A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.


Background
Infection with Entamoeba histolytica results in 34 million to 50 million symptomatic cases of amoebiasis worldwide each year, causing 40 thousand to 100 thousand deaths annually [1]. E. histolytica, the pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. dispar [1] and E. moshkovskii [2], except in rare cases of invasive disease when E. histo-lytica trophozoite may contain ingested red blood cells [3]. Estimates of intestinal E. histolytica infections have been primarily based on microscopic examination of stools, which has a sensitivity of only 60%, even under optimal standards [4,5]. The microscopic examination of stool alone, however, fails to differentiate E. histolytica from those of morphologically similar non-pathogenic species such as E. dispar and E. moshkovskii. Stool culture followed by isoenzyme analysis enables the differentiation of E. histolytica from E. dispar [6]. However, this method of culture when followed by isoenzyme analysis requires one to several weeks to obtain the result and also special laboratory facilities, making it impractical for use in the routine diagnosis of intestinal amoebiasis.
Various approaches are being followed for specific identification and detection of E. histolytica in stool specimens such as detection of E. histolytica coproantigen in stool samples by enzyme-linked immuno-sorbent assay (ELISA) and detection of E. histolytica DNA in stool samples by polymerase chain reaction (PCR). Currently few commercial ELISA kits are available for detection of E. histolytica/E. dispar coproantigen in stool. These include TechLab Entamoeba test to detect E. histolytica/E. dispar [5], Alexon ProSpecT ELISA to detect E. histolytica/E. dispar and Giardia lamblia [7] and Triage parasite panel to detect antigen of E. histolytica/E. dispar, Giardia lamblia and Cryptosporidium parvum in stool specimens [8]. The main limitation of all these ELISA kits is that they can identify the amoebae only as E. histolytica-E. dispar complex but not specifically as E. histolytica, E. dispar or E. moshkovskii. However, a monoclonal antibody based Tech Lab E. histolytica II ELISA is commercially available for the specific detection of E. histolytica antigen directly in stool specimen [9], but this kit can neither detect E. dispar nor E. moshkovskii in stool specimen.
Recently, a nested PCR targeting 16S-like rRNA gene has been reported from the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh (ICDDR,B) [2] as well as from our laboratory at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) hospital, Puducherry, India [10] to detect and differentiate E. histolytica, E. dispar and E. moshkovskii directly in stool specimens. But the identification of the amoeba in the stool specimens either as E. histolytica, E. dispar or E. moshkovskii was carried out by performing nested PCR each time separately for individual species which was tedious. To overcome this disadvantage, the main objective of the present study was to develop and evaluate a nested multiplex PCR targeting the 16S-like rRNA gene for simultaneous detection and differentiation of E. histolytica, E. moshkovskii and E. dispar directly in stool samples.

Nested multiplex PCR Quantification of DNA in stool specimen
The DNA yield was found to be approximately 49 µg/ml. The purity of DNA extract from stool specimen was found to be satisfactory as the value of ratio of readings at 260 nm and 280 nm (OD 260 /OD 280 ) was approximately 1.8.  Figure 2).

Cross checking the results of nested multiplex PCR
The cross checking of the results of nested multiplex PCR was satisfactory as the same results were reproduced in randomly selected samples showing a mixed infection when subjected to individual species specific PCR in separate tubes.

