RESEARCH ARTICLE


The Frequency of the Intestinal Parasites Giardia Lamblia and Entamoeba Histolytica in Pediatric Diarrhea Specimens from Central Iran



Elnaz Abbasi1, Alireza Amouzandeh-Nobaveh1, Ehsanollah Ghaznavi-Rad1, 2, *
1 Department of Microbiology & Immunology, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran
2 Molecular and Medicine Research Center, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran


Article Metrics

CrossRef Citations:
3
Total Statistics:

Full-Text HTML Views: 4015
Abstract HTML Views: 1865
PDF Downloads: 753
ePub Downloads: 667
Total Views/Downloads: 7300
Unique Statistics:

Full-Text HTML Views: 2100
Abstract HTML Views: 1002
PDF Downloads: 490
ePub Downloads: 437
Total Views/Downloads: 4029



Creative Commons License
© 2020 Abbasi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Correspondence: Address correspondence to this author at Molecular Research Center, Faculty of Medicine, Arak University of Medical Sciences, Arak, IR Iran;
Tel: + 098-8634173526; E-mail: ghaznaviehs@yahoo.com


Abstract

Introduction:

Intestinal parasitic infections, particularly those caused by Giardia lamblia, are among the major health problems that exist worldwide, especially in developing countries. This study aimed to investigate the prevalence of the intestinal parasites Giardia lamblia and Entamoeba histolytica that were isolated from samples of infectious diarrhea in pediatric patients from Central Iran.

Methods:

This descriptive cross-sectional study included 230 samples of infectious diarrhea that were collected from May 2015 to February 2016. Direct observation, the formalin-ether sedimentation method and the technique using the Polymerase Chain Reaction (PCR) of β-giardin and EH primers were used for the identification of Giardia lamblia and Entamoeba histolytica.

Results:

Out of 230 samples of infectious diarrhea, five cases (2.1%) of Giardia lamblia and no cases (0%) of Entamoeba histolytica were identified using the formalin-ether sedimentation method and the same result were obtained using PCR technique. Of the five patients who had Giardia lamblia, three (60%) were male and two (40%) were female. The most common clinical symptoms in these patients were stomach ache and diarrhea (100%) and mucus in the stool (80%).

Conclusion:

Giardia lamblia was introduced as a parasitic agent causing diarrhea from Central Iran. The results indicate that pediatricians and, even more importantly, experts in laboratories should pay special attention to the identification of this parasite to treat the patients as effectively and as quickly as possible.

Keywords: Giardia Lamblia, Entamoeba histolytica, Intestinal parasites, Pediatric, Infection, Diarrhea, Iran.



1. INTRODUCTION

Being contaminated with parasitic infections is now believed to be inextricably intertwined with personal hygiene, individual conduct, and the social scales of people in terms of their sanitary awareness. Consequently, hygiene and health scale evaluation are considered to be among the yardsticks of measurement, specifically for those in developing countries [1]. In spite of the enormous efforts made by the World Health Organization (WHO), the exposure to parasitic infections is

highly prevalent in developing countries and is particularly widespread in communities where there are poor sanitary conditions and economic and social disadvantages [2].

According to reports issued by the WHO over the past 2010 years, almost two-thirds of the world’s population is contaminated by parasitic infections, of which the most prevalent is Giardia lamblia [3]. Among the parasitic infections of the digestive system, Giardia lamblia and Entamoeba histolytica are principal pathogenic protozoa. The number of those who are afflicted with Giardia lamblia has in fact reached epidemic proportions worldwide, and it is the most commonly found parasite in excrement samples collected in Iran [4]. The prevalence of this protozoan in developing countries fluctuates between 2% and 30%, whereas in industrialized countries the reported figures vary from 2% up to just 7% [5]. Giardia lamblia is capable of causing asymptomatic contamination or spectral diseases, and infections range from acute to chronic, with clinical symptoms such as acute watery diarrhea, abdominal cramps, maladaptation syndrome, and weight loss among both infants and children [6].

