title: Development and evaluation of molecular methods for detection of Cryptosporidium spp. in human clinical samples.
abstract: Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. Detection of Cryptosporidium spp. in human clinical samples is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. A non-radioactive, genus specific DNA dot blot hybridization assay was developed using Digoxigenin (DIG) labelled probes to detect Cryptosporidium DNA in human clinical samples. Four hundred fifty (n = 450) clinical samples were subjected to microscopic examination, Polymerase Chain Reaction assay (PCR), Dot blot hybridization assay and Real Time PCR assay. A total of forty-one (n = 41) samples were positive by microscopy, forty-two (n = 42) by both PCR assay and dot blot hybridization assay and forty-three (n = 43) by Real Time PCR assay. Dot blot hybridization assay with a sensitivity of 95.5% and specificity of 99.75% could be an ideal choice for routine investigation of a large number of samples in a clinical setting as well as field.
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Shehla
authors.initials: S
authors.lastname: Khalil
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India. Electronic address: mirdhabr2078@gmail.com.
authors.forename: Bijay Ranjan
authors.initials: BR
authors.lastname: Mirdha
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Jaishree
authors.initials: J
authors.lastname: Paul
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Panda
authors.affiliation: Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Govind
authors.initials: G
authors.lastname: Makharia
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Rama
authors.initials: R
authors.lastname: Chaudhry
authors.affiliation: Shinjini Bhatnagar, Pediatric Biology Centre, Translational Health Science and Technology Institute, Gurgaon, India.
authors.forename: Shinjini
authors.initials: S
authors.lastname: Bhatnagar
mesh_terms: D000328:Adult; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000517:prevention & control; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D055815:Young Adult
chemical_list: D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S
keywords: Cryptosporidium; Detection; Digoxigenin-labelling; Microscopy; PCR; Real-time PCR