الفهرس 
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Abstract
Protozoan infections are still the most widely spread infections around the world. Intestinal protozoal infections remain a major health problem in tropical and subtropical areas of the world, particularly in rural areas. They may cause malnutrition, anemia and other physical and mental impairments in infected individuals, particularly in children.
The present study was carried out in the Parasitology Laboratory of Medical Research Institute and molecular laboratory of Mustafa Kamel military hospital. Children from El-Missery village, El-Nahda sector, Al- Ameriya district of West Alexandria, Egypt were included in the study after obtaining an oral informed consents from parents/guardians.
The aim of the present work is to evaluate multiplex PCR and conventional microscopic examination for detection of enteric pathogenic protozoa in pediatric diarrheic patients.
In this study, a total sample of 100 diarrheic children ( aged range < 16 years of age ) attending El-Hares health care facility, were included. A questionnaire sheet including demographic, clinical manifestation and housing environmental data was obtained from each parent/guardian. Each stool sample was divided into three portions :-
- First portion of fresh stool samples was examined by direct wet mount smear.
- Second Portion of each stool sample was preserved in 10 % formalin then prepared for formol ethyl acetate concentration technique, one DROP from concentrated sample was permanently stained using MZN stain .
- Third portion of the unpreserved fresh stool samples (about 200 mg each) was kept in a clean labeled plastic container and stored at -20oC for DNA extraction and amplification by multiplex real time PCR, using (RIDA gene parasitic stool panel II) for E. histolytica, G. lamblia, C. parvum.
The percentage of parasitic infection in diarrheic children detected by microscope are 67%, the most common parasite was Giardia.lamblia (23 cases) followed by Blastocystis hominis (17 cases), E. histolytica / dispar (12 cases), E.coli (9 cases), C.hominis/parvum (5 cases) and Cyclospora spp ( one case).
Result showed that 42 % of diarrheic stools were positive (G.lamblia 40%, and C.parvum 2%) among multiplex real time PCR
Regarding the prevalence of intestinal protozoa infection (G.lamblia, E.histolytica, C.parvum) in relation to the diagnostic methods used among hundred diarrheic children. It was found that overall prevalence of intestinal protozoa infection (G.lamblia, E.histolytica/dispar, C.hominis/parvum) among diarrheic children was 35 % detected by wet mount iodine smears, 5 cases detected by Ziehl-Neelsen stain ( C.parvum ), i.e pooled data of coproscopic methods showed infection rate 39 %, and 42 % by multiplex real-time PCR for detect Giardia lamblia, E.histolytica, and C.parvum.
• Multiplex real time PCR showed sensitivity of (74.36 %) and specificity of (78.69 %) when compared to microscopic examination.
• Among E.histolytica/dispar was diagnosed by microscopy as 12 % whereas showed negatively to Entamoeba histolytica by Multiplex Real-Time PCR.
• Cryptosporidum spp (5 %) detected by microscopy while only 2 cases diagnosed by PCR.
• No significant difference in infection rate according to gender and age .
• Regarding housing environments, drinking from unfiltered private tap or general tap water, tank water storage, using shared latrines were associated with increased risk of acquiring intestinal protozoal infection.
• Regarding studied healthy practices, not washing fruits and vegetables or washing them with water only, not washing hand before eating or after defecation, not covering stored water, irregular tanks cleaning and their cleaning with only water was associated by increased risk of intestinal protozoal infection.
• Multiplex real time PCR for detection of G.lamblia, E.histolytica, C.pavum gives sensitive and specific, yet expensive method for simultaneous detection of these protozoa overcoming limitations of the traditional microscopic examination.
Conclusion:
from the present study, it could be concluded that:
• The percentage of parasitic protozoan infection in diarrheic children was (67%). The most commonly detected parasite was Giardia.lamblia ( 23 case ) followed by Blastocyst hominis ( 17 case ) by microscopic examination.
• Results showed that ( 42 % ) were positive, G.lamblia in (40%) and C.parvum in (2%) detected by multiplex real time PCR by using RIDA gene parasitic stool panel II in stool samples of diarrheic children.
• E.histolytica was not detected in any of 100 diarrheic stool children samples examined by multiplex real time PCR
• The Multiplex Real-Time PCR showed moderate agreement versus pooled data coproscopic methods as a diagnostic gold methods for diagnosis of intestinal protozoa.
• Multiplex real time PCR showed sensitivity (74.36 %) and specificity (78.69 %) when compared to microscopic examination.
• Cryptosporidum spp (5 %) detected by microscopy, while only 2 cases were diagnosed by PCR.
• No significant difference in infection rate according to gender and age .
• Regarding housing environments, drinking from unfiltered private tap or general tap water, tank water storage, using shared latrines were associated with increased risk of acquiring intestinal protozoal infection.
• Regarding studied healthy practices, not washing fruits and vegetables or washing them with water only, not washing hand before eating or after defecation, not covering stored water, irregular tanks cleaning and their cleaning with only water was associated by increased risk of intestinal protozoal infection.
• Multiplex real time PCR for detection of G.lamblia, E.histolytica, C.pavum gives sensitive and specific, yet expensive method for simultaneous detection of these protozoa overcoming limitations of the traditional microscopic examination.
Recommendations:
Based on the finding of the present study, the following suggesting items could be recommended :-
1) Estimation of the burden of intestinal protozoa in human and stock animals.
a) Efforts should be directed to increase clinicians and laboratory personnel knowledge on the role of intestinal protozoa as causative agents of gastrointestinal related symptoms
b) The combination of both microscopy and PCR for diagnosis of intestinal protozoa parasites should be used to optimize sensitivity. The performance of both techniques has to be evaluated against serological assays.
c) The use of specific techniques PCR for differentiation of E.histolytica from E.dispar should be encouraged to avoid unnecessary treatment of patients infected with nonpathogenic species.
d) Large scale community based studies are needed to identify possible risk factors for intestinal protozoa which would be important to design appropriate strategies for their control and prevention.
2) Further large-scale epidemiological studies should be conducted on wide range of host among food handler and any worker in contact with animals in different localities in Egypt to assess the true prevalence of intestinal protozoa and to :-
• Provides significant improvement in the quality of the management of intestinal protozoa.
3) Improving prevention and control measures of intestinal protozoa infection.
- Health education about the risk of infection and route of transmission among school children, workers and personnel handlers food, the waste products of infected animals .
- Formulating national guidelines and organized programs aimed to prevention and control of intestinal protozoa infections