Cystic Fibrosis: Practical Updates for the Clinical Microbiology Lab

WV FALL SCACM MEETING

Stephanie L. Mitchell, Ph.D., D(ABMM) Director of Clinical Microbiology at UPMC Children’s Hospital of Pittsburgh Assistant Director of Clinical Microbiology at UPMC-Presbyterian Assistant Professor, Department of Pathology, University of Pittsburgh Disclosures

• None to declare. Cystic Fibrosis (CF)

. Inherited autosomal recessive genetic disorder . Occurs when mutations are present in the CF Transmembrane Conductance Regulator (CFTR) . Normal function of CFTR: • regulates the transepithelial ion flow • maintains the proper ionic composition and volume of airway surface fluid CF Pulmonary Function and Microbiology

Chirico et al. HMGBI and lung function decline in cystic fibrosis. 2015 Clin Microbiol Infect CF Microbiology

CF Foundation. CF Patient Registry Annual Report. 2016. CF Pathogens S. aureus (SA)

. Studies have shown CF patients infected with MRSA have . Longer and more frequent hospital stays . More likely to be co-infected with Aspergillus fumigatus

CF Foundation. CF Patient Registry Annual Report. 2016. Small colony variants (SCV) . Auxotrophic isolates of S. aureus . Auxotrophy for thymidine . Hemin and menadione may also occur but less frequent SA SCV . Treatment with trimethoprim- sulfamethoxazole (SXT) can select for this phenotype

. Some can require CO2 . Small, non-hemolytic, non-pigmented often with a “fried egg” appearance . Sometimes have greening of the agar around colonies SA/SCV Detection

Mannitol Salts Agar (MSA) CHROMagar Staph aureus 89.4% sensitive 100% sensitive

Include either MSA or a chromogenic agar for Adequate for recovery of SCVs SA for all CF cultures

May not recover SCVs but has not been studied SCV Antimicrobial Susceptibility Testing (AST)

. SCV CLSI M100 Ed28 2018 -”Cefoxitin MIC and disk diffusion tests performed on media other than CAMHB or un- supplemented MHA do not reliably detect mecA-mediated resistance in isolates of S. aureus that do not grow well on these media (eg, small colony variants).”

-”Testing for PBP2a using induced growth (ie, growth taken from the zone margin surrounding a cefoxitin disk on either BMHA or a blood agar plate after 24 hours

incubation in 5% CO2) or mecA should be done.”

- Also true for those isolates that terminate on commercial systems. P. aeruginosa (PA)

. Prevalence has been steadily declining . 19.7% never positive for PA

. Mucoid strains commonly recovered . Associated with chronic infection . Eradication assumed to no longer be possible . Recommend to report isolates that are mucoid

CF Foundation. CF Patient Registry Annual Report. 2016. PA Detection . Non-mucoid strains can be identified if exhibiting classic characteristics . Non-lactose fermenting on MAC . Metallic sheen . Green diffusible pigment . Grapelike odor . Oxidase positive

. Different morphotypes that do not exhibit the characteristics above are often isolated . Isolate and identify by commercial system or MALDI-TOF MS

. While mucoid PA identification can be based on morphotype, there are other non-fermenting Gram- negative rods that exhibit a mucoid phenotype . Recommend identification for mucoid isolates PA AST . Different morphotypes can have different susceptibility patterns . Perform susceptibility testing on all morphotypes . Efforts to separate different morphotypes should be undertaken . If multiple attempts to isolate fail, consider reporting AST from mixed inoculum

. AST should be performed by disk diffusion or Etest . Commercial systems were found to have very high very major error rates . AST frequency? . UNC Chapel Hill performs AST no more frequently than once every 3 months for outpatients; upon admission for inpatients

. PA auxotrophs . May grow slowly and fail to grow upon repeated subculture B. cepacia complex . 721 patients were reported to the CF Registry as having B. cepacia complex isolated from culture

. Discordant with reported isolates from the B. CF Foundation. CF Patient Registry Annual Report. 2016. cepacia Research Lab and Repository at University of Michigan . The Complex Emerging pathogen?

LiPuma, J.J., Micobial Epidemiology in CF. 2010 CMR

Hauser, A.R., et al. Microbes and outcomes in cystic fibrosis. 2011. CMR B. cepacia complex

. Accurate identification is important . B. cepacia selective agar (BCSA) is superior to P. cepacia agar (PC agar) and OFPBL agar . Commercial systems commonly mis-identify B. cepacia complex and misidentify B. gladioli and Ralstonia pickettii as B. cepacia . Report as presumptive, send to CF Lab for final identification by molecular methods B. cepacia complex AST

. MIC method (gradient diffusion) and/or disk diffusion should be performed . Levofloxican by MIC only

. Test or do not test? . CLSI recommends to not test as drugs may test susceptible for which the organism is IR . Do not test, how do you report? 1. Do not report 2. Report as “NT” with comment 3. Always report as “R”, no MIC B. cepacia complex AST . CLSI Intrinsic Resistance Table in M100 . Some of these drugs may not be “intrinsically resistant” across all the species . CLSI currently evaluating data to determine if some drugs are no longer considered intrinsically resistant for some species

Modify for certain species?

