Estimation of Performance Characteristics of Analytical Methods for Mycobacterium avium subsp. paratuberculosis Detection in Dairy Products

Abstract

Paratuberculosis is a chronic enteric infection, caused by Mycobacterium avium subsp. paratuberculosis (MAP), affecting virtually all ruminants as well as other animals. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. In surveillance studies, different analytical methodologies were employed to detect MAP, showing different results and incidence in dairy products. The aim of this study was to evaluate the performance characteristics of three analytical methods [culture, quantitative PCR (qPCR) and peptide-mediated magnetic separation (PMS) phage-based assay] for MAP detection in raw, heat-treated and powdered milk. The methods were evaluated according to performance characteristics defined for qualitative methods in ISO 16140-2:2016. To estimate sensitivity (including trueness) and LOD, 720, and 900 test portions, respectively, were blind tested by two laboratories. Considering all matrices, different sensitivities, expressed as the percentage of positives from the total of true positive test portions, were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%). Trueness, expressed as results correctly assigned (including positive and negative) to the reference value, was 93% for the IS900 qPCR method, 89% for culture and 49% for the PMS-phage. The LODs obtained in this study were similar to the LODs previously published for cultural and qPCR methods. However, for the PMS-phage method, the obtained results showed higher LOD values compared to the limited data available in the scientific literature. Our results highlight that while the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices should be treated with a lot of caution as performance characteristics obtained were lower than for the two other methods tested. qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable MAP, but qPCR, which is widely used in analytical and surveillance studies, can be considered a suitable and recommendable alternative to cultural methods for screening, if confirmation of MAP’s viability is not requested.

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

“…However, when researchers in other laboratories have attempted to adopt the optimised phage assay or the PMS-phage assay, technology transfer has generally not been a smooth process (e.g. Butot et al 2019 ), and considerable training and troubleshooting has been needed from QUB researchers. We acknowledged some time ago (Foddai and Grant 2017 ) that the PMS-phage assay has a complex, multi-step protocol that does not lend itself well to high-throughput testing of milk samples.…”

Phage Amplification Assay for Detection of Mycobacterial Infection: A Review

“…For the usage of PMS-PA for milk, it is crucial to know that: (i) maximal numbers of MAP cells were found to sediment in the pellet fraction upon centrifugation at 2500 g for 15 min at ambient temperature; (ii) milk specimens should be refrigerated at 4 °C after collection and MAP testing should commence within 24 h, or, when not possible, specimens can be frozen at −70 °C for up to one month without significant loss of MAP viability [ 92 ]. However, the PMS-PA performance characteristics obtained by Butot et al [ 94 ] were inconsistent with those published by Foddai and Grant [ 43 ]. Using the most probable number enumeration technique to determine the reference value, the LOD 50 of PMS-PA was assessed to be 3.7 log 10 CFU/50 mL of both raw and heat-treated milk.…”

“…Different paramagnetic beads coated with antibodies and/or peptides were evaluated with the aim of determining the capture efficiency and specificity for MAP cells [ 91 , 94 ]. The most common beads for MAP capture are MyOne Tosylactivated Dynabeads, which were used in all studies summarized in Table 5 and Table 6 .…”

Bacteriophage-Based Methods for Detection of Viable Mycobacterium avium subsp. paratuberculosis and Their Potential for Diagnosis of Johne's Disease

“…In practice, potential false positive results may arise with plaque-based phage assays due to ineffective viricide treatment, meaning that some plaques would be due to non-inactivated D29 phages, or some cells bursting before plating in agar happens, also releasing D29 phages that will interact with M. smegmatis lawn to form plaques. Early adopters of the phage-PCR assay or PMS-phage assay have run into such issues, and have found the multiple steps and transfers involved, the timed incubation steps, the need for molten agar and an M. smegmatis culture, and one or two overnight incubations tedious ( 32 ). In my experience of transferring our optimized Phage assay or PMS-phage assays to a number of other laboratories, this has rarely proved to be a straightforward process and a lot of follow-up troubleshooting activity has ensued.…”