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Year : 2021  |  Volume : 11  |  Issue : 2  |  Page : 122-125  

Molecular characterization of Entamoeba, Blastocystis and Cryptosporidium species in stool samples collected from Jordanian patients suffering from gastroenteritis


1 Department of Medical Laboratory Sciences, Faculty of Applied Health Sciences, The Hashemite University, Zarqa, Jordan
2 The Centre of Biosecurity and One Health, Harry Butler Institute, Murdoch University, Perth, Western Australia, Australia

Date of Submission13-Mar-2021
Date of Decision13-Mar-2021
Date of Acceptance18-May-2021
Date of Web Publication20-Oct-2021

Correspondence Address:
Nawal Hijjawi
Department of Medical Laboratory Sciences, Faculty of Applied Health Sciences, The Hashemite University, PO Box 150459, Zarqa, 13115
Jordan
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DOI: 10.4103/tp.TP_106_20

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   Abstract 


Little is known about the prevalence of intestinal protozoa in patients suffering from diarrhea in Jordan. The present study aimed to detect and speciate Entamoeba, Blastocystis, and Cryptosporidium species in a total of 159 human patients with diarrhea from November 2014 to October 2016. The overall prevalence for the three parasites was 19.5% (31/159). Entamoeba spp. (Entamoeba. dispar and/or Entamoeba histolytica), Blastocystis hominis, and Cryptosporidium parvum subtype IIaA15G2R1 were detected in 12.6%, 6%, and 0.6 of samples, respectively. This is the first molecular study in Jordan to confirm the diagnosis of Entamoeba species and to discriminate between E. histolytica and E. dispar.

Keywords: Blastocystis hominis, Cryptosporidium parvum, Entamoeba dispar, Entamoeba histolytica, human patients, Jordan, polymerase chain reaction


How to cite this article:
Hijjawi N, Zahedi A, Ryan U. Molecular characterization of Entamoeba, Blastocystis and Cryptosporidium species in stool samples collected from Jordanian patients suffering from gastroenteritis. Trop Parasitol 2021;11:122-5

How to cite this URL:
Hijjawi N, Zahedi A, Ryan U. Molecular characterization of Entamoeba, Blastocystis and Cryptosporidium species in stool samples collected from Jordanian patients suffering from gastroenteritis. Trop Parasitol [serial online] 2021 [cited 2021 Nov 28];11:122-5. Available from: https://www.tropicalparasitology.org/text.asp?2021/11/2/122/328688



Globally, diarrhea remains the second most common cause of death, especially among children <5 years old, with 1.7 billion reported cases of diarrheal diseases every year.[1] Enteric parasites are major contributors to the global diarrheal disease load, infecting >67.2 million people annually.[2] In the Middle-Eastern country of Jordan, despite improvements in health care, diarrhea is still a major public health issue with an average of 117,894 cases of diarrhea per year reported to the Ministry of Health between 2000 and 2010, although this is likely to be greatly underestimated.[3]

The protozoan parasites: Entamoeba, Blastocystis, and Cryptosporidium are major causes of diarrhea worldwide.[3] All three parasites are transmitted by fecal-oral contamination through the environmentally resistant oocyst/cyst stages. There are many species in the genus Entamoeba and at least eight of them are infectious to humans, namely, Entamoeba histolytica, Entamoeba dispar, Escherichia coli, Entamoeba moshkovskii, Entamoeba hartmanni, Entamoeba polecki, Entamoeba gingivalis, and Entamoeba bangladeshi, but E. histolytica is the only species which has been universally recognized as a cause of intestinal and extraintestinal disease in humans.[4],[5] Cryptosporidium species infect a wide range of hosts and cryptosporidium infections can cause acute diarrhea, nausea, vomiting weight loss, and in infants, can lead to malnutrition, growth retardation, and impairment in cognitive function.[6] In immunocompetent humans, cryptosporidiosis is usually self-limiting, however, in patients with compromised immune systems, it can be the cause of chronic diarrhea, cachexia, lack of appetite, and malnutrition.[6] Humans are susceptible to a wide range of Cryptosporidium species, with C. hominis and C. parvum, the main species infecting humans globally, and so far, there is no effective treatment for cryptosporidiosis in immunocompromised populations.[7]

Blastocystis is a strict anaerobic single-celled parasitic protist which colonizes the gastrointestinal tract of domestic and wild animals worldwide (>1 billion individuals).[8] The pathogenic potential of Blastocystis is under debate, however, several studies have reported the presence of Blastocystis hominis in patients with acute and chronic diarrhea and its colonization is significantly higher among irritable bowel syndrome patients compared to patients with other gastrointestinal disorders (diarrhea, abdominal pain, irritable bowel syndrome, constipation, and flatulence), suggesting a possible association between this parasite with irritable bowel syndrome and its pathogenesis.[8]

The previous studies that have investigated the etiology of diarrhea among Jordanians, based mainly on microscopy, have reported prevalence of 0.1%–80.7% for Entamoeba, 1.5%–37.3% for Cryptosporidium, and 0.6%–60% for Blastocystis hominis.[9],[10] However, detection of enteric protozoa by microscopy lacks specificity and sensitivity, and therefore, the aim of the present study was to use PCR and sequencing to detect and speciate Entamoeba, Blastocystis, and Cryptosporidium parasites in Jordanian patients suffering from diarrhea and gastroenteritis.

