Entamoeba histolytica,
E. dispar, and
E. moshkovskii are morphologically identical but biochemically and genetically different. These parasites colonize the human gut, but only
E. histolytica is thought to be capable of causing disease. Amebiasis, defined as asymptomatic, invasive intestinal or extraintestinal disease due to
E. histolytica infection, is one of the most common parasitic infections worldwide, infecting about 50 million people, frequently in developing countries, resulting in 40,000 to 100,000 deaths per annum (
21). Asymptomatic cyst passage is the most frequent manifestation of intestinal
Entamoeba infection. All
E. dispar and
E. moshkovskii infections and 90% of
E. histolytica infections are asymptomatic (
21). The studies conducted to date that have used methods capable of differentiating between the two species suggest that, in general,
E. dispar is much more prevalent than
E. histolytica (
9,
10,
11) and that only a small proportion of individuals specifically infected with
E. histolytica will progress to having amebic disease (
8). The necessity to identify and treat asymptomatic carriers of
E. histolytica is emphasized by the observation that 10% of them develop invasive amebiasis in due course (
6). Additionally, asymptomatic carriers are more likely to spread the disease than symptomatic persons with invasive diseases, as the latter individuals seek medical attention (
12).
In nearly all of the studies of asymptomatic amebic cyst passers in which the distribution of the two species has been investigated (
1,
3,
5,
11,
16,
18),
E. dispar has been found to be much more prevalent than
E. histolytica; however, in a few studies, including two from Mexico (
14,
15) and one targeting mentally retarded individuals in Japan (
20), either
E. histolytica occult infections were more common than
E. dispar infections or
E. histolytica was the sole
Entamoeba sp. found.
Several microscopy-based epidemiological studies in Iran have shown
Entamoeba sp. infection rates of 2.2% to 30% (
13,
17). The current study, therefore, was designed to address the distribution of
E. histolytica and
E. dispar in regions of Iran where previous microscopy-based studies showed a high prevalence of infection and to compare the commercially available TechLab
E. histolytica II kit to PCR.
A total of 1,037 single fresh stool samples were collected from apparently healthy persons in rural and semirural settings of western (Luristan), northwestern (West Azerbaijan), and northeastern (Golestan) Iran, where the standards of hygiene are suboptimal. Stool samples were collected from persons that had given their informed consent prior to the collection according to the Iranian Ministry of Health, Treatment and Medical Training Protection Code of Human Subjects in Medical Research.
Ritchie's fecal concentration method of formalin-fixed specimens was performed on formed stool specimens, and the cysts were stained with Lugol's iodine and identified by microscopy (
4). The stool samples were tested within 24 h after collection, or the samples were kept at −20°C for later analysis.
The TechLab E. histolytica II test (TechLab, Inc., Blacksburg, VA) was performed on stool samples containing E. histolytica/E. dispar cysts according to the manufacturer's instructions. This diagnostic kit is an antigen-based enzyme-linked immunosorbent assay and is designed to detect specifically E. histolytica and not the closely related nonpathogenic E. dispar. The well strips were measured in an automatic microtiter plate reader (Organon Teknika, Salzburg, Austria) at 450 nm. Positive results were defined as an optical density reading of ≥0.05 after subtraction of the negative control optical density.
Fecal specimen sediments (0.2 g) were used for DNA extraction by use of a QIAamp DNA stool mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.
Genomic DNA (5.0 μl) was subjected to PCR using primers (Alpha DNA, Quebec, Canada) P1 (5′-TAA AGC ACC AGC ATA TTG TC-3′) and P4 (5′-TTA ATT CCA TCT GGT GGT GG-3′), which were described previously (
19). A second set of primers (TAG Copenhagen, Denmark), HF (5′-AAG AAA TTG ATA TTA ATG AAT ATA-3′) and HR (5′-ATC TTC CAA TTC CAT CAT CAT-3′), located within the fragment replicated by P1 and P4, making this a nested PCR, were designed, resulting in a 374-bp fragment for the axenic isolate HM1:IMSS. Amplification consisted of 35 cycles of 30 s at 94°C, 45 s at the primer-dependent annealing temperature of 54.5°C, and 1 min at 72°C, with a final extension of 7 min at 72°C. This step was followed by the nested PCR using 1 μl of the initial PCR product as the template DNA. The annealing temperature was raised from 54.5°C to 57°C for the nested PCR, while the other parameters remained unchanged. Aliquots of 15 μl of PCR products were digested with 1 μl of the restriction endonuclease HinfI (Roche Diagnostic GmbH, Mannheim, Germany) according to the supplier's instructions. Digested DNA was fragmented and visualized on 2% agarose (Merck, Darmstad, Germany).
The samples that were positive by microscopy but negative by using both the TechLab antigen detection kit and PCR for the presence of
E. histolytica were subjected to a nested PCR with the
E. moshkovskii-specific small-subunit (SSU) rRNA gene nested primers (Operon, Alabama) Em1 (5′-CTC TTC ACG GGG AGT GCG-3′), Em2 (5′-TCG TTA GTT TCA TTA CCT-3′), nEm1 (5′-GAA TAA GGA TGG TAT GAC-3′), and nEm2 (5′-AAG TGG AGT TAA CCA CCT-3′) to detect the presence of concurrent
E. moshkovskii infections (
2). In the initial PCR (total volume, 50 μl), 5.0 μl of stool DNA was used. The PCR included 30 cycles, each consisting of 92°C for 1 min, 55°C for 1 min and 15 s, and 72°C for 1 min, followed by a final extension of 7 min at 72°C. In the nested PCR, 1 μl of the first PCR product was used as the template DNA, the annealing temperature was increased to 62°C, and the other parameters were left unchanged.
