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 [
4‐
17]. 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 1–2 and 5–6 [
11,
12,
16,
17]. In addition, the existence of genetic variation in non-protein-coding loci 1–2 and 5–6 [
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.
Discussion
Mutations and other polymorphisms in genes, gene systems, or whole genomes may play an important role in Entamobea. DNA sequencing is considered the gold standard for identifying such mutations. Nevertheless, DNA sequencing method is cumbersome and costly when large numbers of samples need to be rapidly analyzed. The method, therefore, is not always suitable for use in epidemiological studies.
In the present study, PCR products of 16S-like rRNA gene of E. histolytica, E. moshkovskii and E. dispar were subjected to screening for mutation by riboprinting and SSCP analysis. These products were of a relatively small sizes, 553 bp for E. moshkovskii, 439 bp for E. histolytica and 174 bp for E. dispar, and were found to be particularly suitable for SSCP analysis.
Riboprinting analysis detected a mutation in only one of the E. histolytica samples, from a stool specimen (Stool- 5); the alteration in the RFLP pattern (i.e. the PCR product remained undigested) was due to substitution of G by A in the recognition site of N1aIV. The inability of riboprinting to detect mutations in a majority of Entamoeba isolates is due to the limitation that RFLP detects mutation only when they occur in restriction endonuclease cut sites. However, SSCP analysis detected mutation in a total of five E. histolytica and three E. moshkovskii isolates from stool and one E. histolytica isolate from saliva. This may be attributed to the ability of SSCP, unlike RFLP, to detect any mutation in a DNA sequence. The SSCP analysis results that were suggestive of mutations were reproducible (i.e. SSCP analysis showed identical profiles in three separate experiments).
In this study none of the 171 E. dispar isolates showed characteristics suggestive of mutation by SSCP analysis, perhaps due to the screening of only a 174 bp region of the gene. The chances of finding a mutation in such a small region is less than in larger regions studied for the other two species.
Although it was observed that riboprinting was useful for screening a large number of samples, especially when looking for a specific single nucleotide polymorphism, the method has some inherent disadvantages. In this method, a large amount of amplicon (15 μl) is needed for digestion with each of several enzymes, and each clinical sample ends up being electrophoresed on several gels, making it a very laborious and time consuming procedure.
SSCP analysis, on the other hand, was found to be simple and convenient in the present study. The results were obtained from only 2 μl of PCR product on a single SSCP gel, which in the long run turned out to be much more economical. The total time needed for PCR-SSCP was less than 24 hours. The SSCP technique developed and evaluated in our laboratory provides us with a rapid tool for detecting mutations in Entamoeba. This method could conveniently supplement other methods of mutation detection analysis on large-scale. The limitation of SSCP, although minor, is that it detects the occurrence of single base mutations in a segment of DNA but does not give any information on the type or location of the base changes, which have to be confirmed by nucleotide sequencing.
In the present study, the results showed that SSCP analysis was able to detect even single nucleotide differences (Figure
5, Figure
7, and Figure
8). Nucleotide sequences were verified three times by sequencing of products of different PCR reaction from the same source of DNA to rule out any discrepancy due to sequencing errors. The sequencing analysis identified four new
E. histolytica genotypes and three new
E. moshkovskii genotypes (Figure
4 and Figure
6). Recently, a report from Bangladesh [
16] studied clinical specimens using six tRNA-linked STR loci, and detected 85 genotypes in 111 unrelated samples. Another report from Bangladesh [
15] also studied clinical samples but used a nested PCR-RFLP of the SREHP gene. Twenty five genotypes among 42 intestinal isolates and 9 genotypes among 12 ALA samples were found. Out of 9 genotypes from liver abscess samples, 8 were unique to the ALA samples investigated. In our study no unique genotypes among any of the liver abscess samples and urine samples from ALA cases were found, but out of 5
E. histolytica genotypes from stool samples 4 were unique to the stool samples investigated. In addition, our study also detected one unique
E. histolytica genotype from a saliva sample from an ALA case and three new
E. moshkovskii genotypes from stool samples. Clark and Diamond [
6] combined results of both PCR-RFLP of SREHP and amplification of the SSG locus to report 16 different genotypes among 18 isolates of
E. histolytica from varied geographical locations. Haghighi et al. [
11] reported a total of 53 different genotypes among 63 isolates of
E. histolytica, mostly from Japan and Thailand, using sequencing of four loci (two tRNA-linked STR loci, chitinase, and SREHP). The high level of diversity reported from different geographic locations suggest that the rapid generation of new
Entamoeba variants is taking place. The occurrence of new
E. histolytica and
E. moshkovskii genotypes in the area of Puducherry, India, may be a unique finding and the close relationship of these genotypes with a recognized human pathogen like
E. histolytica should prompt further studies.
In this study, the presence of mutations was explored in a region of 16S-like rRNA gene of E. histolytica, E. dispar and E. moshkovskii by applying riboprinting and SSCP analysis. Further studies are needed to extend mutation detection to the complete ribosomal RNA gene of these species, which will possibly reveal much more about genetic variation in Entamoeba.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SCP supervised and coordinated the study, and helped to draft the manuscript. KK carried out the experimental work, and drafted the manuscript.