Nested multiplex PCR
The nested multiplex PCR developed and evaluated in the present study showed that the size of diagnostic fragments of PCR products was clearly different for all the three Entamoeba species, the species-specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively ( Figure 3).  Table 1). The result of the nested multiplex PCR as compared with microscopy and culture is also summarized in Table 1.
Sensitivity of PCR for detection of minimum number of E. moshkovskii (EM), E. histolytica (EH) and E. dispar (ED) cells     [17], the study reported that out of 27 stool samples positive for Entamoeba spp. by microscopy only 7 were successfully identified at species level by PCR, which included 1 positive for E. histolytica and 6 for E. dispar, but no amplification of E. moshkovskii was observed in a Thai population [17]. In contrast, our study has shown the presence of E. moshkovskii in the Indian population. The negative result for E. moshkovskii in Thai population may be attributed to the small sample size used in the study. A detailed comparative study between these two newly described PCR techniques may yield useful information especially in the field of molecular-based diagnosis of intestinal amoebiasis.
Nested PCR was used in the present study because it increases sensitivity [18]. Clinical specimens such as stool often contain PCR inhibitors even after purification steps. The two rounds of PCR might have assisted in compensating the effects of inhibitors present in clinical specimens. The first PCR product may be in too low concentration for detection with ethidium bromide stained gels using a UV transilluminator. The detection limit of agarose gel electrophoresis with ethidium bromide stain using an UV transilluminator is approximately 10 ng of DNA [19]. The product of first PCR may be just enough to provide adequate templates for the synthesis of second PCR product in the nested reaction to be detected by ethidium bromide staining.
The nested multiplex PCR was negative in 12 out of 202 stool specimens that were positive for E. histolytica/E. dispar /E. moshkovskii complex trophozoites/cysts by microscopy and/or culture. The negative result due to inhibition of PCR in all these 12 stool samples was ruled out by spiking with standard DNA of Entamoeba followed by nested multiplex PCR amplification.
The negative PCR result in these 12 stool samples may be due to the presence of other Entamoeba species. However, we feel that this supposition needs to be proven by further development of molecular tools to confirm the presence of other Entamoeba species commonly found in humans, such as E. coli, E. hartmanni or other similar looking Entamoeba species. Till then these negative results may be imputed to the sensitivity limitation of the nested multiplex PCR technique.  Nested multiplex PCR was positive for E. histolytica DNA in 6 stool specimens which were negative for coproantigen by TechLab ELISA. The possible reason for such occult infection (PCR-positive, ELISA-negative stool specimens for E. histolytica) may be due to degradation of lectin antigen in stool specimen due to prolonged storage (approximately 30-60 days) at -20°C prior to testing; thus resulting in a negative test for amoebic coproantigen by the ELISA.
In the present study the overall correlation between the results of nested multiplex PCR and that of TechLab E. histolytica II ELISA to detect E. histolytica in stool specimen was greater than 90%. This agreement between these two techniques shows clearly that either of the techniques may be used alone to yield an accurate assessment of the presence of E. histolytica in a stool specimen, but not for the detection of either E. dispar or E. moshkovskii in stool specimens by TechLab E. histolytica II ELISA.
The inability of TechLab E. histolytica II ELISA to detect either E. dispar or E. moshkovskii in stool specimens is the noted disadvantage of the test. The nested multiplex PCR, on the other hand appears to be more useful for simultaneous detection of all the three species,E. histolytica, E. moshkovskii and E. dispar when performed directly on the stool specimens. This is the main advantage of this test, and is of importance due to the fact that there is an increasing documentation of both E. moshkovskii and E. dispar from different parts of the world [2,6,9,[11][12][13][14][15][16][17], including from Puducherry, the southern union territory of India [10]. The coexistence of non-pathogenic E. dispar and E. moshkovskii as mixed infection or solely as monoinfection amongst the patients showed an increased possibility of faulty diagnosis when the identification of E. histolytica was based primarily on morphology by microscopic examination of stool. The high PCR positivity of E. moshkovskii among the study population supports the view that humans are a true host for this free-living amoeba [2].

Conclusion
The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid; results of the test are available within 12 hours of receipt of stool specimens.

Microscopic examination of stool
Both saline and iodine wet mounts of fresh unpreserved stool samples were examined microscopically for demonstrating E. histolytica/E. dispar/E. moshkovskii complex cysts and trophozoites as previously described [22]. Briefly, saline wet mounts were made by mixing approximately 2 mg of stool with a drop of physiological saline on a glass microscope slide and placing a cover slip over the stool suspension. Similarly, iodine wet mounts were prepared by adding approximately 2 mg of stool to a drop of Lugol's iodine (diluted 1:5 with distilled water) on a glass microscope slide and placing a cover slip on the stool suspension. These wet mounts were microscopically examined initially by using a low-power (10×) objective and then using a high-power (40×) objective of a compound light microscope. The wet mount was read in approximately 5 min to view at least 100 fields per slide. Each stool sample was screened by at least three well trained microscopists before reporting negative results in our laboratory.

Stool culture
Stool samples were cultured for Entamoeba species in Locke-egg (LE) medium (NIH modification of Boeck and Drbohlav's medium) within 6 h of collection as previously described [23,24].

