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Investigating transmission of Mycobacterium abscessus amongst children in an Australian cystic fibrosis centre

  • Jennifer Yan
    Correspondence
    Corresponding author at: Department of Microbiology, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia
    Affiliations
    Department of Microbiology, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Infection Prevention and Control, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia
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  • Ajay Kevat
    Affiliations
    Department of Respiratory Medicine, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Murdoch Children's Research Institute, 50 Flemington Rd, Parkville, Victoria, Australia
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  • Elena Martinez
    Affiliations
    Institute for Clinical Pathology and Medical Research, Centre for Infectious Diseases and Microbiology, Level 3, ICPMR Building, Westmead Hospital, Westmead, NSW, Australia
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  • Nicky Teese
    Affiliations
    Department of Microbiology, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia
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  • Kareena Johnson
    Affiliations
    Department of Infection Prevention and Control, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia
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  • Sarath Ranganathan
    Affiliations
    Department of Respiratory Medicine, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Murdoch Children's Research Institute, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Paediatrics, The University of Melbourne, Victoria, Australia
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  • Jo Harrison
    Affiliations
    Department of Respiratory Medicine, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Murdoch Children's Research Institute, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Paediatrics, The University of Melbourne, Victoria, Australia
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  • John Massie
    Affiliations
    Department of Respiratory Medicine, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Murdoch Children's Research Institute, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Paediatrics, The University of Melbourne, Victoria, Australia
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  • Andrew Daley
    Affiliations
    Department of Microbiology, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Infection Prevention and Control, Royal Children's Hospital, 50 Flemington Rd, Parkville, Victoria, Australia

    Department of Paediatrics, The University of Melbourne, Victoria, Australia
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Open ArchivePublished:March 07, 2019DOI:https://doi.org/10.1016/j.jcf.2019.02.011

      Highlights

      • Mycobacterial cross-infection between paediatric CF patients occurs.
      • Transmission in the outpatient setting is a significant risk.
      • Aerosol generating activities such as respiratory function testing may be higher risk.
      • CF patient discharge cleaning protocols should include mycobacterial active agents.

      Abstract

      Background

      Mycobacterium abscessus is an emerging pathogen in cystic fibrosis (CF) lung disease. Hospital transmission of M. abscessus has been described. This paper details the investigation into possible cross-transmission of M. abscessus locally at our paediatric hospital CF centre, and the subsequent infection control response.

      Methods

      Whole genome sequencing (WGS) of M. abscessus respiratory isolates with epidemiological linkage analysis using hospital electronic medical records.

      Results

      6.7% (22/328) of CF patients had M. abscessus isolated from respiratory specimens.
      WGS revealed a cluster of three patients with genomically related isolates that differed by <7 single nucleotide polymorphisms (SNPs), suggesting a shared recent ancestor and probable cross-transmission.
      Epidemiological investigation revealed multiple potential crossovers between patients with genomically similar M. abscessus isolates.

      Conclusions

      Cross-infection of NTM occurs in CF hospital patients. Hospital infection control practices should be upgraded to reflect this. Consensus is needed between centres.