Entamoeba histolytica, which is mainly a cause of amoebiasis (amoebic bloody diarrhea), involves primary pathogenic protozoans in the human body [4]. Studies conducted between 1988 to 2009 found a prevalence of less than 1.3% in different parts of Iran [7]. However, identifying intestinal parasites in clinical detection labs is not carried out efficiently, and there is no specific information concerning the abundance scale of parasites, such as Giardia lamblia and Entamoeba histolytica in Iran’s central region. Therefore, this study is being conducted to examine the in depth abundance scales of Giardia lamblia and Entamoeba histolytica, by examining samples of diarrhea from pediatric patients who were referred to the pediatric educational and therapeutic center at the Arak University of Medical Sciences (Amir Kabir Hospital).

2. MATERIALS AND METHODS

2.1. Collecting Samples

For this sectional descriptive study, 230 samples of diarrhea were collected from pediatric patients who were referred to the Amir Kabir Medical Center from the beginning of May (the second month of the year in accordance with the solar calendar) 2015 to the end of February (The eighth month of the year in accordance with the solar calendar), 2016. This study was approved by the Ethics Committee of the Arak University of Medical Sciences under No. 93-176-10.

2.2. Microscopic Observation

The procedure was carried out using direct and formalin-ether sedimentation methods and according to WHO protocol, using samples of diarrhea that were collected in less than 10 minutes. The formalin-ether sedimentation method was used as described elsewhere [8].

The clinical sample that was available through Valiasr Hospital’s part of parasitology was used during this procedure as a positive control.

2.3. DNA Extraction and PCR Analysis

DNA extraction from samples of feces, using the QIAamp DNA Stool Mini Kit by Qiagen GmbH Hilden Germany, was conducted as per the protocol. The amount and purity of the extracted DNA were measured with a NanoDrop apparatus. The primers B-giardinand EH were deployed as genetic markers to validate the isolated Giardia lamblia and Entamoeba histolytica in the PCR (Table 1) [9, 10].

The eventual volume of PCR used in this study was 25 µl, which consisted of 12.5 µlof the master mix (1X), 0.5 µl of Taq DNA Polymerase (2.5 units), 10 µl of the forward and reverse primers (10 pm), 2 µl of the DNA sample (5 ng), and 8 µl of the twice distilled water. (All the substances were purchased from Yekta Tajhiz Corporation, Iran). From each positive gene, a sample was tested using the sequencing of isolated PCR products (Gene Fanavaran Corporation, Iran), which was verified using Blast analysis.

3. RESULTS

Of the 230 analyzed samples, five of the Giardia lamblia samples (2.1%) and none of the Entamoeba histolytica samples (0%) were found to be positive using direct observation and the Formalin-ether sedimentation technique; this finding was also confirmed by PCR. Four Balantidium coli samples (80%) were obtained from the five Giardia lamblia samples (2.1%) using the direct observation method, and one cyst (20%) was found using the Formalin-ether sedimentation technique.

The average age of the people afflicted with Giardia lamblia was 5 years and 8 months. The youngest diseased person was a 4-year-old boy; the oldest was an 8-year-old girl. Of the five patients (2.1%) afflicted with Giardia lamblia, three (60%) were male and two (40%) were female.

The clinical symptoms among the people suffering from giardiasis included stomachache and diarrhea (100%), mucus spotted in feces (80%), and nausea and anorexia (60%).

4. DISCUSSION

Intestinal parasitic infections are widespread across the world, and they often occur among people in poor social and economic circumstances, miserable living conditions, and densely populated regions deprived of proper sanitary protection, with inappropriate garbage disposal mechanisms, poor water quality, and unsanitary hygiene habits [11, 12].