Nested multiplex-polymerase chain reaction (PCR)
Extraction of genomic Entamoeba DNA The DNA was isolated from stool specimens by a cetyltrimethylammonium bromide (CTAB) extraction method modified from previously described method for DNA isolation from in vitro cultures of Entamoeba [25]. Briefly, 50 mg of stool specimen, was dispersed in 250 µl of lysis buffer (0.25% sodium dodecyl sulfate in 0.1 M EDTA, pH 8.0), and 100 µg/ml of proteinase K was added. The lysate was incubated at 55°C for 2 hours. Then 75 µl of 3.5 M NaCl followed by 42 µl of 10% CTAB/0.7 M NaCl (heated to 55°C) was added. After the components were mixed, the sample was incubated at 65°C for 30 min. This was followed by extractions with equal volumes of chloroform and then phenol-chloroform-isoamyl alcohol, and the DNA was precipitated with ice cold ethanol. The dried DNA pellet was dissolved in sterile distilled water and passed over a DNA clean-up spin column (Bangalore Genei KT-62, Bangalore). The DNA was finally eluted from the spin column in 100 µl of Tris-EDTA (TE) buffer; 2.5 µl of this DNA solution was used in the PCR reaction.

Quantification of DNA in stool specimen
Quantification of DNA in spin column purified DNA extract from stool specimens was determined by UV absorbance using a Cintra 5 double beam spectrophotometer. DNA yields were calculated on the basis of UV absorbance × dilution. The purity of the nucleic acid in the samples was estimated by the ratio of readings at 260 nm and 280 nm (OD 260 /OD 280 ). The quantification of DNA was done only for representative stool specimens to know the DNA yielding capacity of the CTAB-DNA extraction method and also to estimate the purity of extracted DNA for its suitability to be used in PCR.

Primer design
The genus specific primers were designed using nucleotide sequences of Laredo revealed significant differences enough to design species specific primers. The primers were designed using Primer 3 on-line software [26]. The primer sequences used for nested multiplex PCR are shown in table 4.

Primer validation
The primer sequences designed for E. moshkovskii, E. histolytica, and E. dispar were subjected to a Basic Local Alignment Search Tool (BLAST) in the genome database of all organisms [27] and were found to be specific for the study.
The amplified PCR products of all the three species in stool samples were confirmed by getting both the strands of DNA sequenced on ABI PRISM 377 sequencer (Indian Institute of Science, Bangalore, India). Briefly, the ABI PRISM 377 DNA sequencer automatically analyzes DNA molecules labeled with multiple fluorescent dyes. After samples are loaded onto the system's vertical gel, they The conditions for genus specific PCR were as follows The PCR mix was subjected to an initial denaturation at 96°C for 2 minutes, followed by 30 cycles -each consisting of 92°C for 60 seconds (Denaturation), 56°C for 60 seconds (Annealing), and 72°C for 90 seconds (Extension). Finally one cycle of extension at 72°C for seven minutes was performed. In the species specific nested multiplex PCR (which had multiple primer sets in the same tube), only the annealing temperature was changed to 48°C, leaving the other parameters of the amplification cycles unchanged.
Three micro litres of the amplification products were separated by electrophoresis through 1.8% agarose gel (Agarose Low EEO, Bangalore genie products, Bangalore, India) in 0.5 × Tris-borate-EDTA at 120 V for 45 min and were visualized by ethidium bromide staining under UV light for bands of DNA of appropriate sizes (Figure 3). Positive and negative control reactions were included with each batch of samples analyzed by nested multiplex PCR.

Assessment of competition for non-target DNA
To assess the competition of other non-target DNA present in stool samples with target DNA, the nested multiplex PCR was checked with reference DNA (DNA from standard strain of E. histolytica, E. dispar and E. moshkovskii) spiked with DNA from negative control stool samples (negative by microscopy and culture) followed by nested multiplex PCR amplification.

Estimation of minimum number of Entamoeba cells detectable by nested multiplex PCR
To determine the minimum number of Entamoeba cells detectable by nested multiplex PCR, all the three species were studied by Locke-egg (LE) medium (NIH modification of Boeck and Drbohlav's medium) cultures and the amoebae were counted using a standard haemocytometer. A cell pellet containing 10 6 cells was preferred for determining the detection limit of nested multiplex PCR for each Entamoeba species. The cell pellet containing 10 6 cells of E. histolytica, E. dispar and E. moshkovskii was diluted ten folds in Phosphate buffer saline (PBS) to obtain different concentrations of cells, such as 10 5 , 10 4 , 10 3 , 10 2 and 10 cells. The different quantities of cells ranging from 10 6 to 10 cells were added to 0.05 gm of faeces (negative control stool samples) followed by DNA extraction and PCR as per the aforementioned protocol.