Remove? Other Gram-negative non-fermenters Considered to be true pathogens: • S. maltophilia • A. xylosoxidans ID and AST

CF Foundation. CF Patient Registry Annual Report. 2016.

CLSI M100S28 Other Gram-negative non-fermenters Considered to be true pathogens: • S. maltophilia • A. xylosoxidans ID and AST • Pandoraea apista • Can grow on BSCA • Elevated MICs to β-lactams, aztreonam and amingoglycosides CF Foundation. CF Patient Registry Annual Report. 2016. AST: • MIC or disk diffusion method • Commercial systems are not recommended

CLSI M100S28 Other Gram-negative non-fermenters Considered to be true pathogens: • S. maltophilia • A. xylosoxidans ID and AST • Pandoraea apista

Questionable pathogens: • Inquilinus limosus • ~5% prevalence

Chiron, R., et al. Inquilinus sp in CF patients. 2005 JCM

Bittar, F., et al. Inquilinus limosus and Cystic Fibrosis. 2008. Emerg Infect Disease Other Gram-negative non-fermenters Considered to be true pathogens: • S. maltophilia • A. xylosoxidans ID and AST • Pandoraea apista

Questionable pathogens: • Inquilinus limosus • ~5% prevalence • Highly resistant • Penicillins, cephalosporins, kanamycin, tobramycin, colistin, doxycycline and SXT • Has been associated with worsening outcomes in few studies • MIC or disk diffusion recommended

Chiron, R., et al. Inquilinus sp in CF patients. 2005 JCM Other Gram-negative non-fermenters Considered to be true pathogens: • S. maltophilia • A. xylosoxidans ID and AST • Pandoraea apista

Questionable pathogens: ID, consider AST if • Inquilinus limosus predominate or pure culture Unlikely to be pathogens: • Ralstonia spp • Mero R and imipenem S (reverse for BCC) ID only. AST by • R to colistin, ceftazidime, request. amikacin, tobramycin and gentamicin • Pantoea spp • Chryseobacterium spp

Hauser, A.R., et al. Microbes and outcomes in cystic fibrosis. 2011. CMR Trichosporon mycotoxinivorans

• T. mycotoxinivorans being recognized as a true CF pathogen • Raised, wrinkled colonies • Appears mold-like (eg fuzzy) • Oval budding yeast • Have arthrocondidia • Urease positive

• Identification should be performed • MALDI-TOF is promising but sometimes gives “No ID” due to crumbly, adherent colonies • ITS or D1/D2 sequencing Nontuberculous Mycobacteria species (NTM) • 6-13% prevalence in sputa • Appears to infect those with milder lung disease • 20% of patients with bronchiectasis and/or pulmonary NTM infection had undiagnosed CF • Recommendation if patient presents with bronchiectasis and positive culture for NTM, screening for CF should occur regardless of age • More than 1 positive culture with NTM is recommended to start treatment • ~70% patients only have 1 culture CF Foundation. CF Patient Registry Annual Report. 2016. positive • Swabs are insufficient, must be from sputum • Recommended frequency • Annually • Testing before and 6 months after starting azithromycin or macrolide therapy • Then annually thereafter Nontuberculous Mycobacteria species (NTM) • M. avium complex and M. abscessus complex most commonly recovered • M. gordonae 3rd most common but is also a common laboratory contaminate • NTM should be identified • MALDI-TOF with in-house validated RUO database • Sequencing of hsp65, rpoB

Hauser, A.R., et al. Microbes and outcomes in cystic fibrosis. 2011. CMR CF Foundation. CF Patient Registry Annual Report. 2016. Culturing for AFB from CF samples The problem: • Normal flora can overgrow and reduce isolation and recovery of NTM • Decontamination helps but isn’t perfect

Options: 1. AFB culture with 7H9 broth (ideally MGIT) and LJ slants 2. BSCA media with extended incubation to 14 days 3. RGM media (not commercially available in US) -based on Middlebrook 7H9 + oleic acid-albumin- dextrose-catalase with Fosfomycin, colistin, amphotericin B and C-390 Culturing for AFB: RGM v BCSA

Plongla.R, et al. Evaluation of RGM media for isolate of nontuberculous mycobacteria from respiratory samples from patients with Cystic Fibrosis in the United States. 2017. JCM Treatments and AST Requests Antibiotics and their uses in CF • IV antibiotics are commonly used for exacerbations • Despite improvement in pulmonary function and nutritional status, there has been minimal reduction in the use of IV antibiotics for exacerbations

CF Foundation. CF Patient Registry Annual Report. 2016. Antibiotics and their uses in CF • Inhaled antibiotics are used throughout • Eradicate organisms prior to becoming chronic • Reduce burden of organism load after becoming chronic

CF Foundation. CF Patient Registry Annual Report. 2016.

