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Context: a customer in united states notified biomérieux of an increase of false positive gram negative target with the genmark eplex bcid when testing blood samples inoculated in one lot of bact/alert fa plus (plastic) - reference (b)(4) (lot# 0004102286 - expiration date: 11 dec 2024).The customer indicated that the issue concerns five (5) patients for different blood culture bottles but same lot number.Summary: results - patient 5 (bottle id: (b)(6)).Eplex result (lot#91660177 - exp date: 1-5-2025): gram negative target.Gram staining result: gram positive cocci clusters.Documented culture results : staphylococcus capitis.Investigation: as biomérieux is not the manufacturer of the genmark eplex bcid product, any investigational testing has been performed using the biofire bcid 2 panel.Investigation results, root cause analysis and conclusion: based on the evaluation of manufacturing, quality control, bcid2 panel testing, and the information provided by the customer, root causes for nucleic acid interference with fa plus pertaining to complaint (b)(4): materials: customers obtained positive bottle results with fa plus culture bottles.Samples from the positive bottles were tested with gram stain and subculture.Gram stains resulted with gram positive cocci (e.G., staphylococcus, streptococcus).The customers transferred sample from positive pf plus/fa plus bottles and tested the sample with a bcid molecular test.One of the results had a positive detection for a gram-negative organism (identification unknown).Bact/alert culture bottles: raw materials for bact/alert product is a contributing source for non-viable nucleic acids.The blood culture bottles contain media components that originate from animal/plant where non-viable nucleic acids (e.G., serratia marcescens) will be evident as a result of processing these raw materials.This will not impact the functionality of the bact/alert culture bottle; it is part of the bottle design.Bcid2 panel testing of raw materials as part of incoming inspections requirements has been implemented.However, distribution of non-viable nucleic acids within the raw material lots is an unknown.An industrialization/engineering scientific study (approved on 04jan2024) was an informational evaluation of media samples from twenty (20) locations across the filling process of a bact/alert pf plus lot and tested with bcid2 panels.The fill lot was selected to analyze the cumulative effects of media flow on possible detection of microorganism levels.Samples taken were from collection sites, prior to the autoclave process.Several samples from filtered media had positive detections for nucleic acids (e.G., serratia marcescens).The autoclave process exposes the bottles to high temperature, higher than the survival temperature of serratia marcescens, resulting in non-viable dna.Pf plus/fa plus quality control (qc) testing with bcid2 panel: all the qc records were reviewed for the pf plus and fa plus lots involved in this investigation.All bcid2 panel testing of samples from the fill lots was performed as per the release procedure, whereas not all bulks for the fill lot were represented within the sampling plan.Refer to the methods section below for further details.The results of the bcid2 panel tests of all the finished goods lots for the pf plus/fa plus lots were ¿no detection¿ except for pf plus lot 0004101718.Qc bcid2 panel tests for this lot showed detection of serratia marcescens.Further testing of this lot was conducted, and the impacted bottles were packaged into a different lot number.The blood culture bottles contain media components that originate from animal/plant where non-viable nucleic acids will be evident as a result of the original processing of these raw materials.The manufacturing process of the bottles exposes them to higher temperature than the survival temperature of serratia marcescens.The bcid2 panel will detect nucleic acids whether viable or non-viable.The bcid2 testing is recommended to be used with other clinical and laboratory results to aid in the diagnosis of microbial components within patient samples.Materials are a contributing factor to this complaint.Method: a review of the bact/alert® fa plus culture bottle instructions for use (document (b)(4)) provides the following adequate guidance pertaining to non-viable organisms with an bact/alert bottle.The laboratory procedure section states: "smear and subculture all positive bottles.¿ the limitations of the test section states ¿a gram-stained smear from a negative bottle may sometimes contain a small number of non-viable organisms that were derived from culture medium components¿¿ as per the biofire® instructions for use (rfit-asy-0841-05 july 2023) for bcid2 test panel, the following information is provided to alert the user that possible false positive results can occur due to detection of non-viable organisms/nucleic acids when using this product: ¿ laboratory precautions: (2) ¿blood culture media may contain non-viable organisms and/or nucleic acid levels that can be detected by the biofire bcid2 panel.