الفهرس 
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Abstract
Giardia is an intestinal flagellate that infects a wide range of vertebrate hosts. Giardia intestinalis is the only species found in humans; it is also found in other mammals, including pets and livestock. It produces robust cysts, which are voided in the faeces and transmitted directly through faecal/oral contact, or by ingestion of contaminated water and food. G. intestinalis is assigned to at least eight distinct assemblages (A to H) based on protein or DNA polymorphisms. Molecular characterization of Giardia carried out in humans at several loci, gdh, bg, ssu-rRNA, and tpi genes, have revealed that two major genetic assemblages, designed as A and B are responsible for causing the majority of human infections.
Although several clinical and epidemiological studies of giardiasis have been conducted in Egypt, few studies have addressed the molecular analyses of this intestinal parasite and its association with the symptomatology manifested in children. In the present study, prevalence and molecular characterization of G. intestinalis detected among children in some governorates of West Delta region were studied and analysed in relation to epidemiological and clinical data collected.
The present study was conducted on 315 randomly selected sample of children (2-16 years) from three different localities namely; Abd El-Kader village in Alexandria, El- Bakawat village in El-Beheira and Arab El-Mahder village in Kafr El-Sheikh. Participants were asked to submit stool samples that were divided into two portions. The first part was used for microscopic examination and the other part was used for DNA extraction and PCR analysis. Genotyping was performed using two PCR approaches addressing the tpi and the gdh genes. tpi assemblage specific primers were used for detection of mixed infection and gdh PCR-RFLP approach was used for sub-typing of G. intestinalis isolates.
Among the examined children, parasitological examination of faeces revealed that the overall prevalence of parasitic infections was 58.1%. The frequencies of the parasites in a decreasing order were: Blastocystis sp. (46%), followed by G. intestinalis (18.1%), Entamoeba coli (6.6%), E. histolytica / E. dispar complex (2.9%), Ascaris lumbricoides (2.5%), Hymenolepis nana (1.5%), Enterobius vermicularis (0.9%) and Schistosoma mansoni (0.3%). The infection rates of G. intestinalis were significantly different in the three studied areas. Kafr El-Sheikh and El-Beheira showed higher infection rates (29.5% and 16.2%, respectively) compared to Alexandria (8.6%). The prevalence of Giardia was higher among the younger age group (21.1% vs 15.6%) and males (32% vs 25%), though the difference was not statistically significant.
DNA extraction was done according to the kits’ instructions with some modifications in the form of using fresh stool samples, boiling with InhibitEx buffer for 1 hr at 95oC and incubation with proteinase K for 1 hr at 70oC to allow for better quality of DNA yield. Amplification of both tpi and gdh genes revealed that tpi gene gave better results for Giardia detection than gdh gene.
tpi approach revealed that mixed assemblages A and B was the predominant infection form (47.4%) followed by assemblage B (36.8%), then assemblage A (15.8%). Mixed assemblages A and B infection was significantly higher in El-Beheira (82.4%) compared to Alexandria and Kafr El-Sheikh (11.1% and 38.7%), while assemblage B showed a significantly higher rate in Alexandria (77.8%) than El-Beheira (17.6%) and Kafr El-Sheikh (35.5%).
Summary, C onclusions and Recommendations
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Assemblage A infection rates were not significantly different in the three studied areas (11.1%, 0% and 25.8% in Alexandria, El-Beheira and Kafr El-Sheikh; respectively).
PCR-RFLP of gdh gene revealed the presence of sub-assemblages AII, BIII, BIV or mixture of them. Sub-assemblages BIII and BIV were equally detected each in 30.9% of the samples. The remaining 38.4% was equally divided between sub-assemblage AII, mixed BIII & BIV and mixed AII & BIII. Assemblage B was responsible for the majority of infections (74.5%). In Alexandria, the percentages of various sub-assemblages were nearly similar to the above mentioned overall percentage. In El-Beheira, sub-assemblage BIII was more frequently detected (41.2%) compared to other sub-assemblages, while sub-assemblage AII single infection was not detected in any sample. In Kafr El-Sheikh, sub-assemblage BIV was the dominating sub-assemblage (37.9%), followed by BIII (24.1%), mixed BIII- BIV infection was detected in 13.8%, while AII-BIII mixed infection was detected in 3.4%.
Comparison of genotyping results of PCR-RFLP of gdh gene and tpi assemblage specific primers revealed that mixed A and B infection was more commonly detected through tpi PCR protocol (47.4%) than PCR-RFLP approach (12.7%). gdh gene showed higher sensitivity for assemblage B amplification than assemblage A.
By analysis of demographic characteristics (age, gender, residence) and environmental data (water source, animal contact and hand washing) using tpi approach, no significant associations were observed for age, gender or water source and the detected assemblage. On the other hand, significant association was observed between residence, hand washing and the detected assemblage.
Concerning the clinical manifestations, no significant association was reported between the presence of symptoms and the detected assemblage. However, subtyping revealed a significant association between the presence of symptoms and the detected sub-assemblage. Sub-assemblage BIII showed mostly asymptomatic presentations.