      Keywords

      1. Background

      There is increasing interest in the role of non-tuberculous mycobacteria (NTM) in cystic fibrosis (CF) lung disease. Mycobacterium abscessus is a species of rapidly growing NTM that has been associated with accelerated decline in lung function in patients with CF [
      • Esther C.R.
      • Esserman D.A.
      • Gilligan P.
      • Kerr A.
      • Noone P.G.
      • Noone P.G.
      Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis.
      ]. Treatment is challenging; M. abscessus is often multi-drug resistant and difficult to eradicate despite intensive treatment regimens with significant adverse effects [
      • Koh W.-J.
      • Jeon K.
      • Lee N.Y.
      • Kim B.-J.
      • Kook Y.-H.
      • Lee S.-H.
      • et al.
      Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus.
      ,
      • Jeon K.
      • Kwon O.J.
      • Lee N.Y.
      • Kim B.-J.
      • Kook Y.-H.
      • Lee S.-H.
      • et al.
      Antibiotic treatment of Mycobacterium abscessus lung disease.
      ,
      • Ellender C.M.
      • Law D.B.
      • Thomson R.M.
      • Eather G.W.
      Safety of IV amikacin in the treatment of pulmonary non-tuberculous mycobacterial disease.
      ,
      • DaCosta A.
      • Jordan C.L.
      • Giddings O.
      • Lin F.-C.
      • Gilligan P.
      • Esther C.R.
      Outcomes associated with antibiotic regimens for treatment of Mycobacterium abscessus in cystic fibrosis patients.
      ,
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ,
      • Floto R.A.
      • Olivier K.N.
      • Saiman L.
      • Daley C.L.
      • Herrmann J.-L.
      • Nick J.A.
      • et al.
      US Cystic Fibrosis Foundation and European cystic fibrosis society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis.
      ,
      • Jarand J.
      • Levin A.
      • Zhang L.
      • Huitt G.
      • Mitchell J.D.
      • Daley C.L.
      Clinical and microbiologic outcomes in patients receiving treatment for Mycobacterium abscessus pulmonary disease.
      ].
      Acquisition of M. abscessus was thought to occur from independent environmental exposures [
      • Bange F.-C.
      • Brown B.A.
      • Smaczny C.
      • Wallace R.J.
      • Bottger E.C.
      Lack of transmission of Mycobacterium abscessus among patients with cystic fibrosis attending a single clinic.
      ,
      • Jonsson B.E.
      • Gilljam M.
      • Lindblad A.
      • Ridell M.
      • Wold A.E.
      • Welinder-Olsson C.
      Molecular epidemiology of Mycobacterium abscessus, with focus on cystic fibrosis.
      ] due to its ubiquitous presence in soil, water and dust [
      • Falkinham J.O.
      Environmental sources of nontuberculous mycobacteria.
      ]. However, recent reports describe the emergence of dominant worldwide clones and acquisition through patient cross-transmission [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ].
      In this context we sought to describe the burden of NTM at our paediatric CF centre, and investigate our M. abscessus patient isolates for genetic relatedness and evidence of cross-transmission.

      2. Methods

      The Royal Children's Hospital in Melbourne, Australia is a large tertiary hospital that provides care for paediatric patients with CF. We performed a retrospective laboratory database review of all respiratory specimens (sputum and bronchoalveolar lavage) from patients with CF sent for mycobacterial culture over the period January 2013 to March 2017, including phenotypic antibiotic susceptibility data. Since 2013, routine sputum collection for mycobacterial culture has been performed once per year for all patients able to expectorate, and more frequently in the presence of clinical suspicion. Mycobacterial culture of bronchoalveolar lavage samples was performed for patients with high clinical or radiological concern for infection. The CF patient cohort included all patients managed at the Royal Children's Hospital over this time period. Correlation with chest CT findings and investigation of epidemiological linkage was performed by review of the hospital electronic medical record.
      Whole genome sequencing (WGS) was performed on M. abscessus isolates from respiratory specimens collected prospectively and maintained by serial subculture on Brown and Buckle slopes. Initial patient isolates of M. abscessus were collected; three same-patient repeat isolates were also included to evaluate within-patient genetic relatedness. Sequencing was performed using Illumina NextSeq500 to a coverage depth > 65×. Analysis was done with Nullarbor pipeline (https://github.com/tseemann/nullarbor). Trimmed reads were assembled with MEGAHIT v1.1.3 [
      • Li D.
      • Liu C.-M.
      • Luo R.
      • Sadakane K.
      • Lam T.-W.
      MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.
      ]. Core alignment and pairwise core single-nucleotide polymorphism (SNP) distance was calculated with Snippy v3.1 (https://github.com/tseemann/snippy), against M. abscessus ATCC 19977 reference genome (GenBank Accession No NC_010397), with minimum 10× read depth and a > 90% consensus for each isolate [
      • Seemann T.
      • Goncalves da Silva A.
      • Bulach D.
      • Schultz M.
      • Kwong J.
      • Howden B.
      Nullarbor. Github n.d.
      ]. Maximum likelihood phylogenetic trees were estimated with FastTree [
      • Price M.N.
      • Dehal P.S.
      • Arkin A.P.
      FastTree: computing large minimum evolution trees with profiles instead of a distance matrix.
      ] using the general time reversibility model. SNPs from closely related isolates were manually curated. Genomes of 1272 international isolates previously published by Bryant et al. [
      • DaCosta A.
      • Jordan C.L.
      • Giddings O.
      • Lin F.-C.
      • Gilligan P.
      • Esther C.R.
      Outcomes associated with antibiotic regimens for treatment of Mycobacterium abscessus in cystic fibrosis patients.
      ] were downloaded from the European Nucleotide Archive [
      • European Bioinformatics Institute
      European nucleotide archive 2018.
      ]. These isolates were run through the Nullarbor pipeline to reconstruct phylogenetic associations and to test possible relatedness with our cohort of isolates. Isolates were analysed separately by subspecies. Genomic variation of <20 SNPs between isolates was considered suggestive of probable cross-transmission [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ]. Possible mutations associated with drug resistance for clarithromycin, amikacin and ciprofloxacin were evaluated by variant calling with Snippy. Figures were created with FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Raw short read sequence data was submitted to the Sequence Read Archive at the National Centre for Biotechnology Information (NCBI) (BioProjects PRJNA439313).
      Ethical approval was granted by The Royal Children's Hospital Melbourne (study reference number 37247A).

