This document describes a study that used nested PCR to differentially detect Entamoeba dispar and Entamoeba moshkovskii in stool samples. The study found an overall infection rate of 15.36% among 59 samples tested, with E. dispar detected in 10.17% of samples and E. moshkovskii in 5.1% of samples. Nested PCR proved to be more sensitive than other diagnostic methods for differentially detecting these morphologically identical parasites. Further research is needed to determine the pathogenic potential of E. dispar and E. moshkovskii.

1. Differential Detection ofDifferential Detection of
Entamoeba disparEntamoeba dispar andand
Entamoeba moshkovskiiEntamoeba moshkovskii UsingUsing
Nested PCRNested PCR
Hala Amin IssaHala Amin Issa
2062163220621632

2. Outline
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgement

3. Introduction
 E. dispar and E. moshkovskii
are morphologically identical
to pathogenic E. histolytica.
 The two Entamoebas which
were discovered recently,
inhabit the large intestines.
 Been mistaken for E.
histolytica for decades,
which led to worldwide
increase in its prevalence.
Cysts of E. histolytica/E. dispar
stained with trichrome.

4. Aim of the Study
 Differentially detecting the two
morphologically identical species of
Entamoeba; E. dispar and E. moshkovskii.
 Determine the rate of infection of both
Entamoeba species in our study group.

5. Materials and Methods
 Sample details:
 A total of 59 samples were collected from Sharjah
municipality
 Age range between 20-52
 Different nationalities
 No mentioned symptoms
 Sample processing and Extraction:
 Extraction was performed using QIAGEN kit for DNA
purification from stool samples.
 Extracted specimens stored at -20°C

6. Materials and Methods
 Nested Polymerase Chain Reaction (PCR):
 Standard strains as positive control:
 E. dispar SAW 760
 E. moshkovskii Laredo
 2 rounds of PCR:
 1st
PCR round
 Nested PCR round
 Ethidium bromide stained Agar gel visualization under
UV light.

7. Materials and Methods
Fig (A): 1.2% ethidium bromide stained garose gel in 1X TAE buffer;
Lane-1; 100 bp Ladder, Lane-3; positive E. moshkovskii control (500 bp),
Lane-5; negative control,
Lane (7-15); samples 9-19 negative for E. moshkovskii excluding 13 &
17
15 7 5 3 1
Agar Gel Visualisation
under UV light

8. Results and Discussion

9. %of total E. dispar and E. moshkovskii
infections in tested stool samples using PCR
Types of
Entamoeba
Species
No. of positive
samples by
nested PCR
(%)
E. moshkovskii 3/59 5.1%
E. dispar 6/59 10.17%
Table (1)

10. Rate of mixed infections in the studied
samples
Mixed Entamoeba
infections
Results of nested PCR
E. dispar + E.moshkovskii
(mixed)
1/59
E. dispar + E. histolytica
(mixed)
2/59
E. histolytica +
E.moshkovskii (mixed)
0/59
Table (2)

11. Differential detection of E. dispar and E.
moshkovskii by nested PCR on stool samples under
UV light..
The E. dispar (ED) and E. moshkovskii (EM) bands were 174 and 553
bp respectively
12391113
1 2 3 7

12.  The overall rates of infection
of the two parasites in the
study were found to be
15.36% (9/59)
 Rate of total infection with E.
dispar was higher than that
of E. moshkovskii.
 Though the sample size was
small. But other studies
yielded same results in
different countries.
5.1
10.16
0
2
4
6
8
10
12
E. moshkovskii E. dispar
Pecentage

13. Correlation of different studies with
the present one
E. dispar E. moshkovskii
Stark et al
(Australia)
70.8% 61.8%
Khairnar et al
(India)
49.5% 1.0%
Hamzah et al
(Thailand)
20% 0%
Ibne Karim et al
(Bangladesh)
35.8% 21.1%
Entamoeba
Studies

