ABSTRACT
Cryptosporidium is a leading cause of persistent diarrhea in developing countries where it is reported to be more common in malnourished children with more severe consequences of the disease than in well nourished ones. PCR which is the most sensitive and specific diagnostic method for detecting and genotyping Cryptosporidium still has some drawbacks. The present study aimed at evaluating melting curve analysis after real-time PCR in diagnosis and genotyping Cryptosporidium as an alternative to traditional multiplex PCR followed by gel electrophoresis. Seventeen naturally infected immun°Compromised patients suffering from Cryptosporidium diagnosed by both microscopy and immun°Chrommatographic strip assay provided the study isolates. All samples were subjected to traditional multiplex PCR followed by gel electrophoresis and real-time PCR followed by melting curve analysis. Melting curve analysis real-time PCR proved 100% sensitivity and specificity when compared to traditional multiplex PCR. It had the advantage of being less time consuming [1 hr], less liable for post amplification contamination by carry-over as the pr°Cess is completed in a closed tube. Genotyping of Cryptosporidium on the basis of melting curve analysis revealed that C. hominis showed two distinct peaks at 85.5°C and 88.5°C while C. parvum showed two distinct peaks at 80.1°C and 88.5°C. Interestingly, one isolate proved to be a mixed infection showing three peaks at 80.1°C, 85.5°C and 88.5°C. DNA melting curve analysis real-time PCR offers a step forward in the detection and differentiation of Cryptosporidium spp. by circumventing disadvantage of traditional PCR