      3. Results

      3.1 Isolates

      30.5% (157/514) of the respiratory specimens sent for mycobacterial culture were positive for NTM. M. abscessus was most frequently isolated, followed by M. avium complex (Table 1).
      Table 1NTM species from respiratory isolates, 2013–2017.
      Species# Patients# Isolates
      M. abscessus22118
      M. avium complex1333
      M. gordonae33
      M. chelonae11
      M. alsiense11
      M. lentiflavus11

      3.2 Patients

      30.2% (99/328) of children had mycobacterial cultures performed. Of those tested, 22.2% (22/99) had M. abscessus isolated, including two patients with M. abscessus subsp. massiliense; 59.0% (13/22) of these individuals had M. abscessus cultured on more than one occasion. The prevalence of any NTM species from the total population was 11.0% (36/328) and the prevalence of M. abscessus was 6.7% (22/328). Six patients had more than one species of NTM isolated at different time points.
      Mean age at first isolate was 13 years (range 6–17), and 41% (n = 9) were female. 95% (21/22) of patients with M. abscessus had bronchiectasis at the time of first isolate and 77% (17/22) had nodular changes evident on chest CT imaging.

      3.3 Genomic analysis

      WGS was performed on 17 M. abscessus isolates obtained from 14 different patients. Multilocus sequence typing (MLST) was used to categorize the isolates into subspecies [
      • Macheras E.
      • Konjek J.
      • Roux A.-L.
      • Thiberge J.-M.
      • Bastian S.
      • Leão S.C.
      • et al.
      Multilocus sequence typing scheme for the Mycobacterium abscessus complex.
      ]. 15 isolates were identified as M. abscessus subsp. abscessus, and two as M. abscessus subsp. massiliense. Genotypic profiling showed resistance to amikacin for three isolates, conferred by mutations in the rrs gene (1375 A > G). This was confirmed by phenotypic antibiotic susceptibility testing.
      In the M. abscessus subsp. abscessus cohort of isolates, there were three clusters of closely related genomes (Fig. 1). Two isolates from the same patient obtained eight months apart differed by two single nucleotide polymorphisms (SNPs) [Cluster 1, isolates Melb-004 and Melb-007]. Two isolates from a second patient obtained three years apart showed same-patient difference of less than seven SNPs [Cluster 2, isolates Melb-005 and Melb-017]. Three isolates collected from different patients between 2014 and 2016 also differed by less than seven SNPs [Cluster 3, isolates Melb-002, Melb-009 and Melb-014]. Two further patients had isolates that differed by 20–35 SNPs to those in Cluster 3 [isolates Melb-008 and Melb-013]. One patient had an initial isolate that formed part of Cluster 3, but a repeat isolate three years later that was not genomically related. The remaining M. abscessus subsp. abscessus isolates were not genomically related.
      Fig. 1
      Fig. 1M. abscessus subsp. abscessus: Maximum likelihood core genome SNP phylogenetic tree of local (Melb-) and international (ERR-) isolates. Local isolates are shown as dots, as they relate to global isolates, enlarged for detail in boxes A, B and C. The three sets of clustered local isolates are identified by the arrows, with cluster 1 (Box A) and cluster 2 (Box B) being pair isolates from the same patient, while cluster 3 (Box C) are from all different patients. 9 of the 15 local isolates were grouped within the two major global clusters.
      When compared to international isolates, isolates from Cluster 1 and Cluster 3 were found to be part of two large international clusters of M. abscessus subsp. abscessus isolates (Fig. 1, Box A and C). Our Cluster 2 patient isolates were closely related to only three international isolates (Fig. 1, Box B). Only one isolate [Melb-001] was not genomically close to any of the local or international isolates included in this study.
      The two M. abscessus subsp. massiliense isolates [Melb-003 and Melb-011] were not closely related. However, they formed part of a cluster with the international isolates (Fig. 2).
      Fig. 2
      Fig. 2M. abscessus subsp. massiliense: Maximum likelihood core genome SNP phylogenetic tree. Local isolates (Melb-) shown as they relate to international isolates.

