Our main objective in this study was to evaluate the utility of our recently developed E. histolytica genotyping system in determining whether parasite genotypes correlate with the outcome of infection. We found evidence of a nonrandom distribution of parasite genotypes among the sample groups. However, there are some limitations in this study, which are described below, and these may have some impact on the observed population differentiation.
(i) All our asymptomatic and diarrhea/dysentery samples were from preschool children from Mirpur, and we do not know whether their genotypes are representative of those from all age groups and of the strain diversity across Bangladesh. All ALA samples were from adults in other areas of Dhaka. No adult or geographically matched asymptomatic or diarrhea/dysentery control groups were available, and so comparisons between the Mirpur and ALA samples must be interpreted with caution.
(ii) The genotypes were assigned on the basis of the estimated PCR product sizes in gels, and as a result some assignments could be incorrect. Nevertheless, sequencing of approximately half of the products at two loci (unpublished data) suggests that an incorrect STR type assignment is rare. The individual short tandem repeat units in the various loci are generally 8 or 9 bp in length, which means that a difference of one repeat more or less is usually clearly detectable under our gel conditions.
(iii) Our genotyping is based on PCR product size differences, but products of the same size do not necessarily have the same sequence. Sequencing has confirmed that this does occur. However, unless both sequence types show the same unequal distribution among sample populations, the combination of two different sequences in the same STR type is likely to reduce rather than increase any population differentiation signal that exists.
Using the six tRNA-linked STR loci, we detected 85 genotypes in 111 unrelated samples, a level of diversity which is comparable to that described in previous reports. Ayeh-Kumi et al. (2
) also studied clinical samples from Bangladesh but used a nested PCR of the SREHP gene coupled with restriction digestion. They found 25 genotypes among 42 intestinal isolates and 9 genotypes among the isolates from 12 ALA samples. Eight of their nine genotypes among the liver abscess samples were unique to the ALA samples investigated. We also found that most of our ALA genotypes were unique and not detected among the intestinal strains. Clark and Diamond (6
) observed 16 different genotypes among 18 isolates of E. histolytica
from diverse geographical locations using the combined results of restriction digestion of SREHP and amplification of the SSG locus. Haghighi et al. (9
) used sequencing of four loci (two tRNA-linked STR loci, chitinase, and SREHP) to investigate 79 isolates of E. histolytica
, mostly from Japan and Thailand, but failed to find an association between the parasite genotype and the outcome of infection. The high level of diversity seen suggests that the rapid generation of new variants is occurring. If this is the case, then it may be that we have detected population differentiation only because our samples came from a geographically restricted population (Mirpur) and all of our samples were collected over a relatively short period of time (2 years).
The high degree of polymorphism detected in E. histolytica could have been problematic, but we did find a statistically significant difference in genotype representation among the three sample populations, suggesting that the parasite genome does influence the outcome of infection. Other approaches will be needed to identify the genes responsible for this result, as it is highly unlikely that the tRNA-linked STR loci are directly responsible in any way. It is also important to know whether these observations made in Bangladesh can be replicated in other parts of the world. To investigate this, extensive sample collections from additional areas of endemicity are needed.
Explaining the link between parasite genotype and infection outcome presents us with some theoretical problems. Existing evidence points to E. histolytica being a clonal organism; therefore, meiotic recombination and reassortment of genes should not be contributing to genotype differences. If this is true, a novel mechanism for generating genetic variation may exist. This could involve a recurring genome instability that leads to the repeated generation of the same genotypes through intragenomic reorganization and is reflected in turn in the tRNA-linked STR polymorphisms detected by our method. Only future genome-wide analyses of chromosome organization can address this possibility.