Molecular-Genotyping Detection of Entamoeba histolytica in Diarrheic Patients

1. Introduction

Entamoeba histolytica is an anaerobic, pseudopod-forming nonflagellated protozoan pathogenic parasite classified in the Entamoebidae family that belongs to Amoebozoa phylum of Eukaryota ( 1 ). This parasite distributed worldwide, but the estimation of global prevalence is complicated due to the latent disease as well as limited capacity for detection and surveying among many endemic regions ( 2 ). Several reports estimated that 90% of amebiasis are asymptomatic ( 3 ). However, there is a high prevalence of infection occurs most commonly in poor socioeconomic population with lowered public health due to increasing the risk factors of infection such as the fecal – oral transmission, poor hand hygiene, and defecation into water sources that being in close proximity with animals ( 4 ). Post excretion of trophozoite in terminal part of ileum, colonization occurs in large bowl, but in responding to unknown stimuli, trophozoite moves and initiates pathogenesis ( 5 ). The parasite can induce tissue damage by three main events including direct host cell death, inflammation and parasite invasion to cause an intestinal amebiasis that characterized by diarrhea, amebic dysentery and amebic colitis ( 6 ). In rare cases, trophozoites enter the bloodstream resulting in an extra-intestinal amebiasis typically in liver causing amebic liver abscesses, or in brain causing amebic brain abscesses ( 7 ). Hence, the National Institute of Allergy and Infectious Diseases (NIAID) classified E. histolytica as a category B priority biodefense pathogen due to lowered dose of infection, resistance to chlorine, stability at different environments and properties that having a threat of simple spreading by contaminating of water and feed resources ( 8 ).

In laboratory, microscopic examination of stained (wet mounts) or concentrated fresh stool is the commonest method for morphological identification of cysts and trophozoites of E. histolytica; however, nonpathogenic Entamoeba species make this method is not always easy and possible ( 9 ). In the last decades, advancing in molecular detection techniques have supported our knowledge which leading to reorganization and isolation of pathogenic from nonpathogenic Entamoeba ( 10 ). As well as, apparent variation in lipophosphopeptidoglycan and lipophosphoglycan-like molecules was confirmed throughout avirulent and virulent E. histolytica strains ( 11 ). DNA typing of polymorphic genetic loci can aid in closely examination the polymorphic structure of field isolates ( 12 ). Serine-rich E. histolytica protein (SREHP) is abundant immunogenic surface protein, which strongly implicated in E. histolytica phagocytosis ( 13 ) Great genetic polymorphism in both coding and encoding loci was detected in different E. histolytica isolates ( 14 ). To demonstrate if there is any association between E. histolytica and clinical outcomes of infections, reliable diagnostic assays are needed to genotype the parasite. For this reason, different diagnostic techniques have been developed to investigate variation in E. histolytica strains among many years, but a polymerase chain reaction (PCR)-based approaches have revealed a high sensitivity and discriminatory ( 15 ). This study was aimed to molecular identification of E. histolytica in children using the qualitative nested (nPCR) through targeting 18S rRNA gene, and then, a genotyping of positive E. histolytica isolates using the quantitative PCR (qPCR) assay through targeting SREHP gene in Iraq.

2. Materials and Methods

2.1. Sample Collection

A total of 50 bloody diarrheic patients who referred to the Al-Zahraa’ Teaching Hospital and Alkut Hospital for Gynecology, Obstetric and Pediatrics (Alkut, Wasit, Iraq) were subjected to the present study from September to December 2021. Stool samples were collected from all patients into disposable plastic containers that transported under cold temperature conditions to be subjected for molecular analysis.