      3.4 Epidemiological linkage

      On epidemiological investigation, there were multiple potential opportunities for cross-transmission between the three patients with genomically similar M. abscessus isolates [Cluster 3]. These included same day pulmonary function testing and physiotherapy, and multiple episodes of same day outpatient visits with the same clinical team. There were periods of overlapping inpatient stay, although patients were assigned to separate, single rooms and on different wards (Fig. 3). There were two occasions with inpatient stays to the same room (patient 1 followed by patient 2). These admissions were not on consecutive dates, and in each case were separated by a time period of greater than seven months. Patient 1 had first demonstration of acquisition in 2014 followed by patients 2 and 3, each a year later. There were no identified social links between patients with similar isolates; the patients were not siblings and did not undertake social activities together nor attend the same school.
      Fig. 3
      Fig. 3Hospital attendance for the three patients with isolates <7 SNP difference in Cluster 3 (Patient 1 had isolate Melb-002, Patient 2 isolate Melb-009 and Patient 3 isolate Melb-014). Dots represent outpatient visits. Longer horizontal lines represent inpatient stays. Cross-overs in hospital attendance are shown by the grey vertical bars.

      4. Discussion

      In this paper, we demonstrate a NTM prevalence of 11% and M. abscessus prevalence of 6.7% from culture of respiratory specimens in a paediatric CF cohort. Findings from WGS are suggestive of cross-transmission, with closely related M. abscessus isolates obtained from three different patients. Globally predominant clones were represented amongst local isolates. There were multiple crossover periods in hospital attendance lending evidence to the possibility of cross-transmission, despite standard infection control practices. This has prompted a revision of our infection control measures, as described below.
      Our reported prevalence of NTM is similar to that reported from other CF centres [
      • Binder A.M.
      • Adjemian J.
      • Olivier K.N.
      • Rebecca Prevots D.
      Epidemiology of nontuberculous mycobacterial infections and associated chronic macrolide use among persons with cystic fibrosis.
      ,
      • Olivier K.N.
      • Weber D.J.
      • Wallace R.J.
      • Faiz A.R.
      • Lee J.H.
      • Zhang Y.
      • et al.
      Nontuberculous mycobacteria: I: multicenter prevalence study in cystic fibrosis.
      ]. Of note, the frequency of M. abscessus within our patient cohort may be an underestimate; many patients, particularly younger children with CF, were unable to expectorate an adequate sputum sample for mycobacterial culture. Bronchoalveolar lavage specimens were also not routinely sent for surveillance mycobacterial culture.
      M. abscessus has an estimated mean mutation rate of 1.8 SNPs per genome per year; hence, genomic differences of <20 SNPs between isolates are considered to represent close genomic relatedness [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ]. This is consistent with our finding of highly similar genomic sequences in same-patient isolates, differing by <7 SNPs over a maximum of three years. Another patient had two isolates taken three years apart that had significantly different sequences and were not genomically related, suggesting that clearance and reinfection with a different strain or genetic recombination may have occurred. Unfortunately, only 14 of the 22 patients with M. abscessus had initial respiratory isolates available for WGS, as some specimens were not able to be retrieved from storage following initial M. abscessus identification.
      Three of the 14 patients with M. abscessus isolates that underwent WGS had genomically similar isolates. This cluster of isolates with a difference of <7 SNPs (i.e. equivalent to or less than same-patient isolate difference) suggests a shared recent ancestor and high probability of cross-transmission. Antibiotic susceptibility profiles were also similar. Interestingly, these three patients with closely related M. abscessus isolates had nil or scanty acid fast bacilli detected on repeated sputum examination (no AFBs detected on smear in 60% (18/30) of culture positive isolates), implying a lower sputum bacillary load and lower risk of transmission outside of aerosol generating activities.
