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Cryptosporidium species are obligate apicomplexan intracellular parasites that infect the microvilli of the gastrointestinal epithelial cells of a wide range of vertebrate hosts, including humans. Cryptosporidiosis is a global public health problem that has been increasingly reported world-wide in immunocompetent and immunocompromised individuals causing a spectrum of diseases depending on the immune status of the host.
Human cryptosporidiosis had been reported as a prevalent and virulent agent of diarrhea in Egyptian patients especially in childhood with varied prevalence: 0% - 47%. Accurate determination of the incidence of infection is hampered by the lack of sensitive diagnostic techniques.
Conventional direct microscopic diagnosis using acid-fast stains is time consuming, labor-intensive and requires a skilled technologist. In addition, it has relatively low diagnostic sensitivity. For this reason new highly sensitive diagnostic methods have been developed to complement or even replace microscopic approaches.
Immunological based detection methods including ELISA and ICT are simple, rapid and offer a less subjective method than microscopy. However, antigenic variability within clinical isolates can result in some infections remaining undetected. These methods also can not be used for species differentiation. In order to solve the problem, better techniques with higher sensitivity and specificity are necessary.
Molecular techniques for laboratory diagnosis of cryptosporidiosis showed excellent specificity and sensitivity, compared with antigen detection and microscopy. This facilitated early detection of Cryptosporidium, aided in the epidemiologic investigations and provided new insights into the diversity of Cryptosporidium spp. with better understanding of population genetics.
Knowing that the basis of molecular diagnosis is DNA extraction, a proper extraction method should be achieved to improve sensitivity of molecular techniques. Stool is chemically complex and DNA extraction from stool samples is extremely difficult. Hemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can interfere with the PCR reaction inhibiting DNA amplification and produce false negative results.
Stool samples are sometimes required to be stored for variable periods for epidemiological studies, also to achieve lower cost for PCR, processing of the samples is better done collectively than individually.
An effective DNA extraction method is needed to isolate DNA either from fresh stool as quickly as possible, or the stool in question should be appropriately preserved; so preservation time and conditions are important fac¬tors in the isolation of DNA from stool.
The present study was conducted to evaluate five different preservation conditions and to detect the most suitable one for preservation of stool samples and subsequent DNA extraction and amplification via nested PCR for detection of Cryptopsporidium species.
In this study, 380 fresh stool samples were collected during the time interval from July 2013 to December 2014 from patients attending the Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University, the Pediatric department of El-demerdash hospital, Ain Shams University, Abo-elreesh hospital, Cairo University and the fever hospital in El-abbasya.
The fresh samples were examined microscopically and stained with MZN. Positive results were confirmed by Coproantigen detection using RIDA®QUICK Cryptosporidium/Giardia ICT cassettes. The outcome of ten positive samples were applied to the five preservation conditions; -20ºC, 70% ethyl alcohol, 10% formalin, 2.5% K dichromate at 4ºC, and 2.5% K dichromate at room temperature (RT).
DNA extraction was done using QIAamp Stool Mini Kit from the fresh stool samples, then from the preserved samples after 10, 20 and 30 days. DNA amplification was done by nested PCR targeting the COWP gene, and the PCR results were visualized using gel electrophoresis and UV transillumination.
Using the DNA extraction from fresh stool samples as a nominated gold standard, After 10 days of preservation, -20°C and K dichromate at RT showed the highest sensitivity (100%), followed by K dichromate at 4°C with sensitivity 80% and alcohol 40%. After 20 days of preservation, -20 °C and K dichromate at 4°C showed the highest sensitivity (80%), followed by K dichromate at RT (60%), then alcohol (40%). After 30 days of preservation, -20°C and K dichromate at 4°C remained the most potent conditions (60%), followed by K dichromate at RT (40%) and alcohol (20%). Formalin was of very poor performance (0%) along the three extraction intervals.
The overall sensitivity showed that -20°C and K dichromate at 4°C had the highest sensitivity (80% and 73.3% respectively), followed by K dichromate at RT (66.7%), then alcohol (33.3%), while formalin was (0%).
There was a highly significant difference between different preservatives as regard sensitivity after 10, 20 and 30 days and along the whole preservation period.
There was a significant difference between -20°C which gave the best outcome compared to formalin signifying the poor effect of formalin as a preservative, while there was a non significant difference compared to alcohol, K dichromate at RT and K dichromate at 4°C which signifies their potent preservative effect with variable degrees.
Comparing K dichromate as a preservative solution in its different conditions; RT and 4°C along the whole preservation period revealed a non significant relation which signifies the potency of K dichromate persay as a preservative.
It’s obvious that the efficiency of the preservation decreases along time; each preservative gave best results during the first 10 days followed by the second 10 days and the least in the third 10 days. This means that DNA in the preserved samples is degraded by time and this rate of breakdown is affected by the used preservative.
Comparing the preservatives with positive outcome, regarding their potency along different intervals of extraction revealed a significant difference between the outcomes of K dichromate at RT along the whole preservation period, which signifies it is not a stable preservative. While there was a non significant relation between the performance outcomes of alcohol, K dichromate at 4°C and -20°C which signifies their stability as preservatives.
The study highlighted freezing at -20°C as the most suitable condition for the preservation of Cryprosporidium spp. DNA in stool samples. K dichromate proved also to be an efficient preservative, but extra washing of the stool sample should be done for the complete removal of the preservative before DNA extraction to avoid its inhibitory effect on PCR and false negative results.
Formalin 10% had a very poor preserving efficiency. The study recommends its exclusion as a preservative for Cryptosporidium spp. DNA in stool samples.