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Context: a customer in ireland notified biomérieux of an instrument false negative result in association with bact/alert mp 100 btls (ref.419744, batch number 0001060658, expiration date 15-aug-2024) for one patient on the bact/alert 3d instrument for a mycobacterium positive sample.Investigation: root cause analysis and conclusion materials: the mp culture bottle provides media with suitable nutrients to recover mycobacterial species.It is used in conjunction with the mp antimicrobial supplement (mas) kit that reduces the presence of bacteria (break-through contamination) after the digestion-decontamination process of non-sterile specimens.The antimicrobial supplement (as) contains lyophilized powder four antibiotics (nalidixic acid, polymyxin b, trimethoprim, and fosfomycin) along with one antifungal (amphotericin b) that suppresses unwanted respiratory flora from non-sterile samples (e.G., sputum, bronchial lavage, bronchial wash).The as is reconstituted with the nutrient supplement (ns) until dissolved and mixed gently before use.The customer obtained a bronchial lavage sample type from the right middle lobe with no blood present in (b)(6) 2023.Article 1: diagnostic value of bronchoalveolar lavage and bronchial washing in sputum-scarce or smear-negative cases with suspected pulmonary tuberculosis: a randomized study; y.W.Kim, b.S.Kwon, s.Y.Lim, y.J.Lee, y-j.Cho, h.I.Yoon, j.H.Lee, c.-t.Lee and j.S.Park; 1198-743x/©2019 european society of clinical microbiology and infectious diseases.Published by elsevier ltd.All rights reserved.Https://doi.Org/10.1016/j.Cmi.2019.11.013.Key points in this article pertaining to this investigation are listed below: bronchoalveolar lavage (bal) samples are obtained by the placement of the bronchoscope into the infected lung segment then 150ml sterile normal saline injected into the area followed by the recovery of the specimen into a sterile container tests were performed within 24 hours after sample collection bal can provide additional accuracy in the diagnosis of tuberculosis; targeted the infected region of the lung and obtained more abundant volume of specimen bal also provided more culture-positivity for mycobacterium species (m.Tuberculosis and nontuberculosis mycobacterial ntm species) the timing of the bal sample collection from the patient ( on (b)(6) 2023) and the testing of the patient sample with the first mp bottle from lot 0001060658 (bottle loaded on (b)(6)2023) and with a second mp bottle from lot 0001060664 (bottle loaded on (b)(6) 2024) suggest that the sample loaded into the first mp bottle was from the original digestion-decontaminated patient specimen (only if the patient sample was obtained on the last day of (b)(6) 2023) or the sample could have been from the original digestion-decontaminated patient specimen and frozen-thawed for testing if the bal sample collection occurred earlier in the month, nov2023.Typically, a bal sample would undergo decontamination process within 24 hours or refrigerated at 4°c if there was an anticipation of a delay.To preserve a sample for testing at a later date, aliquots of the original bal sample would be frozen, thawed, decontaminated and tested.The customer inoculated the second mp culture bottle with an aliquot of the processed bronchial lavage sample and obtained a positive detection of microbial growth at 23 days.The time between inoculations into the two different mp bottles was approximately 6 weeks, suggesting that the second aliquot was a preserved sample.It is unknown, perhaps most likely, this aliquot was from the original bronchoscopy process to obtain the bal sample immersed in sterile saline and then underwent a separate decontamination process before being inoculated into the second bottle.The customer¿s digestion-decontamination process resembles a modified petroff¿s method, adequate to reduce the contamination rates for non-sterile samples (e.G., bronchial lavage) and isolate mycobacteria for detection testing.The myco antimicrobial (mas) kit p/n 414997, lot number 1010161620 (nutritif supplement 2234510, antimicrobial supplement 2234730), expiry date 30oct2024 was stored in refrigerator as instructed by the mp ifu prior to use.The customer confirmed that the mp bottles were stored at room temperature before use and mp lot 0001060658 was passed internal quality control procedures using mycobacterium tuberculosis atcc strain 25177 on (b)(6) 2023 (after bottle was loaded on (b)(6) 2023).Article 2: cryopreservation of mycobacterium tuberculosis complex cells; z.Shu, k.M.Weigel, s.D.Soelberg, a.Lakey, g.A.Cangelosi, k-h lee, j-h chung, and d.Gao.Journal of clinical microbiology (p.3575-3580); doi:10.1128/jcm.00896-12.The following key points pertain to this investigation: viability of some bacteria (e.G., m.Tuberculosis) can be affected by shipping and storage conditions the samples were human sputum spiked with mycobacterium bovis (m.Bovis) cells with a known concentration for first part of study the three different cryopreservation media were phosphate buffered saline (pbs), difco middlebrook 7h9 broth (7h9), and 7h9 plus glycerol after media addition to the cells, the cells (all at the same density in each media type) were kept at room temperature for 15 minutes for equilibration prior to cryopreservation 0.5ml cell suspension was aliquoted into 2ml cryogenic freezing vials two cooling methods were used: slow cooling- vials placed in polyethylene layer box providing a thermal insulation and placed into a -80°c freezer overnight before immersed into liquid nitrogen: cooling rate of -1 to -3.5°c/min was observed rapid cooling-vials were immersed into liquid nitrogen: cooling rate -100 to -400°c/min observed the frozen cell samples were removed from the liquid nitrogen and immersed into a 37°c water bath and gently agitated