      Review of hospital attendances found multiple episodes of overlap in outpatient clinic attendance and inpatient stays. Inpatient admissions were to separate single rooms on different wards when patients were hospitalised together, and sequential admissions to the same room were separated by a period of several months. Notably, there were no identified social links between these patients with similar isolates. This implies that transmission is most likely to have occurred within the hospital grounds, with greater concern for transmission in the outpatient setting.
      The only prior paediatric-specific study of M. abscessus transmission published in 2015 concluded that there was no evidence of hospital cross-transmission in a single-centre CF patient cohort. In this study, WGS was performed on M. abscessus isolates from 16 paediatric patients with M. abscessus; two clusters of genomically similar isolates (<25 SNPs) were identified, with one in a sibling pair and another in two patients who had no discernible epidemiological links [
      • Harris K.A.
      • Underwood A.
      • Kenna D.T.D.
      • Brooks A.
      • Kavaliunaite E.
      • Kapatai G.
      • et al.
      Whole-genome sequencing and epidemiological analysis do not provide evidence for cross-transmission of mycobacterium abscessus in a cohort of pediatric cystic fibrosis patients.
      ].
      More recent publications describe the presence of dominant worldwide clones including two major clusters of M. abscessus subsp. abscessus and a major cluster of M. abscessus subsp. massiliense [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ]. These clones demonstrated high genomic similarity across different CF centres and different countries, with multiple isolates showing <20 SNP difference. Our findings confirm the presence of global circulating clones in our local population.
      Though we were unable to prevent cross-transmission, we postulate that the relatively low number of clustered cases at our centre may be due to the pro-active infection prevention and control practices already in place, informed by national and international CF infection control guidelines [
      • Cystic Fibrosis Australia
      Infection control guidelines for cystic fibrosis patients and Carers 2012.
      ,
      • Fitzgerald D.A.
      Cystic fibrosis standards of care, Australia.
      ,
      • Saiman L.
      • Siegel J.D.
      • LiPuma J.J.
      • Brown R.F.
      • Bryson E.A.
      • Chambers M.J.
      • et al.
      Infection prevention and control guideline for cystic fibrosis: 2013 update.
      ]. From here, we detail the possible routes of transmission, infection control practices currently in place at our centre and the key risk areas identified on which to focus further infection prevention and control efforts (Box 1).
      Addressing potential M. abscessus transmission at the Royal Children's Hospital
      • -
        Improve surveillance by requesting mycobacterial culture on routine annual bronchoalveolar lavage and sputum specimens
      • -
        Improve staff adherence to cleaning and hand hygiene:
        • Clinical staff perform surface and equipment cleaning between patients in outpatient clinic
        • Clarify responsibilities of cleaning staff versus clinical staff for cleaning equipment and room fixtures and fittings
        • Ongoing education, auditing and feedback of compliance
      • -
        Inpatient discharge cleaning for all CF patients to include agents with activity against mycobacteria
      • -
        Optimise infection control practices and processes in Physiotherapy and Pulmonary Function Laboratory
      • -
        Change to new clinic rooms midway through the clinic session after initial patients leave and before others arrive, to minimise exposure to shared spaces
      • -
        Patients wear a surgical mask when in the outpatient room to reduce droplet nuclei generation and environmental contamination
      • -
        Minimise cough-inducing and aerosol-generating procedures in outpatient rooms by collecting sputum and cough swab specimens at home prior to appointment
      • -
        Consider infrastructure modifications to improve ventilation and air exchange
      • -
        All patients with M. abscessus (any subspecies) have inpatient admission to negative pressure rooms, if available
      • -
        Cohort separation or similar ‘single outpatient’ process to minimise outpatient clinic exposure and waiting room time for other specialty appointments e.g. endocrinology, gastroenterology clinics
      • -
        Increase usage of telehealth consultations where possible

