Author name:
صبا فاضل علي مهدي معلة
Supervisor name:
علي حسين مكي الكبيسي
Abstract:
This study was conducted on the 1350 stool samples from diarrhea - suffering patients in three provinces of middle Euphrates region : Karbala, Babel and Al - Najaf (450 samples in each province). Patients ranged in age from 1 to more than 60 years and from both sexes and different regions who were attended to some hospitals, dispensaries and private laboratories in these provinces during the period from the first of May 2013 till the end of April 2014. All collected fresh samples were examined microscopically by wet - mounts method for G. l a mbl i a and E n ta m oeba histolytica detection One third of positive samples in microscopic examination (which was selected to be representative for all positive samples) from each province and ten of negative one as control group were submitted to the nucleic acid (DNA) extraction. Molecular diagnosis of G. l ambl i a and E. h i s to l yti ca were performed using PCR reaction with amplification of the 532 - bp and 355 - bp fragments from the internal transcribed spacer region1 ITS1 for each parasite respectively. The eluted DNA of each positive sample in ITS1 test with its specific primer was also submitted to a traditional PCR and RT - PCR techniques for detection two genes in G. l a m b l ia { Glycoprotein A Repetitions Predominant (GARP) and Adenosine Triphosphate Guanine Tri Phosphate (ATP/GTP) } and two genes in E. histolytica {Acetatekinase (ACK) and Methionine GammaLyase (MGL)} using PCR and RT - PCR techniques. Five positive PCR products in ITS1 test of G. la m blia were used for sequencing. Based on the results of this study, microscopic examination showed that the total percentages of infection with G. l am bl ia were (14.7, 17.3, 16) % and with E. histolytica were (15.1, 13.3, 16.7) % in Karbala, Babel PDF created with pdfFactory Pro trial version www.pdffactory.comand Al - Najaf respectively. The infection with G. l am bli a and E. histolytica was more frequent in males than in females and the prevalence of both parasites in rural areas was higher than the urban areas P < 0.05 in all studied provinces. The highest incidence of infection with G. lamblia occurred in age group 11 - 20 years (P < 0.01) and that with E. histolytica occurred in age group (1 - 10) years (P < 0.05) in all studied provinces. This study was revealed uneven percentages of infection with both parasites during different months of a year (P < 0.01), the highest rate of infection with G. lamb l ia and E. h i s t o l y ti ca were in summer. For the molecular diagnosis of G. l a mbl ia and E. hi s t ol y t i ca, using PCR technique, the prevalence of G. l amb l ia and E. his t o l y ti c a in middle Euphrates region were found to be 14.9 % and 12.8 % respectively. The infection with G. l a m b lia and E. hi stol y t i c a was more frequent in male (62.7, 63.8) % than in female (37.3, 36.2)%, the prevalence of the parasites in rural area (67.2, 69.0)% was higher than the urban area (32.8, 31.0) %, the highest incidence of infection occurred in age groups 1120 and 1 - 10 years with the percentages of (28.4, 29.3) %, the highest rates of infection were in August (20.9%) and July (19.0%) respectively. Significant differences were found between the rate of infection with both G. l a m bl i a and E. hi stol y t i ca and the different parameters (gender, residence, age gropes and months of a year). The results of detection the gens (GARP, ATP/GTP, ACK, MGL) by using PCR technique showed that the total percentages of positive samples were (55.0, 70.0, 45.0, 75.0) %, (62.5, 79.2, 47.0, 70.6) % and (65.2, 73.9, 61.9, 71.4) % in samples of Karbala, Babel and Al - Najaf respectively while those percentages by using RT - PCR were (60.0, 80.0, 50.0, 80.0) %, (66.7, 83.3, 58.8, 82.4) % and (65.2, 78.3, 66.7, 71.4) % respectively. For the detection of genes in G. l am bl ia, sensitivities of 91.8 % and 97.8 % and specificities of 94.6 % and PDF created with pdfFactory Pro trial version www.pdffactory.com81.4% for PCR and RT - PCR respectively were observed when compared to each others, While Sensitivities of 89.9 % and 98.6% and specificities of 97.3 % and 81.8 % for PCR and RT - PCR respectively for the detection of genes in E. hi sto l y t i c a were observed when compared to each others. Multiple sequence alignment analysis and phylogenetic analysis results of this study demonstrated that the G. l ambl ia in Middle Euphrates region - Iraq isolates had nucleotide homology sequence identity percent of 99% with Giardia intestin a l i s (AF199448.1) Australia isolate and with USA isolate Giardia microti
