Evaluation of Different Copro-Preservation Conditions on DNA Extraction and PCR Detection of Cryptosporidium species

Abstract

Background: Cryptosporidium species (spp.) are enteric protozoan parasites considered as a major cause of diarrheal disease. Epidemiologic studies on human cryptosporidiosis are made difficult by the limitation in identifying species using staining and immunodiagnosis, so molecular techniques were developed. Extraction of DNA from stool samples is not simple due to the presence of multiple inhibitors which can affect PCR results. Preservation of stool specimens is required for epidemiological studies and research purposes. So, 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 samples and its use in molecular approaches. Objective: This work aimed to evaluate different conditions and timing of fecal preservation for the best outcome of molecular diagnosis of Cryptosporidium species.

Methodology: I. Preparatory steps for the study:  Sample collection: Setting: 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.  Examination of 380 fresh stool samples collected from patients with diarrhea. Setting: Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University Design: • Two direct wet smears were done for each sample once by saline and the other by iodine. • Samples were concentratetd by formalin-ethyl acetate sedimentation concentration technique. • Stained smears were done with Modified Ziehl-Neelsen technique, revealing a net of 10 positive samples which were used in the study. • Immunochromatographic test (ICT) was applied to validate positivity of staining and microscopy. II. Study of preservation conditions for subsequent DNA amplification:  Preservation of stool samples: Setting: Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University. Design: Each of the 10 stool samples was preserved at five different conditions; -20ºC, 70% ethyl alcohol, 10% formalin, 2.5% potassium dichromate (K dichromate) at 4ºC and 2.5% K dichromate at room temperature (RT).  DNA extraction: Setting: Research and Training Center on Vectors of Disease, Ain Shams University.

Design: • DNA extraction from each sample at zero time (fresh specimens) as the gold standard for the comparison with the preserved samples. • DNA ex¬traction at 10, 20 and 30 days of preservation from each sample in each of the five different conditions.  DNA amplification and visualization of PCR products. Setting: Molecular Medical parasitology Lab, Department of Medical Parasitology, Faculty of Medicine, Cairo University. Design: • DNA amplification using nested PCR, with Cryptosporidium oocyst wall protein (COWP) gene as a genetic marker for the identification of Cryptosporidium species. • Detection of the PCR products using gel electrophoresis and ultraviolet (UV) trans-illumination.

III. Data management and analysis: According to the type of data obtained for each parameter. Results: 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 a very poor performance (0%) along the three extraction intervals. The overall performance 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 performance was (0%).

Conclusions: The study highlighted freezing at -20°C as the most suitable condition for the preservation of Cryptosporidium spp. DNA in stool samples. K dichromate proves 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 it to be excluded as a preservative for Cryptosporidium spp. DNA in stool samples. The study revealed a highly significant difference between different preservatives performance along the preservation period indicating a highlighted outcome between the different preservatives. There was a significant difference between -20°C which gave the best outcome compared to 10% formalin signifying the poor effect of formalin as a preservation condition, 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 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. 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 per say as a preservative solution.

Background: Cryptosporidium species (spp.) are enteric protozoan parasites considered as a major cause of diarrheal disease. Epidemiologic studies on human cryptosporidiosis are made difficult by the limitation in identifying species using staining and immunodiagnosis, so molecular techniques were developed. Extraction of DNA from stool samples is not simple due to the presence of multiple inhibitors which can affect PCR results. Preservation of stool specimens is required for epidemiological studies and research purposes. So, 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 samples and its use in molecular approaches. Objective: This work aimed to evaluate different conditions and timing of fecal preservation for the best outcome of molecular diagnosis of Cryptosporidium species.

Methodology: I. Preparatory steps for the study:  Sample collection: Setting: 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.  Examination of 380 fresh stool samples collected from patients with diarrhea. Setting: Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University Design: • Two direct wet smears were done for each sample once by saline and the other by iodine. • Samples were concentratetd by formalin-ethyl acetate sedimentation concentration technique. • Stained smears were done with Modified Ziehl-Neelsen technique, revealing a net of 10 positive samples which were used in the study. • Immunochromatographic test (ICT) was applied to validate positivity of staining and microscopy. II. Study of preservation conditions for subsequent DNA amplification:  Preservation of stool samples: Setting: Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University. Design: Each of the 10 stool samples was preserved at five different conditions; -20ºC, 70% ethyl alcohol, 10% formalin, 2.5% potassium dichromate (K dichromate) at 4ºC and 2.5% K dichromate at room temperature (RT).  DNA extraction: Setting: Research and Training Center on Vectors of Disease, Ain Shams University.

Design: • DNA extraction from each sample at zero time (fresh specimens) as the gold standard for the comparison with the preserved samples. • DNA ex¬traction at 10, 20 and 30 days of preservation from each sample in each of the five different conditions.  DNA amplification and visualization of PCR products. Setting: Molecular Medical parasitology Lab, Department of Medical Parasitology, Faculty of Medicine, Cairo University. Design: • DNA amplification using nested PCR, with Cryptosporidium oocyst wall protein (COWP) gene as a genetic marker for the identification of Cryptosporidium species. • Detection of the PCR products using gel electrophoresis and ultraviolet (UV) trans-illumination.

III. Data management and analysis: According to the type of data obtained for each parameter. Results: 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 a very poor performance (0%) along the three extraction intervals. The overall performance 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 performance was (0%).

Conclusions: The study highlighted freezing at -20°C as the most suitable condition for the preservation of Cryptosporidium spp. DNA in stool samples. K dichromate proves 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 it to be excluded as a preservative for Cryptosporidium spp. DNA in stool samples. The study revealed a highly significant difference between different preservatives performance along the preservation period indicating a highlighted outcome between the different preservatives. There was a significant difference between -20°C which gave the best outcome compared to 10% formalin signifying the poor effect of formalin as a preservation condition, 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 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. 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 per say as a preservative solution.