      4.1 Infection prevention and control

      Possible routes of transmission include fomite and aerosol spread, particularly from aerosol generating activities such as coughing during physiotherapy and forced expiration testing during visits to the pulmonary function laboratory. Airborne transmission of Mycobacterium tuberculosis through aerosolised droplets is well recognised [
      • Gerberding J.L.
      • Snider D.E.
      • Popovic T.
      • Solomon S.L.
      • Bernhardt J.M.
      • Parker M.S.
      • et al.
      Centers for Disease Control and Prevention coordinating center for Health Information and Service Centers for disease control and prevention. Guidelines for preventing the transmission of Mycobacterium tuberculosis in. Heal settings.
      ], and cough aerosolisation of M. abscessus has also been described [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ,
      • Wainwright C.E.
      • France M.W.
      • O'Rourke P.
      • Anuj S.
      • Kidd T.J.
      • Nissen M.D.
      • et al.
      Cough-generated aerosols of Pseudomonas aeruginosa and other gram-negative bacteria from patients with cystic fibrosis.
      ,
      • Jones A.M.
      • Govan J.R.W.
      • Doherty C.J.
      • Dodd M.E.
      • Isalska B.J.
      • Stanbridge T.N.
      • et al.
      Identification of airborne dissemination of epidemic multiresistant strains of Pseudomonas aeruginosa at a CF centre during a cross infection outbreak.
      ,
      • Knibbs L.D.
      • Johnson G.R.
      • Kidd T.J.
      • Cheney J.
      • Grimwood K.
      • Kattenbelt J.A.
      • et al.
      Viability of Pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis.
      ]. Indirect contact transmission may have occurred via environmental contamination and fomite spread on contaminated equipment transferred between patients or via the hands of patients or health care workers. Direct person-to-person droplet (<1 m) transmission is less likely given the physical separation between CF patients described below, minimising contemporaneous contact.
      Infection prevention and control practices for patients with CF at our hospital have evolved over time. Cohorting of outpatient clinic bookings according to a patient's colonising organisms commenced in 2000 following an outbreak of epidemic Pseudomonas aeruginosa [
      • Griffiths A.L.
      • Jamsen K.
      • Carlin J.B.
      • Grimwood K.
      • Carzino R.
      • Robinson P.J.
      • et al.
      Effects of segregation on an epidemic Pseudomonas aeruginosa strain in a cystic fibrosis clinic.
      ,
      • Griffiths A.L.
      • Wurzel D.F.
      • Robinson P.J.
      • Carzino R.
      • Massie J.
      Australian epidemic strain pseudomonas (AES-1) declines further in a cohort segregated cystic fibrosis clinic.
      ] and now includes cohorting for MRSA, P. aeruginosa, P. aeruginosa epidemic strain AES-1, Burkholderia cepacia and non-tuberculous mycobacteria. Moving to a newly constructed hospital facility in 2011 provided single rooms for inpatient and respiratory outpatient care. Single room CF outpatient care at our centre involves allocation of patients to a room which they enter on arrival, minimising shared waiting room time, with the multidisciplinary care team rotating between patients in their individual rooms. Hand hygiene and cough etiquette are regularly reinforced.
      Following reports in 2012–2013 of M. abscessus subsp. massiliense cross-transmission in CF patients and poor patient outcomes [
      • Aitken M.L.
      • Limaye A.
      • Pottinger P.
      • Whimbey E.
      • Goss C.H.
      • Tonelli M.R.
      • et al.
      Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center.
      ,
      • Bryant J.M.
      • Grogono D.M.
      • Greaves D.
      • Foweraker J.
      • Roddick I.
      • Inns T.
      • et al.
      Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.
      ], we recommended admission to negative pressure rooms for inpatients with M. abscessus subsp. massiliense at our hospital. This recommendation did not extend at the time to include all patients with M. abscessus complex. In 2016, hospital CF infection control guidelines were updated, stipulating avoidance of contact between all patients with CF (2 m rule), contact precautions for staff entering the room of a patient with CF, and wearing of surgical masks by patients with CF on hospital grounds when outside their room. Wearing of a surgical mask may reduce the amount of droplet deposition and environmental contamination by patients with respiratory pathogens but is unlikely to eliminate the risk of airborne transmission [
