Age-Stratified Bayesian Analysis To Estimate Sensitivity and Specificity of Four Diagnostic Tests for Detection of Cryptosporidium Oocysts in Neonatal Calves

Abstract

There is no gold standard diagnostic test for the detection of bovine cryptosporidiosis. Infection is usually highest in 2-week-old calves, and these calves also excrete high numbers of oocysts. These factors may give rise to variations in the sensitivity and specificity of the various diagnostic tests used to detect infection in calves of various ages. An age-stratified Bayesian analysis was carried out to determine the optimum diagnostic test to identify asymptomatic and clinical Cryptosporidium sp. infection in neonatal calves. Fecal samples collected from 82 calves at 1 week, 2 weeks, 3 weeks, and 4 weeks of age were subjected to the following tests: microscopic examination of smears stained with either phenol-auramine O or fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium monoclonal antibody, nested-PCR, and quantitative real-time PCR. The results confirmed a high prevalence of Cryptosporidium sp. infection, as well as a high level of oocyst excretion, in 2-week-old calves. The sensitivities of all the tests varied with the age of the calves. Quantitative real-time PCR proved to be the most sensitive and specific test for detecting infection irrespective of the age of the calf. The microscopic techniques were the least sensitive and exhibited only moderate efficiency with 2-week-old calves excreting large numbers of oocysts, the majority of which were diarrheic. It was concluded that, when interpreting the results of routine tests for bovine cryptosporidiosis, cognizance should be taken of the sensitivity of the tests in relation to the age of the calves and stage of infection.

Diagnosis of gastric cryptosporidiosis in birds using a duplex real-time PCR assay

“…The different rates of positivity for C. galli between nPCR/S and duplex real-time PCR may occur because realtime PCR normally exhibits a higher sensitivity than nested PCR (De Waele et al, 2011;Yang et al, 2009;Homem et al, 2012). A significant portion of the positive samples exhibited Ct values between 35 and 39, most likely due to the small amounts of oocysts in the samples, which is common in infections with C. galli (Antunes et al, 2008;Nakamura et al, 2009).…”

“…The real-time polymerase chain reaction (PCR) method has proven to be a valuable tool for the diagnosis of Cryptosporidium (Yang et al, 2009;De Waele et al, 2011). As a result, some studies have developed methods for the detection of several Cryptosporidium species by real-time PCR, including Cryptosporidium spp., Cryptosporidium andersoni, Cryptosporidium hominis, Cryptosporidium parvum and the cluster formed by Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi (Jothikumar et al, 2008;Yang et al, 2009;Hadfield et al, 2011;Homem et al, 2012;Burnet et al, 2012).…”

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

“…Several other authors have also observed higher sensitivity of real-time PCR for the diagnosis of Cryptosporidium spp. compared to the sensitivity of nested PCR (Yang et al 2009;De Waele et al 2011). However, Hadfield et al (2011) reported similar sensitivities for the two techniques.…”

“…Among these methods, real-time polymerase chain reaction (PCR) has a high sensitivity and allows for the diagnosis of clinical cryptosporidiosis as well as for the detection of asymptomatic carriers, making it a valuable tool for epidemiological studies in humans and animals (Tanriverd et al 2002;Fontaine and Guillot 2003;Minarovicová et al 2009;De Waele et al 2011;Hadfield et al 2011).…”

Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing

“…These mimic epitopes might react with the lateral flow assay to produce a false-positive result. False positives due to non-specific reactions in immunoassay tests have been previously reported, and decreased specificity among age groups have been noted in animal studies [24, 26]. Cross-reactivity of either RCA test’s proprietary antibodies with other organisms was not suspected as package inserts included a list of parasites, bacteria, and viruses tested for cross-reactivity during product development, quality assurance, and quality control.…”