This section is targeted to problems identified within the project. We will post problems and answers here, so that other network members, EU officials and other stakeholders or just interested people can find them. We hope that these will solve problems identified by other parties.
Question 1. We are in need of macrolide resistant B. pertussis isolate to be used as a control in Etest/disk diffusion and in WGS based identification. Additionally Etest/PCR protocols would be helpful to set up the methods in our laboratory.
Actions. We have provided both PCR and Etest protocols. In addition, a macrolide resistant B. pertussis isolate (MTA required) was dispatched to be used as a control.
Question 2. For B. pertussis macrolide resistance. There is no cut-off values from EUCAST. What would you recommend to be used as “cut-offs” for sensitive and resistant isolates. In the “Laboraotry diagnosis and molecular surveillance of Bordetella pertussis”, <0.250 ug/mL is recommended for sensitive isolates and >256 ug/mL for resistance. However, our validation results only include values </= 0,125 and 256 ug/mL.
Actions. We recommend the values mentioned in the laboratory handbook (<0.25 and >256). Strains which have other than these MIC values are extremely rare, and not seen in the recent years. Further, all studies from China, where these strains are highly prevalent, are within the same cut-offs.
Question 3A. We are currently validating PCR assays for the confirmation of identification and toxin gene detection in Corynebacterium isolates in suspected Diptheria cases with a view to offering these services for clinical laboratories. However, we are having difficulty sourcing C. ulcerans and C. pseudotuberculosis strains and we were wondering if you could provide us with these strains please.
Actions. We provided one C. ulcerans and one C. pseudotuberculosis isolates (MTA required).
Question 3B. In regards, to the C. ulcerans and C. pseudotuberculosis isolates which you sent us, We have run them repeatedly on our Biomerieux Vitek MS (Maldi) and the C. pseudotuberculosis isolate is mostly giving an ID of C. ulcerans 99.9%. On two occasions, it gave a split ID of C. ulcerans 50%/ C. pseudotuberculosis 50%. The Maldi is correctly identifying the C. ulcerans isolate with a 99.9% identification. We tested both isolates with the Biomérieux API CORYNE kit and it correctly identified the isolates as follows, C. ulcerans 99.9% and C. pseudotuberculosis 99.3%. What might be the problem here?
Action. Unfortunately, the situation is the same with our Bruker MALDI-TOF. The current MALDI-TOF database is not accurate enough to discriminate these two species. However, our German colleagues have lately developed a database, which is able to discriminate these two species. Hopefully, it will be adapted for the “universal” database used by Bruker and Biomerieux (please, see Rau et al. under “publications”).
Question 4. I am reaching out because we urgently need Elek medium and antitoxin disks for the modified Elek test (we already have the newborn bovine serum). Could you kindly inform us of the procedure to obtain these supplies?
Action. Unfortunately, there is no commercial source for ELEK-agar nor for the anti-toxin. However, there is one publication describing manufacturers who provide this mainly for WHO laboratories (see Marshall et al. under “publications”). However, we will provide you with a small batch of both ELEK-agar and DAT, and share current protocol for ELEK-test preparation (see WHO diphtheria manual under “publications”).