Comparison of Direct Microscopy and Molecular Method to Detect Amoebiasis Cases from Stool Specimen and Also Identify the Entamoeba Species Involved In Infection: A Study of Nepal

Direct microscopic examination of stained or unstained wet mount preparations or fixed stained smears of clinical material can often provide the etiological diagnosis of an infectious process. A total of 266 human stool specimens from children fewer than 15 years were examined for the presence of amoebiasis by a combination of microscopic examination and molecular method. In molecular method, nested polymerase chain reaction (Nested-PCR) targeting genomic Entamoeba species was used. Stool specimens were collected from 266 participants in Southern plains of Nepal and analyzed at Kathmandu Centre for Genomics and Research Laboratory. The stool specimens were processed by wet mount method using saline as well as iodine staining and examined via microscopy for the presence of Entamoeba cysts or trophozoites. Furthermore, the stool specimens were characterized using Nested-PCR targeting genomic Entamoeba species. Based on microscopic examination, overall prevalence of Entamoeba infection was 17.6% (47/266). The PCR results showed that 52(19.5%) out of 266 stool specimens successfully generated species-specific amplicons. Males (21.7% in PCR) were more commonly infected compared to females (16.6% in PCR). Comparison by age groups showed that 10-15 years age-group (26.6% in PCR) had higher infection than age-group 5-10 years (16.6%) years and 1-5 years (15.2%). The infection with E. histolytica (100%; 52/266) was the predominant cause of amoebiasis, while infection with E. dispar and E. moshkovskii was not found. PCR is a more effective method for the identification of Entamoeba infection than microscopy. This study also suggests that older children especially male are more prone to Entamoeba infection.


Entamoeba
histolytica is the causative agent of amoebiasis and this is one of the leading parasites that cause human mortality (Haque et al., 2003, Haque et al., 2006and WHO 1997).Each year, the parasite invades approximately 50 million people resulting in 100,000 deaths (WHO 1997).Likewise, the productivity of animals is constrained due to Entamoeba infection that can have extensive consequences, ranging from reduced animal performances to mortality (Sykes, 1994;Waller, 1999).
Although the parasite is distributed worldwide; the prevalence rate is higher in the developing countries (Stanley, 2003).Entamoeba infection spreads through fecaloral route (Blessmann et al., 2003 andFotedar et al., 2007).Open defecation and improper disposal waste water often lead to contamination of food and water sources in developing countries and this is a serious issue contributing to human morbidity (Estevenz et al., 1983 andGyawali et al., 2009).
Similarly, grazing management has been a serious issue behind GI infection in animals since pastures are usually not provided or not properly managed.

Entamoeba
histolytica causes gastrointestinal (GI) diseases in farm resulting in reduced productivity, weight gain as well as milk production (Jittapalapong et al., 2011).
Moreover, in some parts of the world, wastewater and human and animal excreta are used as fertilizer to increase the crop yield.This is normally practiced in China, South East Asia as well as various areas in Africa (Drechsel et al., 2011;Cross, 1985 andTimmer andVisker, 1998) with severe water scarcity.Such practices can easily contaminate food sources and spread GI infection to humans and animals.
Furthermore, lack of hygiene and poor hand washing practices among the farm workers play a major role in increasing the infection rate.
Generally, E. histolytica is the common pathogen that can cause such GI diseases.
The diagnosis of an intestinal infection with E. histolytica is commonly made by microscopic examination of stool, in which one must recognize and differentiate the cysts or trophozoites of E. histolytica from those of morphologically different species.The identification of these cysts and trophozoites requires a lot of skill and patience by the microscopist.To overcome the difficulty in recognizing the pathogens and also know the species of the pathogen, molecular methods are increasingly being used for research purposes.Some molecular methods, including conventional and real-time PCR, have been developed for the detection and differentiation of Entamoeba at species level from clinical samples (Hamzah et al., 2006).
Polymerase Chain Reaction (PCR) assay allows the in vitro amplification of a specific DNA fragment in a cyclic process of denaturation, hybridization of primers, and elongation of the DNA strand using a thermostable DNA polymerase.In nested PCR, amplicons from a PCR are used as the template in a second PCR using one primer or two primers different from those used in the initial PCR and located within the sequence amplified by the first primer set.Nested PCRs are used to increase sensitivity and specificity (Messmer et al., 1997).
The study is supportive to describe the current status of amoeba infection in the endemic areas.The study has few general and specific objectives, in general it detect amoeba infection using microscopy and Polymerase Chain Reaction and also identify the Entamoeba species in the stool specimen and in particular it compares the results of PCR and microscopy for the detection of Entamoeba infection also find the Entamoeba species in stool specimen.

MATERIALS AND METHODS
This cross sectional study was carried from 15 July to 18 January 2017.
For this study, stool specimens were collected from hospitals and health centers in southern plains of Nepal and analyzed in Kathmandu Centre of Genomics and Research Laboratory (KCGRL), Lalitpur, Nepal.Nepal lies in sub-tropical zone of Asia sharing its open border with India.Due to compromise in drinking water pISSN-1978-3000 eISSN-2528-7109 Comparison of direct microscopy and molecular method to detect amoebiasis (Das et al., 2018) | 103 supply and unmanaged human settlements in southern plains, prevalence of amoebiasis is higher especially in young children who play in soil most of the time.
The samples were taken from patients complaining diarrhoea from age 1 to 15 years.A total of 266 specimens were collected and processed at KCGRL.Samples from small kids were taken by the help of their care-takers.The filled and well-labeled containers were received and analyzed by microscopy on the following day; the samples were mixed with formalin and then stored under deep freeze for further processing.