In this study, five patients (2.1%) were infected with Giardia lamblia. In other studies carried out in Iran, the percentages of patients with Giardia lamblia were: 2.3% in Tehran, 4.1% in the northern regions of the country, and 10.9% in Hamedan; in Italy, 6.5% of the patients were infected with Giardia lamblia; in Jordan, 29.6% were infected with it [13-17]. The difference in the prevalence of Giardia lamblia infection reported in different studies is due to the differences

Table 1. Primers used in this study.
References Annealing Temperature Amplicon size (bp) Sequence 5'→3' Primer Target Gene Description
[9] 60 171 F: 5- AAGTGCGTCAACGAGCAGCT-3
R: 5- TTAGTGCTTTGTGACCATCGA -3
β-giardin -F
β-giardin -R
Giardia lamblia
[10] 56 170 F: 5- AAGCATTGTTTCTAGATCTGAG-3
R: 5- AAGAGGTCTAACCGAAATTAG-3
EH -F
EH -R
E. histolytica species

in the time, geographical living areas, and the hygiene habits and lifestyles in which the samples were obtained [17].

Amoebiasis is still reported to be one of the most consequential health maladies in tropical and subtropical areas [18]. The outbreak from Entamoeba histolytica is yet to be recognized, whether in Iran or other major parts of the planet [19].

Many studies have scrutinized the prevalence of Entamoeba histolytica, and its impact on certain groups of people in Iran. There is no evidence of the prevalence of Entamoeba histolytica in our study; however, in other studies conducted in Iran, the prevalence of Entamoeba histolytica was found to be 0.02% in Karaj, 0.1% in the northern region of Iran, and 0.4% in Oromia; the prevalence is 4.7% in Saudi Arabia and 18.8% in South Africa [17, 20-23]. The samples of intestinal parasitic infections collected from people can vary due to differences in their hygiene habits and lifestyles, and differences in the time and geographical living areas in which the samples are obtained [24-26].

The initial technique is to detect Balantidium coli, Giardia lamblia and Entamoeba histolytica within feces using a microscope. A PCR test can also be used for the detection of feces [27, 28]. Of these two methods of microscopic observation and PCR, the sensitivity of the PCR method was higher [27]. PCR can accurately identify parasitic species, and it produces less laboratory waste than microscopic observation methods [27]. In spite of being ultrasensitive and exclusive for detecting Giardia lamblia and Entamoeba histolytica, the use of molecular techniques, such as PCR, is extensively restricted to laboratories [27]. The microscopic method is unable to differentiate Entamoeba histolytica from Entamoeba dispar. Thus, the WHO recommends the development and utilization of new techniques, such as PCR for a specific diagnosis of Entamoeba histolytica infection [28]. It is not possible to distinguish Entamoeba histolytica from commensal species, Entamoeba dispar, with morphological indices and only using specific monoclonal antibodies for each species and genomic testing by PCR is possible [8].

In the present study, the average age of the people contaminated with giardiasis was 5 years and 8 months. In Ghana, the average age infected person was less than a year. The highest prevalence was found in children, under the age of 10, in Karaj, Iran [29, 30]. In the present study, to some extent, men were more often inflicted than women, which was virtually identical to the findings reported in a study conducted in the northern region of Iran [17].

In the present study, the most prevalent clinical symptoms among the afflicted patients were stomachache and diarrhea (100%) and mucus in the feces (80%); in a study conducted in Pakistan, the principal clinical symptoms among the patients were abdominal muscle cramps, nausea, vomiting, flatulence, anorexia, and weight loss. The most common clinical symptom was nausea, which was found in 77.6% of the people; the least common clinical symptom was diarrhea, which was found in 19.7% of the people [30, 31].

