Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection in children with cystic fibrosis (SCVSA): a multicentre, observational study

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Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection in children with cystic fibrosis (SCVSA): a multicentre, observational study Daniel J Wolter, Frankline M Onchiri, Julia Emerson, Mimi R Precit, Michael Lee, Sharon McNamara, Laura Nay, Marcella Blackledge, Ahmet Uluer, David M Orenstein, Michelle Mann, Wynton Hoover, Ronald L Gibson, Jane L Burns, Lucas R Hoffman, on behalf of the SCVSA study group*

Summary

Background Staphylococcus aureus is the bacterium cultured most often from respiratory secretions of people with cystic fibrosis. Both meticillin-susceptible S aureus and meticillin-resistant S aureus (MRSA) can adapt to form slowgrowing, antibiotic-resistant isolates known as small-colony variants that are not routinely identified by clinical laboratories. We aimed to determine the prevalence and clinical significance of S aureus small-colony variants and their subtypes among children with cystic fibrosis. Methods The Small Colony Variant Staphylococcus aureus (SCVSA) study was a 2-year longitudinal study of children aged 6–16 years at five US cystic fibrosis centres, using culture methods sensitive for small-colony variants. Children were eligible if they had a documented diagnosis of cystic fibrosis and a minimum of two cystic fibrosis clinic visits and two respiratory cultures in the previous 12 months at enrolment. Participants attended clinic visits quarterly, at which respiratory tract samples were taken and measures of lung function (percentage of predicted forced expiratory volume in 1 s [FEV1] and frequency of respiratory exacerbations) were recorded. We determined the prevalence of small-colony variants and their subtypes, and assessed their independent associations with lung function and respiratory exacerbations using linear mixed-effects and generalised estimating equation logistic regression models. Analyses included both univariate models (unadjusted) and multivariate models that adjusted for potential confounders, including age, sex, race, baseline microbiology, treatment with CFTR modulator, and CTFR genotype. Findings Between July 1, 2014, and May 26, 2015, we enrolled 230 children. Participants were followed-up for 2 years, with a mean of 6·4 visits (SD 1·14) per participant (range 2–9 visits) and a mean interval between visits of 3·94 months (SD 1·77). Across the 2-year period, S aureus small-colony variants were detected in 64 (28%) participants. Most (103 [56%] of 185) of the small-colony variants detected in these participants were thymidine dependent. Children with small-colony variants had significantly lower mean percentage of predicted FEV1 at baseline than did children without small-colony variants (85·5 [SD 19] vs 92·4 [SD 18·6]; p=0·0145). Small-colony variants were associated with significantly lower percentage of predicted FEV1 throughout the study in regression models, both in univariate analyses (regression coefficient –7·07, 95% CI –12·20 to –1·95; p=0·0068) and in multivariate analyses adjusting for potential confounders (–5·50, –10·51 to –0·48; p=0·0316). Small colony variants of the thymidine-dependent subtype had the strongest association with lung function in multivariate regression models (regression coefficient –10·49, –17·25 to –3·73; p=0·0024). Compared with children without small-colony variants, those with small-colony variants had significantly increased odds of respiratory exacerbations in univariate analyses (odds ratio 1·73, 95% CI 1·19 to 2·52; p=0·0045). Children with thymidine-dependent small-colony variants had significantly increased odds of respiratory exacerbations (2·81, 1·69–4·67; p=0·0001), even after adjusting for age, sex, race, genotype, CFTR modulator, P aeruginosa culture status, and baseline percentage of predicted FEV1 (2·17, 1·33–3·57; p=0·0021), whereas those with non-thymidine-dependent small-colony variants did not. In multivariate models including smallcolony variants and MRSA status, P aeruginosa was not independently associated with lung function (regression coefficient –4·77, 95% CI –10·36 to 0·83; p=0·10) and was associated with reduced odds of exacerbations (0·54, 0·36 to 0·81; p=0·0028). Only the small-colony variant form of MRSA was associated with reduced lung function (–8·44, –16·15 to –0·72; p=0·0318) and increased odds of exacerbations (2·15, 1·24 to 3·71; p=0·0061). Interpretation Infection with small-colony variants, and particularly thymidine-dependent small-colony variants, was common in a multicentre paediatric population with cystic fibrosis and associated with reduced lung function and increased risk of respiratory exacerbations. The adoption of small-colony variant identification and subtyping methods by clinical laboratories, and the inclusion of small-colony variant prevalence data in cystic fibrosis registries, should be considered for ongoing surveillance and study.