until no crystals were present cells were assessed for viability by microbiological culture and live/dead staining second part of study: cryopreservation of m.Tuberculosis cells in sputum whereas sputum cells were spiked with known concentration of m.Tuberculosis and aliquots of 0.5ml into vials were frozen by slow cooling method, rapid cooling method, and directly into a -80°c freezer (cooling rate -10 to -20°c/min was observed) after thawing in a 37°c water bath, samples were treated with n-acetyl-l-cysteine and sodium hydroxide (nalc-naoh) followed by standard digestion -decontamination procedures to determine the effects of nalc-naoh and cryopreservation, control experiments were conducted: difference between control 1 and control 2: effects of centrifugation on cells difference between control 2 and control 3: effects of nalc-naoh process viability of cells after freezing and thawing showed that a lower cooling rate allowed greater cfu recovery in all the freezing media tested fresh cells without cryopreservation showed a higher percentage of intact cells; however, there was no significant difference of intact ¿live¿ cells among different freezing and cooling rates viability of m.Tuberculosis cells after cryopreservation in sputum: treatment of cells with nalc-naoh, centrifugation, and freezing-thaw process contributes to viable cell loss viability of cells was better with slow cooling method sample cooling rates depend on several factors (e.G., container, volume of sample, heat transfer conditions inside freezer) the customer inoculated the lj pyruvate slant lot number 04104780, expiry date 18mar2024 and lj glycerol slant, lot number 04104491, expiry date 23feb2024 with the digested-decontaminated bronchial lavage sample at the same time as the inoculation of the mp bottle.Growth of m.Chimaera was detected at 33 days.A lowenstein-jensen (lj) media slant is used for the cultivation and isolation of mycobacterium species.Typically, the lj slant is used to diagnose mycobacterium infections, testing of antibiotic susceptibility of isolates and differentiate species of mycobacterium (e.G., morphology, growth rate).If organisms grow poorly or not at all, further tests are needed to confirm the presence of mycobacterium.Article 3: 2021: updated experience of mycobacterium chimaera infection: diagnosis and management in a tertiary care center; n.Y.Tan, a.D.Tarabochia, d.C.Desimone, c.V.Desimone, j.W.Wilson, g.Bagameri, c.E.Bennett, and o.M.Abu saleh.Published by open forum infectious diseases®2021; https://academic.Oup.Com/ofid/article/8/8/ofab348/6312669.The following bullet point is a key aspect related to this investigation: mycobacterium chimaera (m.Chimaera) is a slow-growing nontuberculous mycobacteria (ntm) associated with mycobacterium avium complex (mac) it was stated that 0.5mls of processed sample was inoculated into a bact/alert mp culture bottle from lot 0001060658 and had a false negative result.The graph of the mp bottle lot 0001060658 was flat indicating no growth was detected.The presence of the mycobacteria in this mp bottle was most likely not viable and therefore, would not have produced enough co2 for the detection of a growth, a positive result for m.Chimaera.Perhaps the digestion-decontamination process for the first sample was too harsh, creating an unfavorable growth environment in the mp bottle for this organism.Additionally, if the first sample was a frozen-thawed aliquot from the digestion-decontamination of the patient sample originally processed for testing, there would have been conditions for viable loss of cells inhibiting a positive detection of m chimaera.In addition, there was no mention of the ph value after the decontamination process of the sample inoculated into the first mp bottle.The sample could have had a long exposure (e.G., greater than 20 minutes) to naoh, a high ph value, would kill off the mycobacteria and result in a false negative culture bottle.It was unknown if the customer visually inspected the mp bottle (lot 0001060658) for media turbidity or sensor color (yellow) indicating any possible presence of organism growth.Also, there was no mention by the customer that the contents of the false negative mp bottle were subcultured to confirm no organism growth.The customer inoculated a second mp culture from lot 0001060664 with an aliquot of the processed bronchial lavage sample and obtained a positive detection of microbial growth at 23 days.The time between inoculations into the two different mp bottles was approximately 6 weeks, suggesting that the second aliquot was a preserved sample.It is unknown, perhaps most likely, this aliquot was from the original bronchoscopy process to obtain the bal sample immersed in sterile saline and then underwent a separate decontamination process before being inoculated into the second bottle.Materials are a contributing factor to this complaint.Device history record and complaint trends query for deviations showed no adverse trend was present for this false negative issue.Queries on complaint data showed no adverse trend was present for a false negative with a subculture positive result for the bact/alert mp bottle.Conclude product compliance to specification/performance there is no evidence of any bottle malfunction with the bact/alert mp culture bottle lot.Release procedures for bact/alert mp culture bottle and bact/alert mp antimicrobial supplement kit require growth performance testing for several mycobacteria.In addition, recovery results for m.Chimaera are shown for specimen type for bronchial lavage in the ifu for the bact/alert mp product.Monitoring and detection methods for bottle defects associated with potential false negative results are part of the manufacturing and quality control processes for bact/alert culture bottles.
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