14. Pathogenicity
Bangladesh Study:
•High prevalence of E. moshkovskii
•73% were mixed
•4 with diarrhea
•1 of diarrhea was co-infected
with E.dispar and E.moshkovskii
Turkey Study:
•2 symptomatic cases.
•Microscopy: both negative for
Entamoeba species
•PCR: 1/2 was positive for
E. histolytica
•ELISA: both positive for
E. histolytica

15.  Significance of the results:
 Prevalence of each species
 Clinical importance in management of patients’
treatment.
 Epidemiology:
 Common infection in HIV infected individuals
(Tanzania).
 Mental institutes and orphanages
 Homosexual men

16. PCR vs. other diagnostic methods:
 Protocol adapted from a study conducted in India.
 Increased sensitivity of the adapted method
 Drawbacks of other diagnostic tools:
 Microscopy
 Isoenzyme analysis
 Serological methods
 ELISA

17. Conclusion & Recommendations
 PCR has proved to be the best method in differentially detecting
the 3 morphologically identical Entamoebas.
 More studies need to be done to determine the potential
pathogenicity of E. dispar and E. moshkovskii.
 This work is a part of an ongoing study, where the ultimate goal
is to be able to perform a nested multiplex PCR on a broader
sample size.

18. Acknowledgement
 I would like to express my deepest gratitude to the Mr. Ali Al
Bakri, Dr. Ra’ed Abu Odeh , and Ms. Sinda Ezzedeen for
constantly encouraging me and guiding me through out this
project that couldn't have been prepared if it wasn't for them.
 I am indebted to many of my colleagues who supported me and
gave me confidence, especially Sahar Abboud, Nazenin
Mohandesi, Mahra Karima, Diana Nashash, and Lina Darwich.
 Lastly, I offer my regards and blessings to my mother and my
family who supported me in any respect during the completion
of the project.

19. References:
1. Krinsha Khairnar and Subhash C Parija (2007); A novel nested multiplex polymerase chain
reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E.
dispar DNA in stool samples.
2. R. Fotedar, D. Stark, N. Beebe, D. Marriott, J. Ellis, and J. Harkness (2007); PCR detection of
Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in Stool Samples from
Sydney, Australia.
3. Zulhainan Hamzah, Songsak Petmitr, Mathirut Mungthin, Saovanee Leelayoova, and Porntip
Chavalitshewinkoon-Petmitr (2006); Differential Detection of Entamoeba histolytica, Entamoeba
dispar, and Entamoeba moshkovskii by Single-Round PCR Assay.
4. Ali IK, Hossain MB, Roy S, Ayeh-Kumi PF, Petri WA Jr, Haque R, Clark CG (2003); Entamoeba
moshkovskii infections in children, Bangladesh.
5. Mehmet Tanyuksel*, Mustafa Ululikanligil, Zeynep Guclu, Engin Araz, Ozgur Koru, and William A.
Petri, JR. (2008); Two Cases Of Rarely Recognized Infection With Entamoeba moshkovskii.
6. David L. Beck, Nihal Doagn, Venance Maro, Noel E. Sam, John Shao, Eric R. Houpt (2008); High
Prevalence of Entamoeba moshkovskii in a Tanzanian HIV population.
7. Soumaya Ben Ayed, Karim Aoun, Nadia Maamouri, Rym Ben Abdallah, and Aïda Bouratbine
(2008); First Molecular Identification of Entamoeba moshkovskii in Human Stool Samples in
Tunisia.
8. Rashidul Haque,1 I. K. M. Ali,1 S. Akther,1 and William A. Petri Jr.2 (1998); Comparison of PCR,
Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection.
9. Amidou Samie, Larry C. Obi, Pascal O. Bessong, Suzanne Stroup, Eric Houpt, and Richard L.
Guerrant (2006); Prevalence and species distribution of E. histolytica and E. dispar in the Venda
region, Limpopo, South Africa.