A total of 159 fecal specimens were collected from patients (age range: 1 month to 54 years) who suffered from diarrhea and abdominal cramps and were referred to clinical laboratories of major hospitals in five regions of Jordan (Al-mafraq, Amman, Irbid, Maan, and Zarqa) from November 2014 to October 2016. Demographic data regarding age, gender, residency, and medical history were also obtained. Whole genomic DNA was extracted from all samples (n = 159) and was screened for the presence of Entamoeba spp. at 18S rRNA and actin loci, Cryptosporidium spp. at 18S rRNA, and gp60 loci and Blastocystis spp. at 18S rRNA locus.[11],[12],[13]

Amplified DNA fragments for each species were then separated by agarose gel electrophoresis and purified for sequencing at each locus using an ABI Prism™ Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California) according to the manufacturer's instructions, using the annealing temperatures from the secondary PCRs. Sanger sequencing chromatogram files were imported into Geneious Pro 8.1.6, and the nucleotide sequences were analyzed and aligned with reference sequences from GenBank using Clustal W (http://www.clustalw.genome.jp).

The prevalence of Entamoeba, Blastocystis, and Cryptosporidium species (which were also confirmed by sequencing analysis of the 18S rRNA locus) was expressed as the percentage of samples positive by PCR, with 95% confidence intervals calculated assuming a binomial distribution, using the software Quantitative Parasitology 3.0.

The overall prevalence for the three parasites (Entamoeba, Blastocystis, and Cryptosporidium) in the 159 samples was 19.5% (95% confidence interval [CI]: 11.1–21.8). Species-specific PCR and sequence analysis at 18S rRNA locus identified Entamoeba spp. in twenty fecal samples (12.6%; 95% CI: 7.9–18.8), comprising 17 E. dispar isolates, two E. histolytica, and one mixed E. histolytica and E. dispar infection [Table 1]. Data obtained at the 18S rRNA locus were also confirmed at actin locus, with the exception of one E. dispar sample, which failed to amplify at the actin locus. Blastocystis hominis was detected in 6% (10/159; 95% CI: 3.1–11.3) of samples and Cryptosporidium parvum (subtype IIaA15G2R1) was identified only in one sample 0.6% (1/159; 95% CI, 0–3.5) [Table 1]. There were no mixed infections with the three tested parasites (Entamoeba, Blastocystis, and Cryptosporidium) in any patient; however, coinfection with E. histolyica and E. dispar were observed in one sample [Table 1].
Table 1: Prevalence and molecular characterization of Entamoeba, Blastocystis, and Cryptosporidium from Jordanian patients suffering from diarrhea and gastroenteritis

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   Conclusions Top


Overall, Entamoeba spp. were the most prevalent of the three parasites screened (12.6%–20/159), with most of the cases identified from Irbid and Amman areas (north and mid-Jordan). Previous studies in different parts of Jordan reported large differences in the prevalence of Entamoeba, Blastocystis, and Cryptosporidium, which might reflect differences in detection methods, microscopist skill levels, sample size, targeted patients, and area of collection.[9],[10] Several previous studies in various areas of Jordan based on microscopic examination have reported prevalence of 0.1%–80.7% for E. histolytica.[3] However, as no molecular typing was conducted, it cannot be confirmed if E. histolytica, E. dispar, or another Entamoeba species was actually present. To the best of our knowledge, this is the first molecular study conducted in Jordan to differentiate Entamoeba species in clinical samples and to confirm that most of the infections are caused by the E. dispar (17/20). Previously, E. dispar was thought to be nonpathogenic, however, it has been identified in patients suffering from diarrhea and may also be involved in the development of lesions in the human intestine and liver.[14] Therefore, further research is needed to better understand its pathogenic potential.

Few previous studies in Jordan have identified the species and subtypes of Cryptosporidium infecting Jordanian populations.[15],[16] In the present study, only one positive (C. parvum, subtype IIaA17G2R1) (0.6%) was detected. The previous studies in Jordan have reported prevalence of 19%, 14.4%, and 11%, respectively,[15],[16],[17] and the reason for the low prevalence in the present study is unknown. IIaA17G2R1 is a common subtype worldwide and was the predominant subtype reported in a previous study in Jordan on cryptosporidiosis in pediatric oncology patients.[16]

Of the relatively few previous studies in Jordan, Blastocystis prevalence ranging from 0.6% to 60% has been reported, and of these, only one previous study by Jadalla et al.[18] used PCR for its diagnosis. The present study is only the second one in Jordan to use molecular tools in the diagnosis of Blastocystis with B. hominis detected in 6.3% (10/159) of samples.

The transmission dynamics for the Entamoeba, Blastocystis, and Cryptosporidium species identified in the present study are unknown. However, a recent study revealed that contaminated water supplies, inadequate sanitation services, and lower socioeconomic status contributed to diarrhea in the human population in Jordan.[19] The problem is compounded by the fact that Jordan suffers from water scarcity and is now considered the third most water insecure country in the world.[20] To identify the risk factors involved in enteric parasite transmission, a well-designed case–control study, with a detailed collection of data on water, sanitation, food sources, and animal and human contact is required. Other infectious agents such as bacteria and viruses may also be responsible for diarrheal diseases among Jordanians but were not screened for in the present study. Large-scale molecular epidemiological studies on the causes of infectious diarrhea in different Jordan populations, particularly in children, the elderly, and immunocompromised patients are urgently required. This could be accomplished using the US Food and Drug Administration-approved multiplex PCR assays for simultaneous detection of Cryptosporidium, Entamoeba, and other common enteric protozoan parasites (as well as bacteria and/or viruses). A limitation of targeted pathogen-specific assays is that other potential causes of diarrhea may be overlooked. Another approach would be next-generation amplicon-based sequencing of bacteria, protozoa, helminths, and viruses simultaneously, although the cost is still impractical for many countries. Increased investment in water and sanitation infrastructure and more targeted hygiene education campaigns are also important.

Financial support and sponsorship

The research study was funded from the Deanship of Scientific Research/The Hashemite University.

Conflicts of interest

There are no conflicts of interest.



 
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