Abstract
Entamoeba histolytica is pathogenic parasite that causes asymptomatic infection mostly; however, it may also cause invasive intestinal amoebiasis and liver abscess, leading to significant rates of human mortality globally. The clinical outcome of the infection with the parasite is variable and evidence suggested the contribution of genetic diversity within E. histolytica to human disease. The information documented the whole-genome sequence of the E. histolytica reference laboratory strain (HM-1:IMSS) and the development of sophisticated molecular technique potentiate ability to identify strains of E. histolytica that may lead to insights into the population structure, virulence, pathogenesis, clinical outcome of the disease and epidemiology of the organism.
Keywords
- Entamoeba histolytica strains
- genetic diversity
- inter-species variability
- E. histolytica genetic biomarker
1. Introduction
The differences reported in the clinical outcome of the infections with
It has been suggested that the presence of these genetic alterations could have either permitted the parasite to more readily evade host immune responses or be related to higher virulence, causing more severe clinical presentations [8].
2. Epidemiology of amoebiasis
Evaluating the global burden of
The global impact of amoebic infection and amoebiasis remains significant, nevertheless challenging to quantify with accuracy given several epidemiologic and methodologic difficulties, but prevalence rates persist as high as 40% in certain populations.
3. Whole-genome sequences of Entamoeba
The genome assembly of
The genome shows impressive evolutionary features, in particular, the presence of substantial number of genes (at least 68) that seems to have been acquired by horizontal gene transfer from bacteria. Most of these transferred genes appear to have been ancient and implicated in metabolic processes specific for the anaerobic lifestyle of the organisms [27].
Genomic structure and architecture of Entamoeba are still not well characterized. For example, it is unknown whether there is a natural ploidy or haploid number of chromosome, although both are estimated [28]. The processes of genetic reassortment are common in
There are complex patterns of
4. Study strain variation of E. histolytica
4.1 Isoenzymes or zymodeme analysis
The first observation of the variation within
4.2 PCR-restriction fragment length polymorphism
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is molecular-based technique to differentiate species within the genus or to show the genetic diversity of certain species or strain. The method involves the digestion of PCR products of specific gene with restriction enzymes to produce fragments of different numbers and sizes based upon the differences in the number and location of restriction sites present in the amplicon. The selections of the target genes and restriction enzymes are based on the way that, for instance, different species of certain genus produce the same-size amplicon but show different banding size or patterns for different species by gel electrophoresis after been digested with specific restriction enzyme [39].
Molecular typing based on polymorphic genetic loci has been confirmed to aid in precise examination and identification of population structures of
4.3 Serine-rich E. histolytica protein (SREHP )
The majority of the biological functions are initiated through proteins, identifying certain proteins and estimating their functions enabling their potential use as markers for studying population nature and genetic diversity. The highest immunogenic protein among all identified proteins in
Furthermore,
4.4 Short tandem repeats (STRs)
The tRNA genes of
A number of primers lying in the non-coding regions were used for the identification of
4.5 Chitinase
The repeat-containing region of
4.6 Use of retrotransposons as genetic markers
4.6.1 Transposon display
The genomic sequence of
Amplified fragment length polymorphism (AFLP) is a highly sensitive technique for detecting polymorphisms in DNA, the method based on restriction enzymes that cut the DNA and adaptors attached to the ends of the fragments. The DNA fragments are then amplified using PCR, and their varying lengths can then be visualized on gel after been electrophoresed. Transposon display (TD) is a modified AFLP technique uses specific primer that anchors in a transposon to simultaneously identify up to several hundred markers in the genome [56, 57]. TD consists of amplification of sequences flanking the transposon by ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. Transposon display has been used to investigate and explore the behaviour and stability of transposable elements in plants [58]. The technique has also been effectively used to display yeast mutants conferring quantitative phenotypes [59].
Laboratory strains of
4.6.2 REP PCR
The repetitive element palindromic-polymerase chain reaction (REP PCR) was first devised for strain and serotype identification in enteric bacteria [61]. REP PCR is commonly used in clinical laboratories for detecting strains of bacteria, fungal and dermatophytes [62]. The method depends on the targeting of interspersed repetitive consensus sequences in the genome that enables amplification of diverse-sized DNA fragments and may be present in both the orientations on the chromosome.
The designed PCR primers must target ‘read outward’ from the repeats, amplifying the region between two such elements in either direction. These primers are complementary and attached to dispersed repeated sequences. This may result in varying band patterns when the repetitive sequences are located in different positions in the genome of different strains. This principle was employed successfully using EhLINEs/EhSINEs dispersed in the
4.7 Single-nucleotide polymorphism
Single-nucleotide polymorphism (SNPs) is the simplest form of variation in the genomic DNA sequence on bases of single nucleotide. To investigate the genetic diversity of
4.8 Microarray
Microarray is a genomic tool used to detect the expression of thousands of genes simultaneously from a sample. Microarray assay has been used successfully for the identification of
Besides conserved genes, like rRNA and
The DNA microarray assay has been used in large-scale expression profiling of
5. Conclusions
Several powerful molecular techniques and genetic biomarker are available nowadays, such as chitinase, SREHP polymorphisms, SNPs, STRs, retrotransposons and microarray all provide informative understanding to study parasite biology. For examples, studying STR regions in tRNA genes of
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