intra-species genetic variation also exists in E. histolytica, Entamoeba
dispar and Entamoeba moshkovskii from a population in a
restricted geographic area like Puducherry, India, still
remains unknown.
The rRNAs, especially the 16S rRNA, have been widely
used for studying genetic variation because of their
conservative nature and universal distribution [20]. In the
present study an attempt has been made to study genetic
variation in regions of the 16S-like rRNA gene of E.
histolytica, E. dispar and E. moshkovskii using riboprinting and
single strand conformation polymorphism (SSCP)
analysis followed by confirmation by nucleotide sequencing.
Methods
Sample details
The study was conducted at the Jawaharlal Institute of
Postgraduate Medical Education and Research (JIPMER)
hospital, Puducherry, India, during the period from July
2004 to July 2006. Informed consent was obtained from
the patients. The study was approved by the Institute
Human Ethics Committee (JIPMER, Puducherry, India).
Stool
Fresh unpreserved stool samples from 202 patients with
complaints of gastrointestinal discomfort and positive for
E. histolytica, E. dispar, or E. moshkovskii by microscopy or
culture were collected in sterile capped containers and
stored at -20C until used.
Liver abscess pus
The liver abscess pus aspiration was performed on (...truncated)