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Validation of the MALDI-TOF for the Identification of Neisseria Gonorrhoeae Proposal Submitted by the State Hygienic Laboratory at the University of Iowa

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Validation of the MALDI-TOF for the Identification of Neisseria Gonorrhoeae Proposal Submitted by the State Hygienic Laboratory at the University of Iowa Validation of the MALDI-TOF for the Identification of Neisseria gonorrhoeae Proposal Submitted by the State Hygienic Laboratory at the University of Iowa 1. Please describe the laboratory’s current MALDI-TOF testing practices including pathogens detected. The State Hygienic Laboratory (SHL) at the University of Iowa acquired the Bruker Microflex LT system in March 2014. SHL currently utilizes the updated MALDI Biotyper Compass BDAL database version 4.1.70, Mycobacteria Library 4.0, filamentous fungi and also has the Bruker Select Agent Database (currently not in-use) for pathogen detection. SHL can identify bacteria from a variety of selective and differential media including Buffered Charcoal Yeast Extract (BCYE) Agar, MacConkey, MacConkey and Sorbitol, MacConkey Agar with Sorbitol, Cefixime and Tellurite (CT-SMAC), Hektoen, Campylobacter CSM Agar, CT-SMAC and chocolate agar. Most bacteria are identified using the direct transfer method. SHL completed a kill study in the summer of 2017 for the extraction of Neisseria meningitidis using a tube extraction followed by filtration using a 0.1 µM filter. A core group of 5 staff were initially trained to operate the instrument and now utilize it on a daily basis to identify bacteria, yeasts, molds, and non-tuberculous mycobacteria (NTM). SHL staff have performed various research studies with the instrument to validate testing methods for fungi and NTM as part of an infectious diseases fellow’s project, for select agent and near neighbor identification, and currently for environmental Legionellaceae. The instrument is used daily to identify between 5-10 organisms per run. We estimate that over the past 3 years, SHL staff have identified over 1000 organisms using the Bruker instrument. 1 | Page 2. Please describe the laboratory’s experience in testing for N. gonorrhoeae including methodologies. The State Hygienic Laboratory has 8 experienced individuals (8 – 25 years of microbiology experience, average 17.5 years) that rotate through the reference bacteriology bench wherein N. gonorrhoeae culture and work-up is performed. With the mainstream use of molecular testing for Chlamydia/gonorrhea (CT/GC) testing, SHL typically receives very few isolates for test of cure by culture. We began reaching out to local county public health departments to send suspect samples from patients presenting to their STI clinic and got 5 new confirmed isolates in the past month. We will expand our outreach and plan to enlist more clinics that participate in the state’s CT/GC screening program to expand isolate collection. SHL staff perform culture for N. gonorrhoeae and confirmation using selective and non-selective media for isolation (MTM, CA), a combination of biochemical testing (CTA sugars, rapid NH), co-agglutination (Phadebact), or DFA. Staff perform N. gonorrhoeae culture and identification on average 1-2 times/month. 3. Please describe the current methodologies for testing non-gonococcal Neisseria species. Current methodologies used for culture and identification of non-gonococcal Neisseria species include biochemical tests, MALDI-TOF, and 16s DNA sequencing. Testing occurs approximately 1-2 times/month by the staff as listed above in #2. Bacterial isolates that are confirmed to be N. meningitidis are serotyped using conventional agglutination methodology. SHL utilizes a number of strategies to mitigate the biosafety risks of working with Neisseria meningitidis. SHL ensures vaccination of staff with both the quadrivalent meningococcal conjugate and serogroup B meningococcal vaccines. All staff are provided with appropriate PPE (gown, gloves, eye protection) when working within the laboratory. All culture manipulations, gram staining and biochemical setup of specimens and referred isolates of suspected Neisseria meningitidis are performed in a Class 2 Biosafety 2 | Page Cabinet. Known or suspected isolates of Neisseria meningitidis are serotyped and processed in a BSC using BSL3 practices (back-closing gown and double gloves). Plates are taped shut prior to incubation to avoid accidental exposure. For general specimen culture work, staff have been trained to recognize potential isolates of meningococcus as well as any select agents and to immediately move all materials inside of a biological safety cabinet for further work-up. Additionally, staff are familiar with the common signs and symptoms of infection and understand that they are to report immediately such indicators to their supervisor and seek medical attention. These staff also carry a medical alert card to inform the health care provider of potential exposure to such pathogens. An annual risk assessment is performed to ensure that both the appropriate environmental and practice controls are in place. All laboratorians must obtain an annual Documentation of Competency in these test methods, which includes observations of safety practices. 4. Please describe the current collection of non-gonococcal Neisseria species. Attached is a list of the number of all Neisseria species currently stored at SHL. 5. Include a completed and signed copy of Appendix B as an attachment. 3 | Page # of isolates Storage Method Moraxella catarrhalis 2 Frozen stock Kingella denitrificans 0 N/A Neisseria gonorrhoeae 448 Frozen stock Neisseria meningitidis 188 Frozen stock N. meningitidis Serogroup A 1 Frozen stock N. meningitidis Serogroup B 73 Frozen stock N. meningitidis Serogroup C 59 Frozen stock N. meningitidis Serogroup W135 8 Frozen stock N. meningitidis Serogroup X 1 Frozen stock N. meningitidis Serogroup Y 33 Frozen stock N. meningitidis Serogroup Z 1 Frozen stock N. meningitidis nontypeable 7 Frozen stock N. meningitidis Serogroup unknown 5 Frozen stock Neisseria bacilliformis 0 N/A Neisseria cinerea 5 Frozen stock Neisseria elongata 0 N/A Neisseria flavescens 0 N/A Neisseria lactamica 1 Frozen stock Neisseria macacae 0 N/A Neisseria mucosa 0 N/A Neisseria polysaccharea 2 Frozen stock Neisseria sicca 1 Frozen stock Neisseria subflava 3 Frozen stock Neisseria flava 0 N/A Neisseria perflava 1 Frozen stock Wade Aldous.
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