التشخيص المجهري الجزيئي لطفيلي الأبواغ الخبيئة Cryptosporidium parvum في محافظة كربلاء المقدسة

Molecular Diagnosis of Cryptosporidium parvum in the Holy Karbala Governorate

Summary
Cryptosporidium parvum is a compulsive intestinal parasite that causes parasitism and causes diarrhea in humans, especially children. The present study aimed to diagnose the Cryptosporidium parasite and identify the extent of the parasite’s spread in people who suffer from diarrhea and its relationship to epidemiological factors in the holy Karbala governorate using three different diagnostic methods. Microscopic examination, which includes staining the samples with the modified acid dye, as well as using the method of flattening with a diabetic Cheddar solution, immuno-chromatography examination using immunoassay slides, and molecular examination using the technique of polymerase chain reaction, the collection of samples was carried out in Al-Hussein Medical City (pbuh) and Karbala Teaching Hospital for children. So, in the laboratories of the College of Veterinary Medicine at the University of Karbala for the period from the beginning of September 2020 until the end of February 2021, 320 stool samples were collected from persons suspected of having the parasite (those with diarrhea and not infected) and who came to Imam Hussein Medical City (PBUH) and Karbala Teaching Hospital. For children, of both sexes, and with age groups from (1-50) years, this was done Each sample was divided into two parts, the first part was used to prepare moderately thick swabs for examination with the modified acid dye and the second part was kept at a temperature of -20 for later use in the molecular examination, as the results of the microscopic examination using the modified acid dye showed that the number of infected samples reached 26 samples as the number of Males have 15 samples, while the number of females reached 11 samples and the overall infection rate was 8.1%, while the infection rate was lower by using immunological tests by 6.8%.
The incidence rate was affected by epidemiological factors below the probability level P≤ 0.05, as males recorded a higher rate of infection compared to females, as the infection rate reached 10% and 6.47%, respectively, while the infection rate recorded significant differences according to the type of housing, as the infection rate reached 12.2% in the countryside and in The city, the infection rate was 6.5%. As the current study recorded different injury rates according to the months of the year, the highest rate of injury was in the month of December by 16%, while the lowest injury rate was concentrated in the month of October and November by 1.8%, 4.7%, and the study recorded The highest rate of injury was in the age group (1-10) by 14.1%, while the current study did not record any injury in the age group (40-31). Drinking water sources played a role in the spread of the Cryptosporidium parasite, as the study showed significant differences in infection rates, as the infection rate for people who rely on tap water was 18.9%, while the lowest rate for people who used sterile water for drinking was 4.8%. As for the companion parasites, the current study recorded that the most associated parasite is Entamoeba histolytica by 3.4%, while the appearance of Girdia lamblia parasite was less by 2.1%.
And a molecular assay was used on the second part of the samples, which was kept at a temperature of -20, for the purpose of confirming infection in the positive samples and others, and to determine the accurate diagnosis of this parasite. Specific primers for the egg wall protein (COWP) gene were used, Cryptosporidium oocyst wall protein. Polymerization, the first stage was the polymerase chain reaction (PCR), where the DNA was amplified using specific primers and the reaction was accomplished using a thermocycler and under optimal conditions. The results revealed a single package (Singal band in agarose gel) with weight Molecular 769 base pairs of nitrogenous bases in the DNA extracted from stool samples of people with Cryptosporidium, which is an indication of the patient’s infection with the parasite, and the second stage in which the technique of nested polymerase chain reaction is used. By using two types of specific primers in the reaction (COWP F and COWP R), the DNA was amplified using specific primers and it was The reaction was carried out using a thermocycler device and under optimal conditions, the results showed the presence of one packet in the acarose gel with a molecular weight of 553 pairs of nitrogenous bases in the DNA extracted from the stool samples of people with Cryptosporidium, an indication of the patient’s infection with the parasite, and the current study showed that the sensitivity The microscopy examination was less than the molecular examination, as the sensitivity of the microscopy examination reached 75%, while the sensitivity of the immunological examination was higher than that of the microscopic examination, and the sensitivity of the immunohistochemical examination was 88%.
It was concluded from this study that the Cryptosporidium parasite is one of the parasites that cause diarrhea in humans, especially children in Karbala Governorate. It can be diagnosed by several diagnostic methods, and the polymerase chain reaction method is one of the best diagnostic methods.