      • Vanden Driessche K.
      • Hens N.
      • Tilley P.
      • Quon B.S.
      • Chilvers M.A.
      • de Groot R.
      • et al.
      Surgical masks reduce airborne spread of Pseudomonas aeruginosa in colonized patients with cystic fibrosis.
      ]. These recommendations were applied to all patients with CF regardless of their organism colonisation status. An ongoing effort has been made to engage patients and their families with infection control practices [
      • Moran F.
      • Reardon N.
      • Glazner J.
      • Morgan A.
      • Daley A.
      • Massie J.
      Responses of parents and children with cystic fibrosis to new infection control practice.
      ].
      Now in the outpatient clinic, physiotherapy setting and pulmonary function laboratory, rooms are left vacant for 30 min between patients, to allow enough time for removal of around 90% of airborne contaminants (outpatient areas at our hospital have 4.8 air changes per hour, compared with 12 air changes per hour in isolation rooms) [
      • Gerberding J.L.
      • Snider D.E.
      • Popovic T.
      • Solomon S.L.
      • Bernhardt J.M.
      • Parker M.S.
      • et al.
      Centers for Disease Control and Prevention coordinating center for Health Information and Service Centers for disease control and prevention. Guidelines for preventing the transmission of Mycobacterium tuberculosis in. Heal settings.
      ,
      • Curry International Tuberculosis Centre
      How long does it take to clear the air in an isolation or high-risk procedure room?.
      ,
      • The Department of Human Services Victoria
      Design guidelines for hospitals and day procedure centres.
      ]. Equipment and surface cleaning between patients is performed using disinfectant wipes (e.g. Tuffie-5™). Inpatient rooms of patients with known M. abscessus receive a routine detergent clean followed by a chlorine-based clean (e.g. Chlora-det™ or Detsol 500™) and treatment with vaporised hydrogen peroxide (Noco-spray/Nocolyse™) [
      • Fonderephar, Oxy'pharm
      Determination of bactericidal, fungicidal, yeasticidal, sporicidal, mycobactericidal and virucidal activity for aerial surface disinfection processes according to the method described in the standard NF T 72-281.
      ] on patient discharge. Inpatient rooms of CF patients without known multi-resistant organisms receive detergent based discharge cleaning only. This was identified as a risk for potential transmission from patients with undiagnosed M. abscessus (e.g. patients who become colonised during the period between testing). Although we did not undertake environmental sampling, contamination of the hospital environment with patient-identical M. abscessus isolates has been shown to occur; a combination of disinfectant wipes, perchlorate solution and vaporised hydrogen peroxide were effective at environmental cleaning [
      • Bryant J.M.
      • Grogono D.M.
      • Rodriguez-Rincon D.
      • Everall I.
      • Brown K.P.
      • Moreno P.
      • et al.
      Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.
      ].

      5. Conclusion

      To our knowledge, this is the first report of clustered cases of M. abscessus in paediatric patients with CF with demonstrated opportunities for cross-transmission within the hospital setting. Having recognised the potential for cross-transmission, preventing acquisition of M. abscessus in children with CF is an important goal. Although each hospital has different challenges and physical resource limitations, a unified approach to infection control practices is needed across hospitals managing patients with CF.

      Declarations of interest

      None.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

      Acknowledgements

      We would like to thank Andrea Bustamante and A. Prof Vitali Sintchenko from The Institute for Clinical Pathology and Medical Research (ICPMR) Westmead for assistance with whole genome sequencing; Maria Globan from the Mycobacterial Reference Laboratory, Melbourne for co-operation in obtaining the isolates, the infection prevention and control team at the Royal Children's Hospital and the patients and families living with cystic fibrosis.

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