Microscopic Examination
Microscopy is inexpensive and rapid technique for the identification of Entamoeba parasite.It is the gold standard tool for the diagnosis of amoebiasis cases and a method of choice for many hospitals.Saline wet mount of fresh unpreserved stool samples were examined microscopically for demonstrating Entamoeba cysts and trophozoites (Parija and Prabhakar 1995).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.Following the extraction of genomic DNA for protozoan parasite from all the stool specimens, the specimens showing the presence of protozoan DNA were further re-checked for Entamoeba species using nested-PCR.
For secondary PCR, the master mix was heated to 96°C for 2 min, followed by 30 cycles of 92°C for 1 min (denaturing), 48°C for 1 minute (annealing), 72°C for 1 minute (extension) and a final extension at 72°C for 7 minutes.For both amplifications, master mix was incubated in the thermal cycler (Palm cycler).The secondary PCR amplicons were subjected to electrophoresis in 2% agarose gel stained with ethidium bromide (EtBr) at 80V for 60 minute and visualized in UV illuminator.

RESULTS AND DISCUSSION
Entamoeba species was detected under microscope on the basis of morphological characteristics of the cysts or trophozoites.The average cyst size ranges from 10-20 μm in diameter.Qualitative data were estimated and presented as frequencies and percentage by using univariate analysis through SPSS statistical tool.

Prevalence of Entamoeba Infection via Microscopy and PCR
A total of 266 stool samples collected were processed through microscopy and PCR methods separately and results thus obtained varied a lot.Out of all the stool samples processed, 47 (17.6%) samples were microscopically positive for Entamoeba cysts (Table 1

Age-wise Prevalence of Entamoeba Infection
Age-wise study of Entamoeba infection detected via microscopy and PCR methods showed following results as shown in Table 3. (26.6%) respectively.But the age-group 1-5 years had least prevalence of 8 (13.6%) and 9 (15.2%) in microscopy and PCR respectively.
The PCR results were positive for only one species of Entamoeba i.e.E. histolytica.Other species i.e.E. dispar and E. moshkovskii were not detected in any of the samples.Thus the chances of mixed infection were also ignored.
Although, the microscopic observation showed 47 positive cases of Entamoeba infection out of 266 total stool specimens, it was not considered overall prevalence in this study population.The present study found an overall prevalence of Entamoeba infection as determined by PCR as 17.6% (52/266).Thus the molecular method has been found more effective in contrast to microscopy.This study also suggests that relying only on microscopy cannot diagnose accurate cases of amoebiasis.
A study conducted on children in Kathmandu, Nepal reported 10% (50/500) of the causative agents of diarrhoea were E.
histolytica (Pokharel et al., 2007) which is consistent with this study.A recent study in India also has more or less similar results of 19% positive Entamoeba infection based on microscopic examination (Nath et al., 2015).In a study conducted by Tuzemen   Ali et al., 2003) and thus it can be pISSN-1978-3000 eISSN-2528-7109 Comparison of direct microscopy and molecular method to detect amoebiasis (Das et al., 2018) | 107 speculated that they may exist in Nepal even though not identified in this study.
Although human cases infected with E. moshkovskii have been reported sporadically from different parts of the world including India (Khairnar and Parija 2007) Bangladesh (Ali et al., 2003) and Australia (Fotedar et al., 2007), they have been attributed to asymptomatic infection most frequently.The global prevalence of E. dispar/E.histolytica is quite uncommon with this study but it cannot be ignored that a single study with limited number of subject's i.e.only children may not be sufficient to assess the overall prevalence of Entamoeba species over certain geographic location.Therefore, future investigation which includes the clinical impact of E. moshkovskii and other Entamoeba species is imperative for a better understanding of a true pathogenic potential of E. moshkovskii.
Sex-wise study shows higher prevalence in males in comparison to females both in microscopy and PCR.This could be attributed to the fact that male members have freedom to participate in outside activities in southern plains of Nepal while the female children have family restriction and need permission of their parents to move out of their house.This situation play a bigger role in transmitting the amoebiasis in male children than female children as one would eat contaminated food or drink water while they are outside their houses.Furthermore, open defecation and eating without washing the hands has further impact of being infected.
Age-wise study shows increasing prevalence of amoebiasis with increasing age of children.The possible reason for this may be more or less the same as sex-wise prevalence.As a child grows older, he can go outside their house to play with their friends, swim in the nearby rivers or lakes which are already polluted by open defaecation.Moreover, there is no provision of clean drinking water in most of the schools of Nepal as well.Contaminated water or food may contain Entamoeba cysts and can easily transmit the infection (Chakraborty, 2001).
The participants of this study were from the southern plains of Nepal where there is no easy access to clean drinking water and living condition is poor.Given the faeco-oral route of amoebiasis, habits related to eating, defaecation, personal hygiene, cleanliness and level of education may have an impact on the prevalence rates (Ngui et. al 2012).Because of such unhygienic practices, the chances of parasitic infections have persisted (Sherchand SB 1997and Maharjan et al., 2007).The majority of the people in Nepal still live in the rural areas where literacy rate is quite lower.Unavailability of medical facilities and social awareness in such regions may contribute to high prevalence of Entamoeba infection.

CONCLUSION
In conclusion, PCR is a more effective method for the identification of Entamoeba infection than microscopy.In the southern plains of Nepal, E. histolytica is the major cause of amoebiasis and male children are more prone to Entamoeba infection.Besides, older children are more vulnerable to amoebiasis than young children.

Table 2 .
Entamoeba infection by sex

Table 3 .
Entamoeba infection by Age-wise

Table 4 .
Molecular technique of detecting other Entamoeba species