Lancet Respir Med 2019 Published Online November 11, 2019 https://doi.org/10.1016/ S2213-2600(19)30365-0 See Online/Comment https://doi.org/10.1016/ S2213-2600(19)30405-9 *Listed in the appendix Department of Pediatrics and Department of Microbiology, University of Washington, Seattle, WA, USA (D J Wolter PhD, J Emerson MD, M R Precit PhD, M Lee BS, Prof J L Burns MD, Prof L R Hoffman MD); Department of Pediatrics, Seattle Children’s Hospital, Seattle, WA, USA (D J Wolter, F M Onchiri PhD, J Emerson, S McNamara RN, L Nay BS, M Blackledge BS, Prof R L Gibson MD, Prof J L Burns, Prof L R Hoffman); Department of Pediatrics, Boston Children’s Hospital, Boston, MA, USA (A Uluer MD); Department of Pediatrics, Brigham and Women’s Hospital, Boston, MA, USA (A Uluer); Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA (Prof D M Orenstein MD); Department of Pediatrics, Texas Children’s Hospital, Houston, TX, USA (M Mann MD); and Department of Pediatrics, University of Alabama, Tuscaloosa, AL, USA (Prof W Hoover MD) Correspondence to: Prof Lucas R Hoffman, Pediatrics, University of Washington, Seattle Children’s Hospital, Seattle, WA 98105, USA [email protected] See Online for appendix

Funding The Cystic Fibrosis Foundation and the National Institutes of Health. Copyright © 2019 Elsevier Ltd. All rights reserved. www.thelancet.com/respiratory Published online November 11, 2019 https://doi.org/10.1016/S2213-2600(19)30365-0

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Research in context Evidence before this study Staphylococcus aureus small-colony variant infections have been hypothesised to result in worse clinical outcomes than do normal-colony S aureus infections in people with cystic fibrosis. Identification of S aureus small-colony variants requires special culturing techniques that are not used by most clinical laboratories, and small-colony variants are therefore not included in registry data, restricting any research on the prevalence of small-colony variants or their clinical associations. To clarify what was known about the prevalence and clinical associations of S aureus small-colony variants in cystic fibrosis, we searched PubMed on Feb 12, 2019, for all studies, with no date or language restrictions, using the keywords “aureus”, “cystic fibrosis”, and “small colony”. Identified studies indicated that S aureus small-colony variants commonly infect the airways of patients with cystic fibrosis in Europe and the USA, with reported prevalences that vary substantially, whereas no small-colony variants were detected in an Australian study. In some studies, small-colony variants were associated with lower lung function. However, these studies were generally short in duration, were single-centre studies, did not focus on small-colony variants or investigate their independent associations with outcomes, and used different culture techniques, restricting their generalisability. We sought to determine the prevalence, risk factors, and independent clinical associations of small-colony variants in a large, multicentre population of children with cystic fibrosis in the USA using a standardised laboratory protocol.

Introduction Cystic fibrosis is a monogenic disease that causes chronic, polymicrobial airway infections that result in obstructive lung disease beginning in early life, and often in infancy.1 Cystic fibrosis lung disease is the most important determinant of morbidity and mortality in cystic fibrosis. Staphylococcus aureus is the bacterium most commonly cultured from cystic fibrosis respiratory tract samples. In 2016, S aureus was detected in over 70% of patients with cystic fibrosis in the USA, with a prevalence of over 80% in children aged 11–17 years, coinciding with a period of decreasing average lung function.2 However, compared with other common cystic fibrosis pathogens, including Pseudomonas aeruginosa and Burkholderia sp, relatively little is known about S aureus with respect to cystic fibrosis pathogenesis. S aureus isolates are often categorised by their susceptibility to the antibiotic meticillin. Meticillinresistant S aureus (MRSA) has been associated with worse outcomes in diverse infectious diseases compared with meticillin-susceptible S aureus (MSSA). In cystic fibrosis, MRSA infection has been associated with increased mortality and worse lung function.3,4 However, whether this association is attributable to the organism

