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A biomérieux internal investigation was performed.The investigation was initiated due to false positive results on chromid® mrsa smart (ref 413050) for 13 staphylococcus aureus strains.The investigation consisted of a review of complaints registered for the impacted reference 413050, a review of impacted lot number files, analysis of the retained sample plates, analysis of the strains returned by customers and a review of the raw materials used in the manufacturing of the product.Review of the impacted lot numbers (1005950400, 1005801390, 10015850250, 1005898640, and 1005860490) revealed the manufacturing process was followed according to the manufacturing techniques and the quality control of the finished product conformed with defined specification for both microbiological performance and the physical parameters of the agar.Quality control tests were performed in order to release the product, the following controlls were carried out: -control of the microbiological state and appearance of the product; the results conformed to specifications.-microbiological activity controls: strains tested included s.Aureus atcc 43300 bmr, s.Aureus 0306055 bmr, s.Aureus atcc 13150, s.Aureus atcc 29213, e.Cloacae atcc 13047, e.Faecalis atcc 29212, c.Albicans atcc 10231, s.Haemolyticus 1105071 bmr, and s.Hominis atcc 700236 bmr.All of the quality controls conformed to specifications.-the ph of the product was controlled and conformed to specifications.Analysis of the retained biomérieux samples involved testing one (1) of the impacted lot numbers 1005898640 (expiry 20oct2017), a lot number close to its expiry date 1005916640 (expiry 02nov2017) and a reference lot number 1006006010 (expiry 14dec2017).Quality control strains were tested according to the technical quality control procedure (019447) by direct inoculation of the plates from a calibrated bacterial suspension.The following quality control strains were tested: s.Aureus atcc 43300 bmr, s.Aureus 0306055 bmr, s.Aureus atcc 13150, s.Aureus atcc 29213, e.Cloacae atcc 13047, e.Faecalis atcc 29212, c.Albicans atcc 10231, s.Haemolyticus 1105071 bmr, and s.Hominis atcc 700236 bmr.All quality controls conformed to specifications.For the mrsa strains, characteristic growth was observed.For the mssa strains, inhibition was observed.Four (4) internal wild mssa strains were also tested on the same lot numbers and the results obtained did not show any growth; the four (4) strains were fully inhibited.As customers reported using an enrichment broth before inoculating the samples on chromid mrsa smart, further analyses were performed on two (2) additional lot numbers, 1005970640 (expiry 28nov2017) and 1006023550 (expiry21dec2017).As in the first analysis, the same atcc quality control strains were tested along with the four (4) internal wild strains and also a strain used during product development.Five (5) strains returned by one (1) customer were also tested as part of this analysis.All of the strains were tested using direct inoculation and also after an enrichment step.Two (2) types of broth were used, bhi + 6.5% nacl and tsb + 6.5% nacl.The plates were read at 24 hours and 48 hours of incubation as one of the customers reported incubating the samples for 48 hours.The results indicated the following: -all of the samples that were tested by direct inoculation onto chromid mrsa smart agar and incubated for 24 hours at 35° c were in accordance with specifications.Indeed, all of the mssa strains coming from the internal biomérieux collection as well as the customer's strains were fully inhibited on chromid mrsa smart agar from a bacterial suspension calibrated at approximately 10e7 cfu/ml.-on the five strains (5) returned by one of the customers, growth of the three (3) mssa strains was observed after enrichment, whatever the broth used, bhi and tsb, at 24 hours of incubation.The results of the above testing also indicated an increase in false positives at 48 hours of incubation, in the case of the research of mrsa strains using a methodology including the enrichment step.However, whatever the protocol used, with and without the enrichment step, chromid mrsa smart must be read only between 18 and 24 hours of incubation, as per the package insert, and not at 48 hours.It is well known that the enrichment step increases the sensitivity of mrsa, but it slightly decreases the specificity of chromid mrsa smart.This remark is also true for all chromogenic media dedicated to the research of mrsa strains in human clinical samples (see publications in the package insert).Certain antibiotics used to manufacture chromid mrsa smart were reviewed for process, supplier and preparation modifications.The review indicated that since jan2017, no modifications were noted for these antibiotics.Additional testing was performed on lots 1005970640 and 1006023550.For these lots, the customer reported using an enrichment step (bhi-t broth) and incubating the plates for 24 hours.In addition, testing was performed on lots 1005970650 and 1005999020.The lot number files were reviewed and results indicated the manufacturing process was followed according to the manufacturing techniques and the quality control of the finished product conformed to defined specifications for both microbiological performance and the physical parameters of the agar.A total of 13 strains were submitted by customers.Identification to staphylococcus aureus was confirmed for all 13.Reference methods were performed to characterize the strains: pcr meca/mecc, agar dilution oxacillin (ad ox), and broth microdilution cefoxitin (bmd fox).There was absence of gene meca and mecc for the 13 strains.Susceptible results were obtained with ad ox.For two (2) strains, ox mic = 0.125 mg/l (s1 and s2).For seven (7) strains (s3, s4, s5, s8, s10, s11 and s12), the results were 0.25 mg/l.For two (2) strains (s6 and s13), results were 0.5 mg/l.For one (1) strain (s7), the result was 1mg/l, and 2 mg/l for another strain (s9).On bmd fox, the 13 strains tested gave fox mic equal to 4 mg/l.Therefore, strains are considered borderline strains (on the breakpoint) for cefoxitin.The different tests performed show the 13 strains of staphylococcus aureus are susceptible to methicillin (mssa).In conclusion: all of the 13 customer strains have cefoxitin mics equal to 4 mg/l and are meca, mecc negative.The hypothesis to explain the issue observed by the customers is that these so-called "borderline" strains are at the mic critical limit for mrsa strains (s.Aureus resistant strains when cmi to cefoxitin > 4 mg/l), which can explain their growth after enrichment on chromid mrsa smart media.As the enrichment step favors the growth of strains in bhi or tsb broth, this may have an inoculum effect on the activity of antibiotic markers of mrsa strains.Indeed, antibiotics in the presence of a strong inoculum from enrichment broth can be insufficient for total inhibition of borderline mssa strains.Dodémont et al.2015 (package insert), emphasizes that enrichment increases the sensitivity of detection of mrsa strains after 48 hours of incubation at the expense of the specificity of the media (the risk of false positive mrsa results can double).It is specified in the package insert that the reading time must be between "18 hours and 24 hours".The package insert also states, "some s.Aureus strains that do not have the meca gene may produce typical colonies." the investigation confirmed that there is no drift in the performance of the media nor a drift in the stability of the antibiotics used in the media.The product is performing within specifications.
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