New study suggest inhaled levofloxacin showed decreased hospitalizations and reduction of PA density in sputa.

“In 2013, the CF Foundation pulmonary guidelines committee determined that there was insufficient evidence to recommend for or against the chronic use of inhaled beta agonists, inhaled anticholinergics, leukotriene modifiers, inhaled colistin, and ibuprofen for adults to improve lung function, reduce exacerbations, or improve quality of life.” AST profiles and outcomes

Hurley.M.N., et al. Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis. 2012. J Cystic Fibrosis “However on no occasion was the empirical antibiotic changed upon receipt of the antibiotic susceptibility profile.” Inhaled Antibiotics

Who performs AST testing for antibiotics (tobramycin, colistin, etc) that are being used as an inhaled formulation?

CLSI breakpoints are based on serum drug concentration and do not apply for inhaled drugs.

Representative of CHP’s CF physicians regarding colistin MIC: “I admit to not having paid any attention to MIC for colistin because I have no feel for lower airway concentrations with inhaled colistin” Inhaled Tobramycin and MIC Correlation

If airway concentrations of ~200mg/dl can be achieved, then ≤64 µg/ml can be considered “susceptible”. ≥128 µg/ml could be considered “resistant”

Representative CHP CF physician CF: “And maybe if it said higher ”

Morosini, M.I., et al. Breakpoints for P. aeruginosa susceptibility in CF. 2005. JCM Synergistic Testing

Patients undergoing exacerbations often will respond to combination therapy to which the organism is resistant in vitro both individually and in combination.

Limited data available showing correlation or utility of synergy testing and outcomes.

US CF Foundation Recommendation: New Antibiotics Mostly for CF exacerbations. No inhaled formulations to date.

Meropenem/vaborbactam

Ceftolozane/ tazobactam

Ceftazidime/avibactam What and when to test?

Mitchell, S. L. and Humphries, R.H. New and novel agents targeting resistant Gram-negative bacteria: A Review for the Clinical Microbiologist. 2018. CMN

Most requests will be for PA and C/T for CF patients. mCIM (+/- eCIM) or another carbapenemase detection test may be helpful in making testing recommendations as C/T does not have activity against carbapenemase producers. C/T susceptibility against CF isolates

Grohs,P., et al. In vitro activity of ceftolozane-tazobactam against multidrug-resistant non-fermenting Gram-negative bacilli isolated from patients with cystic fibrosis. 2017. AAC. How to test?

CLSI Breakpoints FDA Breakpoints FDA-Approved Testing Methods Isolates Testing Available Drug Automated Method Enterobacteriaceae P. aeruginosa Enterobacteriaceae P. aeruginosa Disk Gradient from CDC BMD US AST Diffusion Diffusion AR Bank (Vendor) Systems (Vendor) (Vendor) S I R S I R S I R S I R (Vendor)

Zone (mm) ≥21 18-20 ≤17 ≥21 17-20 ≤16 ≥21 18-20 ≤17 ≥21 17-20 ≤16 Y Y Ceftolozane- Y (bioMerieux, (Thermo- N Y tazobactam (Hardy) Recognizes CLSI Liofilchem*) Fisher) MIC (µg/ml) ≤2/4 4/4 ≥8/4 ≤4/4 8/4 ≥16/4 ≤4/4 8/4 ≥16/4 breakpoints

Zone (mm) ≥21 ─ ≤20 ≥ 21 ─ ≤20 ≥21 ─ ≤20 ≥ 21 ─ ≤20 Y** Y Ceftazidime- Y (BD, (Thermo- N Y avibactam (bioMerieux) Hardy) Fisher) MIC (µg/ml) ≤8/4 ─ ≥16/4 ≤8/4 ─ ≥16/4 ≤8/4 ─ ≥16/4 ≤8/4 ─ ≥16/4

Zone (mm) ≥17 14-16 ≤13 ND Y Meropenem- Y Y No CLSI breakpoints (Thermo- N N- in progress Vaborbactam (Hardy) (Liofilchem) Fisher) MIC (µg/ml) ≤4/8 8/8 ≥16/8 ND

Mitchell, S. L. and Humphries, R.H. New and novel agents targeting resistant Gram-negative bacteria: A Review for the Clinical Microbiologist. 2018. CMN *MTS strips from Liofilchem consistently read 1-2 dilutions higher than BMD for C/T. **Due to lack of intermediate zone for CZA, disk may overall resistance. If using disk, confirm resistant results by an alternative method. Questions?