Molecular epidemiology of human intestinal amoebas in iran.

Many microscopic-based epidemiological surveys on the prevalence of human intestinal pathogenic and non-pathogenic protozoa including intestinal amoeba performed in Iran show a high prevalence of human intestinal amoeba in different parts of Iran. Such epidemiological studies on amoebiasis are confusing, mainly due to recently appreciated distinction between the Entamoeba histolytica, E. dispar and E. moshkovskii. Differential diagnosis can be done by some methods such as PCR-based methods, monoclonal antibodies and the analysis of isoenzyme typing, however the molecular study of these protozoa in Iran is low. Based on molecular studies, it seems that E. dispar is predominant species especially in the central and northern areas of Iran and amoebiasis due to E. histolytica is a rare infection in the country. It is suggested that infection with E. moshkovskii may be common among Iranians. Considering the importance of molecular epidemiology of amoeba in Iran and also the current data, the present study reviews the data currently available on the molecular distribution of intestinal human amoeba in Iran.

Introduction

Cases of transmissions for over 18 different human intestinal protozoa have been reported in Iran. Reports on some of these protozoa such as Microspora spp, Cyclospora cayetanensis, Sarcocyctis hominis, Balantidium coli and Entameoba moshkovskii are as case reports but others including pathogen and non-pathogenic species are more common and prevalent. Human intestinal amoeba contain many species, eight of which (Entamoeba histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, E. hartmanni, Iodamoeba butschlii and Endolimax nana) reside in the human intestinal lumen (1). It is well known that E. histolytica is the only pathological species in humans, that causes about 50 million cases of infections with an annual death rate of over 100,000 worldwide (2). The other human intestinal amoeba are considered non-pathogenic and rarely cause intestinal disease in humans (3,4).

Progress in the molecular epidemiology of protozoa infection has been driven by developments in the laboratory techniques, such as PCR and other molecular biological tools. Application of this method to the epidemiology of human intestinal protozoa (e.g. amoeba) revolutionised the knowledge and concept of epidemiology of parasitic infection. Molecular data on the epidemiology of intestinal protozoa, especially pathogenic amoeba, are important for planning the prevention and control programs.

Up to present, many microscopic-based epidemiological studies on the prevalence of human intestinal pathogenic and non-pathogenic protozoa, including intestinal amoeba, have been performed in different parts of the Iran (5–7). Although several studies (5,7–9) showed the high prevalence of human intestinal amoeba in different parts of Iran, however only a few molecular studies on these protozoa were performed in Iran. This is important due to the recent appreciation on the distinction between E. histolytica, E. dispar and E. moshkovskii and signifying that making differential diagnosis cannot be done by microscopy-based methods.

This review performed to determine and provide an overview of the present information on the molecular epidemiology of human intestinal amoeba in Iran.

Material and Methods

Manual and electronic searches in international and national databases and journals were conducted to find the relevant data reporting molecular studies on human intestinal amoeba in Iran. Searching were performed through in the international database such as PubMed (www.ncbi.nlm.nih.gov), Scirus (www.scirus.com), ISI Web of Science (www.isiknowledge.com), Scopus (www.scopus.com), EMBASE (www.embase.com), Science Direct(www.sciencedirect.com), and Google Scholar (scholar.google.com). National database searched were including: Iranmedex (www.iranmedex.com), IranDoc (), Magiran (www.magiran.com) and Scientific Information Database (www.sid.ir). The search covered the articles published up to the 2011.

In an attempt to identify all related studies, a combination of relevant keywords and MeSH terms including the names of each human intestinal amoeba e.g. “Entamoeba”, “Intestinal Amoeba” and the following keywords: “Iran”, “Molecular” or “genum” were used in search strategy. The searching strategy in Iranian databases was the English and Persian transcription with key words related to the study.

To maximize the sensitivity of search, references of selected articles were checked. Moreover the manual search was carried out in the soft and/or hard copies of articles published in scientific journals, reports of research projects and post-graduate theses, as well as the abstracts of scientific articles related to this topic presented at scientific congresses by Iranian researchers.

Results

Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and E. dispar are two genetically distinct but morphologically identical species. These two species could be differentiated by some methods such as specific DNA probe, PCR-based methods, monoclonal antibodies and analysis of the isoenzyme typing (10). On the basis of biochemical, immunological and genetic data, a formal re-description of E. histolytica was published in 1993 separated this species from the harmless commensal E. dispar (11). E. histolytica is a potentially pathogenic species, while the E. dispar is non-pathogenic one. Hence the differentiation of the two species is important in clinical diagnosis and treatment and also from an epidemiological point of view.