2.2 Molecular Examination by nPCR

Following the manufacturer instructions, DNAs were extracted from the collected stool samples using the Presto^TM Stool DNA Extraction Kit (Geneaid, Korea). After extraction, DNA samples were tested by the Nanodrop spectrophotometer (Thermo-scientific, UK) to estimate their concentrations and purities, and then kept frozen at -20ºC. Targeting 18S rRNA gene, two sets of primers were designed according to Haque, Ali ( 16 ) and provided by the Bioneer Company (Korea) to detect Entamoeba spp. [(F:5´-TTT GTA TTA GTA CAA A-3´) and (R:5´-GTA AGT ATT GAT ATA CT-3´)] and E. histolytica [(F:5´-AAT GGC CCA TTC ATT CAA TG-3´) and [(R:5´-TTT AGA AAC AAT GCT TCT TCT-3´)] at an amplicon size of 900bp and 550bap, respectively. According to the manufacturer instructions of the AccuPower^® PCR PreMix Kit (Bioneer, Korea), PCR MasterMix tubes of each set of primers were prepared at a final volume 20l (5l DNA template, 1.5l F primer, 1.5l R primer and 12l free-nuclease water). The two reactions were performed in thermocycler system (BioRad, USA) at the following conditions: 1 cycle initial denaturation (5 min at 95°C), 30 cycles of denaturation (30 sec at 95°C), annealing (30 sec at 58°C) and extension (1 min at 72°C) and 1 cycle final extension (5 min at 72°C). Stained 1% agarose gel with Ethidium bromide (1g / ml) was used to analysis the PCR products by electrophoresis at 100 volt and 80 Am for 1 hour. DNA fragments were visualized under an ultraviolet transilluminator (Wised, Korea).

2.3. Genotyping by qPCR

This assay was performed for genotyping of positive E. histolytica using the specific primers polymorphic targeting the SREHP gene which responsible for distinguishing of E. histolytica strains. The reaction was performed in two steps; the first conventional nPCR that amplified a 549-bp and the second phase that performed as qPCR using of the SYBR Green I. The 2 sets of primers were designed in according to Haque, Ali ( 16 ) and provided by Bioneer Company (Korea) as following: [(F:5´-GCT AGT CCT GAA AAG CTT GAA GAA GCT G-3´) and [(R:5´- GGA CTT GAT GCA GCA TCA AGG T-3´)] and [(F:5´-TAT TAT TAT CGT TAT CTG AAC TAC TTC CTG- 3´) and (R:5´- TGA AGA TAA TGA AGA TGA TGA AGA TG-3´)]. The qPCR MasterMix tubes were prepared using the AccuPower^® 2X GreenStarTM qPCR Master Mix (Bioneer, Korea) at a final volume of 50l (25µl 2X GreenStar MasterMix , 5µl DNA template 2.5l F primer, 2.5l R primer and 15l DEPC water). The qPCR reaction was performed in MiniOpticon Real-Time PCR system (BioRad, USA) at following conditions: 1 cycle initial denaturation (3 min at 95°C), 45 cycles of denaturation (10 sec at 95°C), annealing/extension detection (scan) (30 sec at 60°C) and 1 cycle melting (30 sec at 80-95°C). The genotyping analysis depend on the SYBR Green I melting curve analysis.

2.4. Statistical analysis

All obtained data were reported and analyzed statistically using the GraphPad Prism Software version 6.0.1. One Way ANOVA was applied to detect significant differences between results of molecular genotyping at P<0.05.

3. Results

Overall, 48% (24/50) positive samples for E. histolytica were detected using nPCR assay (Figure 1). For genotyping, the findings of qPCR assay among 24 positive samples by nPCR were detected an existence of four different genotypes (I, II, III and IV) based on melting curve among with a significant (P<0.05) prevalence of Genotype-II [54.17% (13/24)] when compared to Genotype-I [20.83% (5/24)], Genotype-III [12.5% (3/24)] and Genotype- IV 12.5% (3/24)]. The appearance of different genotypes reflecting different values of melting temperature; 84ºC for Genotype-I, 83 - 83.5ºC for Genotype-II, 82.5ºC for Genotype-III and 81ºC for Genotype- IV (Figure 2, Table 1).

Figure 1. Agarose gel electrophoresis image that shown the Nested PCR product of 18S rRNA gene that using in detection E. histolytica. Where M: Marker (2000-100bp), Lanes 1-10 represented some positive E. histolytica stool samples at a product size of 550bp