¿ ¿the presence of non-viable organisms and/or nucleic acids in blood culture media may lead to false positive test results.Increases in false positive results due to non-viable enterococcus, p.Aeruginosa, proteus and e.Coli have been previously identified in various media types¿¿ ¿ limitations: (3) ¿results from the test must be correlated with clinical history, epidemiological data, and other data available to the clinician evaluating the patient.¿ (4) ¿any blood culture media may contain non-viable organisms and/or nucleic acid at levels that can be detected by the biofire bcid2 panel leading to false positive test results.¿ as per the genmark eplex® blood culture identification (bcid)-gram positive panel, package insert pi1079-c, the following information is provided to the user concerning possible false positive results due to the detection of non-viable organisms/nucleic acids when using this product: ¿ limitations of test: (3) ¿bacterial and fungal nucleic acids may be present in blood, independent of bacterial or fungal viability.¿ (4) ¿the results of the eplex bcid-gp panel should not be used as the sole basis for diagnosis, treatment or other patient management decisions.¿ all the ifus recommend that the product be used with other clinical and laboratory results (e.G., gram stain, subculture) to aid in the detection of microbial components in a patient¿s sample.Polymerase chain reaction (pcr) methods (e.G., genmark eplex, biofire bcid2 panel) have been beneficial in reducing time to identification of bacteria in clinical samples.Variations of molecular methods have been developed to assist in the detection of viable and non-viable organisms in clinical samples.However, there are limitations with the use of these methods in detecting cellular function (e.G., growth of live cells) versus cellular decline (dead cells).Article: dead or alive: molecular assessment of microbial viability; g.A.Congelosi and j.S.Meschke; applied and environmental microbiology, october 2014, volume 80, number 19; doi:10.1128/aem.01763-14.Summarized points that pertain to this investigation are below: ¿ typically, polymerase chain reaction (pcr) tests are inadequate at differentiating dna associated with a viable bacterial cell from a non-viable one ¿ microbiological culture tests detect viable organisms (¿gold standard¿) ¿ viable cells can be described as detection of a number of cells capable of multiplying ¿ non-viable cells can be described as inactivated (e.G., loss of cellular functions due to a puncture to the cell wall, exposure of the cellular functions to disinfectants causing slow decay of cells) qc testing of finished lots: qc release testing for fa plus lots associated with this investigation was reviewed by the investigator.Bcid2 panel testing was performed on all lots as per a release procedure, whereas the sampling plan for bcid2 panel testing requires bottles representing beginning, middle, and end across all media bulks of a fill lot.In this scenario, not all media bulks would have been part of the bcid2 panel testing.As of 03feb2024, the release procedure instructs the sampling plan to include culture bottles per each media bulk of the fill lot to be submitted for bcid2 panel testing.This will allow for a better detection of non-viable nucleic acids in bact/alert product.However, the sampling improvement does not completely eliminate the chances of a customer obtaining a detection when using a molecular test on a positive sample from a bact/alert culture bottle.Pcr tests are beneficial in reducing turnaround times for the identification of microorganisms in a patient¿s sample; however, these tests are inadequate at differentiating dna associated with a viable bacterial cell from a non-viable one.The durham manufacturing site has implemented an improved sampling plan, representing all bulks in the fill lot, for detection for non-viable organisms and/or nucleic acids in bact/alert culture bottles by testing biological raw materials and finished product with bcid2 technology.Method is a contributing factor to this complaint.Retains: finished product samples from fa plus lots were not required for determining root cause for this investigation.Returned products and testing: returned bottles from the finished fa plus lots were not required for determining root cause for this investigation.Conclusion: there is no evidence of any bottle malfunction with the bact/alert fa plus culture bottle lots.The bact/alert bottles detected organism growth as intended.Bcid2 testing is being conducted on designated raw materials and samples representing all media bulks for the fill lots for bact/alert products.Testing of all the media bulks that contribute to the fill lot fills will aid in monitoring product containing nucleic acid residual in an effort to reduce the chances of customers obtaining false positive results with bcid panel testing.
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