E. moshkovskii-specific nested SSU rRNA gene amplification product was digested with restriction endonuclease XhoI for 1 h at 37°C according to the manufacturer's instructions (Roche Diagnostic GmbH, Mannheim, Germany) to verify species identity. All PCR products were separated in 2% NuSieve 1:1 agarose gels (Invitrogen, Carlsbad, Calif.) in 1× Tris-acetate-EDTA buffer and visualized after staining with ethidium bromide (0.2 μg ml
−1).
Of the 1,037 stool samples examined by microscopy, 776, 158, and 103 samples were from western, northwestern, and northeastern Iran, respectively. A total of 88 persons (8.4%) of both sexes were infected by E. histolytica/E. dispar complex cysts. The combined frequencies of E. histolytica/E. dispar according to stool microscopy were 9.3% in Luristan, 5% in West Azerbaijan, and 8.2% in Golestan.
Other intestinal parasites were commonly observed in the persons examined. A total of 335 (32.3%) of the individuals examined were identified as having at least one parasite species, including Entamoeba coli (11.6%), Giardia lamblia (10.6%), E. hartmanni (6%), Iodamoeba butschlii (3.9%), Endolimax nana (0.9%), Chilomastix mesnili (0.7%), Hymenolepis nana (0.5%), Dientamoeba fragilis (0.5%), Taenia sp. (0.2%), and Trichostrongylus sp. (0.09%).
All the samples containing E. histolytica/E. dispar cysts were negative for E. histolytica antigen using the E. histolytica II kit (optical density of <0.05 after the subtraction of the negative control optical density).
An initial 540-bp fragment of genomic DNA was amplified using primers P1 and P4, followed by nested PCR using primers HR and HF to amplify an internal fragment of 374 bp (Fig. ). The PCR products, both primary and nested, were the same size as those of the positive controls (HM1:IMSS strain of E. histolytica and AS2IR strain of E. dispar). After digestion of the nested-PCR product with the restriction enzyme HinfI, two (155-bp and 219-bp) and three (67-bp, 152-bp, and 155-bp) expected fragments were observed for E. histolytica and E. dispar, correspondingly. The 152-bp and 155-bp fragments in E. dispar overlapped and appeared as a single band (Fig. ).
The restriction fragment length polymorphism analysis of the nested-PCR product of genomic DNA from all 88 E. histolytica/E. dispar cysts examined showed the E. dispar pattern (67-, 152-, and 155-bp fragments), while no E. histolytica or mixed-infection electrophoretic patterns were seen. All 88 samples with negative results by TechLab E. histolytica II showed an E. dispar electrophoretic pattern in nested PCR. According to the results of this survey, there was a 100% correlation between the E. histolytica II kit and nested-PCR results.
The reference strain E. moshkovskii Laredo (ATCC accession number 30042) gave the expected band at 258-bp with the E. moshkovskii-specific SSU rRNA gene nested primers, whereas samples from the control strains E. histolytica HM1:IMSS and E. dispar AS2IR were not amplified (Fig. ). To identify species, the nested-PCR products were cut with the restriction enzyme XhoI, and two fragments (236 bp and 22 bp) were produced (data not shown). Only 1 of the 88 (1.1%) microscopy-positive stool DNA samples examined was positive for E. moshkovskii. The child dually infected with E. moshkovskii and E. dispar had a soft stool but no other significant gastrointestinal symptoms.
The most important findings of the present study were that (i) all Iranian asymptomatic cyst passers were infected by nonpathogenic E. dispar, and not pathogenic E. histolytica, in the regions studied; (ii) there was a 100% correlation between the results from stool antigen detection and those from the nested-PCR method; and (iii) E. moshkovskii was identified in one Iranian asymptomatic cyst passer.
In most regions,
E. dispar is the commonest cause of
E. histolytica/E. dispar complex infection in asymptomatic carriers. In Iran, the only previous report on the separate distributions of
E. histolytica and
E. dispar demonstrated that
E. dispar was the prevailing species in Iranian amebic cyst passers (
11). This was true even in the tropical areas of the south, where previous surveys showed that up to 30% of asymptomatic individuals residing in rural areas with poor sanitation were infected by
E. histolytica/E. dispar. By use of an enzyme-linked immunosorbent assay-based test, it was more recently shown that just 8 of the 51 (15.6%) asymptomatic amebic cyst passers examined had anti-
E. histolytica antibodies, supporting the hypothesis that
E. dispar is more prevalent in asymptomatic cyst passers in this country (
7).
The results of the present study showed a 100% correlation between the TechLab antigen detection kit and traditional nested-PCR results, indicating that the TechLab antigen detection kit is a specific and reliable assay for the diagnosis of E. histolytica and E. dispar infections in apparently healthy carriers compared with a sensitive nested PCR. To our knowledge, the current study is the first study to evaluate this commercially available kit for this purpose.
A high prevalence of
E. moshkovskii infection (21.1%) has been detected in preschool children in Bangladesh, indicating that, at least in some parts of the world, it may be a true human parasite (
2). In the present study, a low prevalence of infection with
E. moshkovskii of 1.1% was recorded, showing that this putatively free-living amoeba is a rare human parasitic infectious agent in Iran. It is necessary to consider the presence of
E. moshkovskii infections in asymptomatic cyst passers in order to avoid unnecessary antimicrobial therapy, especially when microscopy is the main route of diagnosis and when other diagnostic methods, including the TechLab kit and PCR, for
E. histolytica and
E. dispar infections, are negative.
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