Figure 2. Prevalence of targeted genotypes among 24 positive samples by nPCR

4. Discussion

Epidemiological investigations regarding an existence of parasites among many regions are commonly aim for identifying a community at risks, as well as description of a disease that having negative impacts on population Shirley, Farr ( 4 ). Worldwide, the clinical symptoms of E. histolytica could be ranged from asymptomatic illness to amebic dysentery or invasive extra-intestinal infection ( 17 ). In comparison to our findings of nPCR assay, E. histolytica was identified molecularly in 6% in Iraq ( 18 ), 3.4% in Australia ( 19 ), 2.6% in Saudi Arabia ( 20 ), 10-16.4% in Colombia ( 21 ), 9.15% in Malaysia ( 22 ), 10% in United Arab Emirates ( 23 ), 44.2% in Yemen ( 24 ), 1.7% in Ethiopia ( 25 ), 0.14% in Iran ( 26 ), 14.7% in Egypt ( 27 ) and 6.38% in China ( 28 ). Difference in incidence of E. histolytica infections could be attributed to variation in socio-economic and environmental characteristics, host risk factors such as age, gender, location, education level, occupational status (working), and existence of other family members affected with the parasite or other Entamoeba species. In developed countries, exact burdens of infection is challenged to be quantified due to sensitivity of diagnostic modality used, incubation period and symptom severity, sample size, study design and geographic area ( 4 ). In Iraq, the rate of morbidities and mortalities correlated to diarrhea is high in particular in children aged 5 years ( 29 ). This elevation in morbidities and mortalities might be due to the multiple challenges of environmental sanitation and basic public health service across Iraq, post many years of war and political instabilities ( 30 ).

Mahmood and Bakr ( 18 ) noted that the disease outcome in endemic areas could reach 30% in the first year of age and elevated up to 90% at 4 years of age. In recently studies, the results showed that the origins of infection remain extremely higher than expected in children of 2 years of age, and monthly application of PCR assays for testing of diarrheal or normal stool samples can reveal positive results for E. histolytica in children of 2 years of age ( 6 , 31 ). E. histolytica infections might be appeared with variable patterns as following; infection with diarrhea, diarrhea with prior or subsequent asymptomatic colonization, and diarrhea without obvious evidences of infection ( 11 ). Several researchers showed that the rate of morbidity was obviously elevated when the testing of children was limited to severe diarrhea ( 32 ). The re-emergence of infections was mentioned in Saudi Arabia in children aged 16 years who referred to the hospital with acutely phase of gastroenteritis, in which, this parasite was the almost enteropathogen related to diarrhea ( 30 ). Laude, Valot ( 9 ) showed that this parasite is one of seven organisms causes dysentery with almost recurrent causes of dysentery due to combination of bacterial and protozoal pathogens. This fact confirmed through utilization of molecular tools and attributed to that, children could exposure for many serious organisms at kindergarten, playground and home ( 31 ). Nonetheless, an existence of multiple infections can complicate diagnosing of a particular organism causes the illness and might be resulted in additional impacts that lead to more severely clinical illness ( 33 ).

Molecular genotyping had proven to help in close testing the structure of E. histolytica field isolates. In targeting of SREHP gene, we detected four E. histolytica genotypes (I, II, III and IV) among 24 positive isolates with a significant prevalence of Genotype-II. The first detection of intra-specific difference in E. histolytica isolates was reported by Gilchrist, Petri ( 31 ) study that confirmed an existence of extensive polymorphisms in size and restriction site of repetitive E. histolytica SREHP gene, with identification of 10 distinct DNA patterns in 18 E. histolytica isolates from different geographic areas. Ayeh-Kumi, Ali ( 34 ) reported that there are 34 distinct patterns of E. histolytica in 54 clinical isolates from endemic areas confirming that polymorphisms are found extensively within the single geographic area. Although, highly degrees of polymorphisms existed in E. histolytica that is pathogenic, Ali, Mondal ( 15 ) found that there is a statistical important variation in genotypes of sample population, proposing the influences of parasitic genomes on the outcome of infections and the existence of the novel mechanism that creating genetic difference. In Turkey, Araz, KORU ( 35 ) identified 3 different genotypes in 16 isolates but without significant differences in diarrheic patient, proposing that association could be observed between clinical outcome and parasite load.

This molecular and genotyping investigation revealed the health burden and uncontrolled widespread prevalence of E. histolytica among children, calling that the public health authorities for informing diarrhea control program among infants and children in Iraq in particular in low-hygienic and poor regions. Application of both qualitative and quantitative PCR assays could have great benefits in detection the causative pathogen(s). Effective hands hygiene could have a significant role in reducing the severity of contamination or even prevention the spreading of infection. Furthermore, extensive studies are required to demonstrate the potential role of E. histolytica or other species of Entamoeba in intestinal and extra-intestinal infections and to detect the effect of multiple enteropathogens in severity of clinical illness.

Authors' Contribution

D. K. K. was collected the stool samples from bloody diarrheic children and extraction of DNAs, both B. A. H. and G. A. were responsible for the work of nPCR and qPCR. All authors participated in genotyping of positive isolates, as well in writing of this manuscript.

Ethics

The study carried out under the license of the Scientific Committee of the College of Medicine, University of Wasit (Wasit, Iraq).

Conflict of Interest

The authors declare that they have no conflict of interest.

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