Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines

摘要

The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheather's flotation (SF) and Sheather's flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheather's flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100min. PCR based diagnosis of a sample took about 7.5-8h for completion and cost of diagnosis was estimated as approximately 7.604 US$ per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative study describing the sensitivity and specificities of four conventional coprological techniques altogether with respect to PCR along with the economic assessment and per sample diagnosis time of all the techniques for the diagnosis of cryptosporidiosis in bovines.