CONCLUSION

To date, detailed investigations about intestinal parasites in various parts of the world have suggested that this type of infection is still a hygiene-related issue, due to the provision of sanitary services, notably in countries lacking basic amenities. In this study, Giardia lamblia was the parasite with the highest number of leading-to-diarrhea protozoans from pediatric patients in the central part of Iran; thus, pediatricians, and especially laboratory authorities, should be exceedingly vigilant in order to detect this protozoan to ensure proper and early medical treatment for affected patients. Due to the high sensitivity of the PCR method, there is a need to use this method in clinical diagnostic laboratories. As no case of Entamoeba histolytica has been observed in this geographical region, which is due to the its increased information and social awareness of hygiene practices, and decreased use of contaminated water supplies and unwashed vegetables.

LIST OF ABBREVIATIONS

WHO = World Health Organization,
DNA = Deoxyribonucleic Acid,
PCR = Polymerase Chain Reaction,
BLAST = Basic Local Alignment Search Tool.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the Ethics Committee of the Arak University of Medical Sciences under the approval no. 93-176-10.

HUMAN AND ANIMAL RIGHTS

No animals were used in the study. All reported human were experimented in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsniki Declaration of 1975, as revised in 2008 http://www.wma.net/en/20activities/10ethics/10helsinki/.

CONSENT FOR PUBLICATION

Informed consent was obtained from a parent and/or guardian for participants under 16 years old. The authors neither use nor access any information that enabled them to identify individual patients.

AVAILABILITY OF DATA AND MATERIALS

The data supporting the findings of the article is available in the manuscript.

FUNDING

This work was financially supported by the Arak University of Medical Sciences (Number: 2116).

CONFLICTS OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the educational assistance of Arak University of Medical Sciences due to its financial contributions to support this study.

