Differential Detection of Differential Detection of Entamoeba disparEntamoeba dispar and and

Entamoeba moshkovskiiEntamoeba moshkovskii Using Using Nested PCRNested PCR

Hala Amin IssaHala Amin Issa2062163220621632

Outline

Introduction

Materials and Methods

Results and Discussion

Acknowledgement

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.

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.

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

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.

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

Results and Discussion

%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. moshkovskii3/595.1%

E. dispar6/5910.17%

Table (1)

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)

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

1 2 3 9 11 13

1 2 3 7

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

Correlation of different studies with the present one

E. disparE. 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%

EntamoebaStudies

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

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

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

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.

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.

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.