Given the criteria mentioned, the epidemiology of amoebiasis has changed since the separation of E. histolytica and E. dispar species. Nowadays it is accepted that E. dispar is the predominant species and much more common than the E. histolytica worldwide (12). However in some regions (e.g. Japan, Mexico, India, South Africa, some Central and South American, and Asian Pacific countries) local prevalence of E. histolytica is especially more common among the male homosexuals (13).

Identification and use of molecular tools for accurate differential diagnosis of Entamoeba spp. such as E. histolytica, E. dispar and E. moshkovskii displays a satisfactory change in the epidemiology of amoebiasis.

In Iran a number of epidemiological studies have been carried out on E. histolytica/E. dispar complex using the routine microscopy stool examination with no separation between them. A prevalence rate of >1% to 30% for E. histolytica/E. dispar complex was reported in different parts of Iran (5). A preliminary comparative study of 15 isolates of E. histolytica/E. dispar isolated from Hamadan, by PCR technique showed that all the isolates were E. dispar (14). A field study on the distribution of E. histolytica/E. dispar cyst passers in northern, central, and southern Iran showed the prevalence of infection with E histolytica/E. dispar was 0.78%, 3.9% and 4.6% for central, northern and southern part of Iran, respectively. The minimum prevalence rate was 0.6% in Tehran, Yazd and Ardekan (central Iran), while the highest rate (8.3%) was seen in rural areas of Ahwaz (southern Iran).This study showed that ratio of E. histolytica/E. dispar was higher in southern (tropical and subtropical regions) than the other regions (15).

Data on differential diagnosis of E. histolytica and E. dispar by molecular methods in different regions of Iran showed that a total number of 92.1% isolates were E. dispar and the remaining 7.9% were E. histolytica or mixed infection (16). This study demonstrated that E. dispar is the predominant species found among the “cyst passers” in Iran (16). The ratio of infection rate with two species in cyst passer in different areas were as follow: In central area (semi temperate zone) 3.5% of isolate were identified as E. histolytica, 93% of isolate were E. histolytica and 3.5% of individuals were infected by both species.

In northern (temperate region) 5.9% of isolates were E. histolytica and 94.1% of isolates E. dispar. In southern (tropical and subtropical region) 7.4% of isolates were E. histolytica and 88.9% E. dispar. 3.7% of person in this area had a mixed infection with two species (16). In Tehran and Karaj, the two major metropolitan areas of central Iran differential diagnosis of 49 isolates of E. histolytica/E. dispar using PCR-RFLP revealed that 46 (93.9%) of isolates were E. dispar, while only 2 (4.1%) E. histolytica. One person (2%) had a mixed infection (10). Another study on 21 isolates of E. histolytica/E. dispar complex in fresh stool by PCR in Tehran showed that 95.45% of isolates were E. dispar and only one isolate (4.55%) was found to be E. histolytica (17). This finding is similar with previous study in this area.

Similar observation that made by Rezaian and Hooshyar(2006),using PCR-RFLP on 21 isolates of E. histolytica/E. dispar in rural areas of Ahwaz and Hamidieh (south of Iran) showed that 19 samples (90.48%) were positive for E. dispar; one (4.76%) positive for E. histolytica and another sample (4.76%) showed mixed infection(5). A local prevalence study in urban and rural areas of Gonbad City (north of Iran) using PCR method showed that 16 samples isolated were E. dispar and none of them showed the E. histolytica pattern (18). A study by PCR/gel electrophoresis, by Haghighi et al. (2009) on eight microscopical-positive E. histolytica/E. dispar samples in Zahedan identified, six of them as E. dispar whereas E. histolytica was not detected there at all (19). A comparative study of stool antigen detection kit and PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from western (Luristan), northwestern (West Azerbaijan), and northeastern (Golestan) part of Iran, showed that all of the 88 samples containing E. histolytica/E. dispar cysts were negative for E. histolytica (20).

All of these studies showed that E. dispar is the predominant species in Iran and amoebiasis due to E. histolytica is a rare infection in Iran (Table 1). Hooshyar et al. (2004) showed that E. dispar is much more frequent than E. histolytica, and also that the E. histolytica/E. dispar ratio is 1:12. Field-based studies in other countries have found the similar ratios: 1:8.5 (Philippines), 1:2.7 and 1:8 (Bangladesh) and 1:46 (Ivory Coast) (16).

