الفهرس 
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Abstract
The majority of the worldwide water-associated outbreaks of parasitic protozoan diseases recently are caused by Cryptosporidium parvum (50.8%). Cryptosporidium is the most persistent in the environment, most resistant to chemical disinfection, and smallest in size, so the most difficult to be removed by filtration. Cryptosporidium is therefore the pathogen of choice as reference for protozoan parasites that use the fecal-oral route in piped water supplies as suggested by World Health Organization in a report about Cryptosporidium in water supplies in 2009. This document was later included in the WHO Guidelines of drinking water quality in 2010.
The diagnosis of cryptosporidiosis rests on the identification of the 5-µm spherical oocysts (or oocyst components) in a sample. In developing countries where resources are limited, microscopic examination of wet preparations, or of dried, fixed, chemically stained specimens, is the most reliable option. Modified Ziehl Neelsen stain has shown superiority in staining this particular parasite.
Immunodiagnostic tests have some serious limitations. Cross-reaction leading to false positive and misdiagnosis is also a problem, especially in regions where more than one parasite is endemic. Since then, other methods evolved in search of better sensitive techniques for the detection of Cryptosporidium oocysts. Flow cytometry is an example that is quite useful as a screening technique, flow cytometry is not sufficiently specific or sensitive to obtain accurate counts of the organisms present.
Polymerase Chain Reaction (PCR) was the first technique to emerge as a molecular-based identification method. The use of gel electrophoresis after PCR paves way to cross contamination and human handling errors in the laboratory. Moreover, PCR inhibitors were present in environmental samples hindering the process of DNA amplification. The lack of quantification by PCR also emerged as a major setback to this method. These disadvantages have led to the evolution of other molecular methods based on PCR, what is now known as real time PCR.
This technique allows the amplification in the PCR to be monitored in real time. The main advantages of real-time PCR are that it allows analysis in a ‘‘closed-tube’’format, not requiring handling. Secondly it gives the quantity of DNA and then the number of oocysts in samples tested.
That is why the present study aimed at using real time PCR technique for the detection and quantification of Cryptosporidia oocysts in different water samples in Alexandria compared to traditional staining techniques. Viability and infectivity of this parasite in the collected samples were assessed.
To fulfill this aim, a total of 56 water samples were collected and examined. Six samples were collected from El Mahmoudeya canal and irrigation water. Three samples were collected from El Mahmoudeya canal, midstream (Canal 1) and sides (Canal 2, Canal 3) and and three samples from water of irrigation from lands in El Mamoura (Irrigation1), Abees (Irrigation2) and El Amreya (Irrigation3). Fifty samples were collected from different districts of Alexandria (El Montaza, Shark, Wassat, Gharb, El Amreya and El Gomrok). Twenty five samples were collected from water tanks and twenty five from pipe tap water.
Samples were directly examined then concentrated. The recovered sediments were preserved in 2.5% potassium dichromate then divided into 1ml pellets for staining techniques, real time PCR and infectivity assays.
Stains included trypan blue for viability and counting, MZN for diagnosis, safranin methylene blue and modified trichrome for exclusion of other parasites and fluorescent stains (phenol auramine and acridine orange) for confirmation.
Real time PCR was done through several steps. First DNA extraction which was accomplished by modifications concerning number of freeze-thaw cycles and sonication. This was followed by gene amplification. Quality control was established by introducing positive, negative and internal controls into the device with the water samples. Detection of positive samples was done during amplification process. Quantification of DNA then oocysts per sample was done in comparison to a standard curve which was drawn earlier through a series of known dilutions of a known positive control supplied in the kit.
Ten swiss albino mice per positive sample were used to assess infectivity. Each mouse was inoculated with 0.1ml from a suspension containing 104 oocysts/ml. After that stool examination was done daily for 7 days and mice were sacrificed after a week for ileal examination by H&E stain.
Sensitivity, specificity, positive predictive value, negative predictive value and accuracy were calculated in comparison to MZN as the gold standard method.
During examination of the water samples with the previous stains other parasites were also detected. Giardia cysts were the most common to find in different water samples. They were found in a total of twelve out of fifty six samples (21.4%), including six drinking water samples and all six irrigation and canal samples. These were all detected by modified trichrome. Cyclospora oocysts came next being found in nine out of fifty six samples (16%), including three drinking and all six irrigation and canal samples. They were best stained by safranin methylene blue. Microsporidia spores were also found in nine out of fifty six samples (16%); three drinking and all six irrigation and canal samples using modified trichrome.
Real time PCR gave the highest number of positive samples (49/56, 87.5%). MZN stain came second in detection giving 36 positive samples. After that was auramine stain (31 positive samples) followed by safranin stain (23 samples) then acridine orange (22 samples) and finally modified trichrome with 17 samples only. Real time PCR also gave the highest number of positive drinking water samples (43/50,86%), followed by MZN (30/50,60%), then phenol auramine (25/50,50%), safranin (17/50,34%), acridine orange (16/50,32%) and last modified trichrome (11/50,22%). Number of positive samples was always higher in tank water than tap water samples originated directly from water pipes. For example, real time PCR showed that 92% of tank samples were positive while 80% were positive in tap water. The same applies to the rest of the stains where the gold standard MZN stain gave 72% of tank samples were positive and 48% of tap water. As regards irrigation and canal waters, all samples were positive by all methods.
Real time PCR showed the highest sensitivity being 100% followed by auramine stain (86%) then safranin (64%), acridine orange and finally modified trichrome. Yet stains took the upper hand in specificity gaining 100% compared to 35% for real time PCR. Interesting to say that real time PCR had the highest negative predicitive value of 100%.
There was a visible difference in concentration between the first 6 samples taken from water source (Mahmoudeya canal) and irrigation waters and the last 50 samples, taken from drinking water. In the latter samples, concentrations showed a large increase ranging from 600 to 1450 oocysts/ml in canal water and irrigation samples. While samples taken from drinking water, if positive ranged from 15 to 600 oocysts/ml in addition to 7 samples being too low to be detected. A slight difference is seen between oocysts counts/ml in tap and tank samples. In tank water counts were higher reaching 600 oocysts/ml while in tap water the maximum count was 450 oocysts/ml. Oocysts were also counted by trypan blue stain per HPF. All samples “too low to be detected” by real time PCR were negative by trypan blue stain and there were 13 samples that had a low number of oocysts by real time PCR and were even missed by staining techniques. In drinking water samples counts ranged from 21 to 66 oocysts/ml. While in canal samples counts were 67, 70 and 73 oocysts/ml and even higher (69, 71 and 75 oocysts/ml) in irrigation samples.
t-test was done and the significance was calculated for each water type. Real time PCR showed to be significantly better than trypan blue for counting Cryptosporidium oocysts in all types of water samples (P<0.05), the value was very significant (<0.001) in drinking water.
Percentages of viable oocysts/ml were calculated using trypan blue stain. Samples taken from irrigation and canal waters gave high figures. They were all in the range between 60% and 70%. Other samples gave different figures as low as 35% and 38%. Other parasites detected as mentioned before by various staining techniques were also found by trypan blue stain; viable Cyclospora oocysts, Giardia cysts and Microsporidia spores.
All positive water samples were infective. Infection appeared in stool samples of mice at the 3rd day of infection, to reach a peak by 4th day and decreased gradually but was still present till the day of sacrifice. Intestinal sections by H&E stain examined under oil immersion lens (x 1000), showed thickening and shortening of the villi with crypt hyperplasia, and non specific inflammatory infiltration of the lamina propria. Cryptosporidia oocysts were seen intracellularly at the brush border and in the lumen as well.