Cryptosporidium parvum parasite microscopic and molecular diagnostics in the Holy Karbala/ Iraq .

Abstract

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 Province using three different diagnostic methods.
Microscopic examination, which includes staining the samples with the modified acid dye, as well as using the molecular examination
using the technique of polymerase chain reaction, the collection of samples was carried out in Al-Hussein Medical City 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 and Karbala Teaching Hospital.
For children, of both sexes, and with age groups from (1-50) years, this was done. The first half of each sample was utilized to produce
reasonably thick smears for evaluation with the modified acid dye, and the second part was preserved at -20°C for subsequent use in
the molecular study., 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 infection rates according to the months of the year, the highest rate of infection 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 infection was in the age group (1-10) by 14.1%, while the current study did not record
any infection in the age group (40-31). Drinking water sources played a role in the spread of the Cryptosporidium parasite, the infection
rate for persons who drink tap water was 18.9%, whereas the lowest rate for people who drink sterile water was 4.8 percent, according
to the research. 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%.
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 oocyst
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, It was concluded from this study that the Cryptosporidium parasite
is one of the parasites that cause diarrhea in humans, especially children in Karbala Province. It may be diagnosed using a variety of
ways, with the polymerase chain reaction approach being one of the most effective.