Table 1:

Prevalence of E. histolytica and E. dispar In Iran

Method of Diagnosis	Year	Region of study (Iran)	No of cases	Prevalence rate (%)			Reference Arthurs (No.)
				E. his	E. dis	Mixed
PCR based Methods	2001	Hamadan	15	-	100	-	Fallah M et al. (14)
	2001	Tehran	8	-	100	-	Hooshyar et al. (22)
	1999–2001	Different regions of Iran	101	4/9	92/1	3	Hooshyar et al. (16)
		Central region		3/5	97	3/5	Hooshyar et al. (16)
		Northern region		5/9	94/1	-	Hooshyar et al. (16)
		Southern region		7/4	88/9	3/7	Hooshyar et al. (16)
	1999–2001	Tehran& Karaj	49	4/1	93/9	2	Hooshyar et al. (10)
	2003	Ahwaz & Hamidieh	21	4/8	90/5	4/7	Rezaian andHooshyar (5)
	2006	Gonbad	16	-	100	-	Nazemalhoseini et al. (18)
	2006–2007	Tehran	21	4/55	95/45	-	Nazemalhoseini et al. (17)
	2008	Zahedan	8	-	100	-	Haghighi et al. (19)
	2004–2008	Tehran, Zahedan, Gonbad	53	3.54	91/37	-	Nazemalhosseini et al. (27)
	2010	Khorramabad	16	-	93/75	-	Kheirandish et al.(28)
	2011	Chelgerd	10	90	10	-	Pesthechian et al.(21)
Stool antigen detection & PCR	2006	Lurestan, West Azarbayijan and Golestan	88	-	100	-	Solaymani-Mohammadi et al.(20)
Zymodem analysis	2001	Tehran	8	-	100	-	Hooshyar et al. (23,24)
	2002–2005	South Iran	23	26	74	-	Sahebani et al. (25)
	2006	Tehran and Kazerun	2	-	100	-	Haghighi A (29.24)

The only molecular study on amoeba in Iran revealed that E. histolytica was more prevalent than E. dispar is a new study that published by Pestechian et al. (2011).In this study From 655 stool samples in Chelgard City (Chaharmahal and Bakhtisry), 11 E. histolytica/E .dispar observed. By PCR 10 of them were E. histolytica and 1 E. dispar (21). This finding is unlike the previous reported data in other parts of Iran and need to more study in this region.

Isoenzyme electrophoresis in starch gel used to distinguish between the E. histolytica and E. dispar. The chosen isoenzymes were malic enzyme (ME), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI) and hexokinase (HK). A zymodeme is defined as a group of amoeba strains that share the same electrophoretic patterns and mobility for these enzymes (22).

By simultaneous the examination of the characteristic banding pattern of PGM and GPI, it was the possible to classify the amoeba in to one of more than 20 zymodemes (12). Twenty-one out of totally of 23 different zymodemes described from human isolates. Eight zymodems pattern proved to be those of the E. histolytica (zymodems: II, VI, VII, X, XI, XII, XIV and XX), while the rest were belong to E. dispar. Prior to the development of DNA-based techniques, isoenzyme (zymodeme) analysis of cultured amoebae was considered the gold standard for the diagnosis of amoebic infection and differential diagnosis of amoeba species.

Study on zymodems pattern of 8 isolates of E. dispar from Hooshyar et al. (2001) study and two E. dispar isolates from Tehran and Kazeroon (AS2IR and AS16IR) by Haghighi (2006) in Japan showed that all of them were zymodem I (23, 24).

Isoenzyme analysis of 23 isolate of Entamoeba in south of Iran, cultured in Robinson's medium by Sahebani et al. (2002–2005) showed that six samples (26%) were diagnosed as Entamoeba histolytica and 17 samples (74%) as E. dispar. Zymodemes of E. histolytica were II and XIV and, zymodemes of E. dispar were I, XVI, XVII and XVIII (25). Based on these results we suggested that zymodeme I of E. dispar similar to other subtropical countries is the predominant zymodem of E. disar in Iran.

Diagnosis of the extra intestinal amoebiasis is important and is most often made by serological methods or detection of the organism at the time of biopsy or autopsy (4). Molecular methods such as PCR are increasingly useful tools for the diagnosis of extra intestinal amoebiasis and can provide rapid diagnosis. The first case of Entamoeba histolytica encephalitis diagnosed by PCR of the cerebrospinal fluid of an Iranian patient was done by Solaymani-mohammadi et al. (20). The patient who was born in the Sistan-Baluchestan province of the southeastern Iran, immigrated to the United States, and 4 months prior to the presentation of disease had visited Iran for 3 months. Finally the patient was treated successfully with metronidazole(26).

Entamoeba moshkovskii

Entamoeba moshkovskii also is not distinguishable from E. dispar and E. histolytica in its cyst or throphozoite forms. For making differential diagnosis nowadays some PCR based methods are available. This organism is considered primarily a free-living amoeba and has been found to be common in sewage, clean and waste-contaminated water worldwide (12). E. moshkovskii is osmotolerant, can be cultured at room temperature, and is resistant to some antiamoebic drugs (e.g. emetine), all characteristics that distinguish it from E. histolytica and E. dispar(30,31,32). Up to present E. moshkovskii has rarely been shown to infect humans (33), however a recent study reported a high prevalence (21.1%) of E. moshkovskii has been reported in young children from Bangladesh (34). Also E. moshkovskii was recently detected in patients between 31 and 50 years of age presented with gastrointestinal symptoms in Australia (35).