REFERENCES

[1] Rokni MB. The present status of human helminthic diseases in Iran. Ann Trop Med Parasitol 2008; 102(4): 283-95.
[2] Schmunis GA, López‐Antuñano FJ. World‐Wide Importance of Parasites 2010.
[3] Organization WH. Working to overcome the global impact of neglected tropical diseases: first WHO report on neglected tropical diseases 2010.
[4] Edrisian G, Rezaeean M, Ghorbani M, Keshavarz M, Mohebali M. Medical protozoalogy 2008.
[5] Minetti C, Chalmers RM, Beeching NJ, Probert C, Lamden K. Giardiasis. BMJ 2016; 355: i5369.
[6] Betancourt WQ, Duarte DC, Vásquez RC, Gurian PL. Cryptosporidium and Giardia in tropical recreational marine waters contaminated with domestic sewage: Estimation of bathing-associated disease risks. Mar Pollut Bull 2014; 85(1): 268-73.
[7] Karambaigi F, Abdi J, Sayehmiri K. Prevalence of Entamoeba histolytica in Iran during 1988 to 2009: Systematic review and meta-analyses Afr J Microbiol Res 2012; 6(17): 3944-7.
[8] John DT, Petri WA. Markell and Voge's Medical Parasitology-E-Book: Elsevier Health Sciences; 2013.
[9] Giangaspero A, Cirillo R, Lacasella V, et al. Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy. Parasitol Int 2009; 58(1): 12-7.
[10] Khairnar K, Parija SC. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples. BMC Microbiol 2007; 7(1): 47.
[11] Adamu H, Endeshaw T, Teka T, Kifle A, Petros B. The prevalence of intestinal parasites in paediatric diarrhoeal and non-diarrhoeal patients in Addis Ababa hospitals, with special emphasis on opportunistic parasitic infections and with insight into the demographic and socio-economic factors. Ethiop J Health Dev 2006; 20(1): 39-46.
[12] Noor Azian MY, San YM, Gan CC, et al. Prevalence of intestinal protozoa in an aborigine community in Pahang, Malaysia. Trop Biomed 2007; 24(1): 55-62.
[13] Zebardast N, Gharavi MJ, Abadi A, et al. Frequency of intestinal parasites in patients with gastrointestinal disorders, in different parts of Iran during 2012-2013. Int J Enteric Pathog 2015; 3(1): 1-5.
[14] Sedighi I, Asadi M, Olfat M, Maghsood AH. Prevalence and risk factors of Giardia lamblia and Blastocystis hominis infections in children under ten years old, hamadan, Iran Avicenna J Clini Microbiol Inf 2015; 2(2)
[15] Grazioli B, Matera G, Laratta C, et al. Giardia lamblia infection in patients with irritable bowel syndrome and dyspepsia: A prospective study World J Gastroenterol 2006; 12(12): 1941-4.
[16] Shakkoury W, Wandy E. Prevalence of Giardia lamblia infection in amman, Jordan. Pak J Med Sci 2005; 21(2): 199-201.
[17] Vahedi M, Gohardehi S, Sharif M, Daryani A. Prevalence of parasites in patients with gastroenteritis at East of Mazandaran Province, Northern Iran. Trop Biomed 2012; 29(4): 568-74.
[18] Tanyuksel M, Petri WA Jr. Laboratory diagnosis of amebiasis. Clin Microbiol Rev 2003; 16(4): 713-29.
[19] Stauffer W, Ravdin JI. Entamoeba histolytica: An update Curr Opin Infect Dis 2003; 16(5): 479-85.
[20] Nasiri V, Esmailnia K, Karim G, Nasir M, Akhavan O. Intestinal parasitic infections among inhabitants of Karaj City, Tehran province, Iran in 2006-2008. Korean J Parasitol 2009; 47(3): 265-8.
[21] Tappeh KhH, Mohammadzadeh H, Rahim RN, Barazesh A, Khashaveh Sh, Taherkhani H. Prevalence of intestinal parasitic infections among mentally disabled children and adults of Urmia, Iran. Iran J Parasitol 2010; 5(2): 60-4.
[22] Zaglool DA, Khodari YA, Gazzaz ZJ, Dhafar KO, Shaker HA, Farooq MU. Prevalence of intestinal parasites among patients of Al-Noor specialist hospital, Makkah, Saudi Arabia. Oman Med J 2011; 26(3): 182-5.
[23] Samie A, Obi LC, Bessong PO, Stroup S, Houpt E, Guerrant RL. Prevalence and species distribution of E. Histolytica and E. Dispar in the Venda region, Limpopo, South Africa. Am J Trop Med Hyg 2006; 75(3): 565-71.
[24] Ramana K. Intestinal parasitic infections: An overview. Ann Trop Med Public Health 2012; 5(4): 279.
[25] Akinbo FO, Ikedje A, Okaka CE. Intestinal parasitic infections among artisans in Benin city, Nigeria. Nig J Basic Clin Sci 2013; 10(2): 66.
[26] Kia E, Hosseini M, Nilforoushan M, Meamar A, Rezaeian M. Study of intestinal protozoan parasites in rural inhabitants of Mazandaran province, Northern Iran. Iranian Journal of Parasitology 2008; 21: 5.
[27] ten Hove R, Schuurman T, Kooistra M, Möller L, van Lieshout L, Verweij JJ. Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR. Clin Microbiol Infect 2007; 13(10): 1001-7.
[28] López-López P, Martínez-López MC, Boldo-León XM, et al. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis. Braz J Med Biol Res 2017; 50(4)e5997
[29] Nkrumah B, Nguah SB. Giardia lamblia: A major parasitic cause of childhood diarrhoea in patients attending a district hospital in Ghana. Parasit Vectors 2011; 4(1): 163.
[30] Elmi T, Ziaie H, Gholami S, Mortazavi M, Tabasi M, Geraili Z. The Prevalence of giardiasis infections among people admitted to Karaj laboratories in 2013: A short report. Majallah-i Ilmi-i Danishgah-i Ulum-i Pizishki-i Rafsanjan 2015; 13(8): 725-30.
[31] Younas M, Shah S, Talaat A. Frequency of Giardia lamblia infection in children with recurrent abdominal pain. J Pak Med Assoc 2008; 58(4): 171-4.