To date, human isolates of this species has been reported from many studies all over the world such as South Africa 13% (36), Australia 24.7% (35), India 1.9%(37). These and other studied showed that, at least in some parts of the world, E. moshkovskii may be a true human parasite. In Iran, a low prevalence of infection with E. moshkovskii of 1.1% was recorded in a healthy person (20). A recent study in Iran reported that out of 3,825 stool samples examined using single-round PCR assay 2(3.5%), 53(91.4%) and 2( 3.5%) were reported positive for E. histolytica, E. dispar and E. moshkovskii, respectively. One person (1.73%) had mixed infection of E. dispar and E. moshkovskii(27). In this study presence of E. moshkovskii was confirmed by sequencing. A latest one study in Iran showed that Out of 16 person infected with E. histolytica/E. dispar/E. moshkovskii cysts by microscopic examination in Khorramabad, single-round PCR showed from sixteen samples that were microscopically positive, 1 (6.25%) was E. moshkovskii, and 15 (93.75%) were E. dispar(28). Considering of this study we suggest that infection with E. moshkovskii may be common among the Iranians. This is an important point in molecular epidemiology of the amoeba in Iran.

Despite using PCR for the detection of E. moshkhovskii in Iran, no attempt was done for its detection by culture methods.

Other intestinal amoeba

Various intestinal amoeba species (e.g. Entamoeba coli, Entamoeba hartmani, Iodamoeba butchlii and Endolimax nana) are frequently found in the stool of human in Iran. A prevalence rate between 1% to >70% was reported in different population around Iran (5, 1, 9). Although these amoebas spp. are considered to be harmless, care should be taken to avoid mistaken diagnosis with E. histolytica, the causative agents of amoebiasis. There is no reliable data on the molecular epidemiology of the nonpathogenic intestinal amoeba in Iran and all surveys previously done were based on the detection of cysts or trophozoites in stools by using light microscopy.

Discussion

Intestinal protozoan infections (e.g. Entamoeba species) are still a public health concern in Iran. There are no comprehensive data on the epidemiology of E. histolytica in Iran especially since the separation of E. histolytica and E. dispar species. The most probable explanation of this condition is due almost to the reliance of all surveys previously done on stool examination by microscopy, not only in Iran, but also in the other parts of the world, because the current epidemiology of amoebiasis is confusing, mainly due to the recently appreciated distinction between the E. histolytica, E. dispar and E. moshkovskii.

A majority of asymptomatic individuals with cysts detected in their stool were actually infected with non-pathogenic E. dispar, Based on molecular study, It seems that E. dispar is the predominant species in Iran and amoebiasis due to E. histolytica is a rare infection in Iran (16, 18, 20), but the definite and true prevalence rate of E. histolytica still has became a matter of speculation. Identification of the true prevalence of E. histolytica infection in the community is very important to predict the clinical burden of ameobiasis. Thus, the future molecular epidemiological study should be done to determine the true prevalence of E. histolytica and E. dispar, especially in the tropical area of Iran. Although previous studies revealed that Zymodemes of E. histolytica were II and XIV and, zymodemes of E. dispar were I, XVI, XVII and XVIII in Iran, subsequent studies suggested that only four zymodems are valuable for isolate typing (Zymodem II, XIV and XIX for E. histolytica and only zymodem I in E. dispar)(12,38,39).The others zymodemes appear to be of bacterial rather than amoebal origin. The remaining zymodemes are due to interaction with bacterial enzymes that present in xenic cultures(40).However zymodems typing have a very important method in recognition of E. dispar as a separate species.

According to the results of the study on E. moshkovskii in Iran we suggest that human infection with this species may be common among Iranians. This may be considered as an important point in the molecular epidemiology of amoeba in Iran.

It has been well established that differential diagnosis of some harmless human amoeba such as E. hartmanni and other intestinal amoeba may be difficult and controversial. So, molecular diagnostic tools can be useful for the accurate diagnosis of infection with these protozoa.

Based on our knowledge of human intestinal protozoa epidemiology, water and waste water has an important role in transmission of amoeba to human, so these amoebas are considered as the waterborne parasites. Investigating the occurrence of Entamoeba species in surface and waste water and also the molecular identification of them in Iran is necessary.

Ethical considerations

Ethical issues (Including plagiarism, Informed Consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.