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Added value of this study This study found that 28% of children with cystic fibrosis were infected with S aureus small-colony variants during a 2-year period. A strong correlation was found between the detection of small-colony variants and worse clinical outcomes—ie, reduced lung function and increased risk of respiratory exacerbations. In multivariate analyses that included small-colony variants, neither of the common cystic fibrosis pathogens Pseudomonas aeruginosa or MRSA had similar associations. The prevalence of S aureus small-colony variants in this population was similar to that identified in previous single-centre studies in Europe and the USA. This observation, combined with the larger sample size and the multicentre design of this study, indicate that the current findings are generalisable to the cystic fibrosis population. Implications of all the available evidence The available evidence indicates that S aureus small-colony variants commonly infect the respiratory tracts of children with cystic fibrosis and are associated with significantly worse respiratory outcomes. Such variants are not routinely identified by most clinical laboratories. The direction of causality—do small-colony variants lead to worse disease or are they simply a marker of worse pre-existing disease (eg, a response to antibiotic use)—is not yet known. In view of our findings, the general adoption of clinical laboratory methods to detect small-colony variants and inclusion of these variants in registry data should be considered to support future research, surveillance, and cystic fibrosis clinical care.

itself or to its association with the higher antibiotic burden that usually accompanies worse underlying disease is uncertain.5–7 Many bacteria undergo genetic changes during chronic infections in patients with cystic fibrosis, as they adapt to the airway environment and to treatments such as antibiotics. Although S aureus adaptations have been less well studied than those of other cystic fibrosis pathogens, slow-growing, antibiotic-resistant S aureus mutants known as small-colony variants have been identified from patients with cystic fibrosis who are chronically infected, and from patients with other chronic infections, including those of bone and infections associated with devices and wounds.8 Small-colony variants can be further categorised accord­ing to the type of metabolic defect they carry.9,10 For example, thymidine-dependent small-colony variants have inactivating mutations in the folate biosynthetic pathway. These mutations are generally thought to be selected by exposure to antifolate antibiotics, especially trimethoprim– sulfamethoxazole, because thymidine monophosphate biosynthesis requires folate, and these metabolic mutations lead to sulfonamide resistance.11,12 Haemindependent and menadione-dependent small-colony variants exhibit defect­­ ive membrane electron transport

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and are known to be selected by both aminoglycosides and growth with P aeruginosa.13 Fatty-acid-dependent smallcolony variants have been selected in vitro with triclosan and other compounds,14 whereas the mechanisms by which small-colony variants depend on carbon dioxide for optimal growth are yet unknown. Small-colony variants with no identified metabolic defects and auxotrophies have also been described.8 Small-colony variants can be formed at the same frequency by MRSA and MSSA.15 Small-colony variants grow poorly on standard laboratory media; as a result, such variants are not routinely identified by most clinical laboratories, which precludes study of their prevalence and clinical associ­ ations from registry data or clinical records. Case reports and prevalence studies have relied on sensitive culture techniques and experience with small-colony variant detection. Thymidine-dependent small-colony variants were described in case studies of patients with cystic fibrosis in the late 1970s,16,17 and a subsequent study identified such variants in 20 (10%) of 200 patients with cystic fibrosis.18 Subsequent single-centre European studies included all subtypes of small-colony variants known at the time, reporting prevalences of 8–33% in patients with cystic fibrosis.19–25 However, relatively few studies have examined the association between detection of small-colony variants and clinical characteristics. In at least four studies, small-colony variants were found to be associated with reduced lung function.15,20,21,23 Patients who were positive for small-colony variants had often been given trimethoprim–sulfamethoxazole;19–21,26 however, associations with respiratory exacerbations were not assessed in these studies. In a prospective, single-centre, US study using culturing methods sensitive for small-colony variants, we identified these variants in 24 (24%) of 100 children with cystic fibrosis over a 2-year period.15 Detection of small-colony variants was associated with reduced lung function and recent treatment with sulfonamide antibiotics, suggesting that antibiotics select for these variants, and that small-colony variants might either lead to worse lung disease outcomes or indicate higher antibiotic exposure due to worse pre-existing disease. However, whether small-colony variants commonly infect children with cystic fibrosis throughout the USA, or whether small-colony variant infection is associated with worse lung function and increased risk of respiratory exacerbations in the broader paediatric cystic fibrosis population, is unknown, given that treatment strategies such as antibiotic regimens differ among providers, centres, and countries.27 We aimed to determine the prevalence and clinical associations of S aureus small-colony variants and their subtypes, and to examine potential risk factors for smallcolony variants, in a large, multicentre, US paediatric cystic fibrosis population. The size and multicentre design of this study, and new information about emerging small-colony variant subtypes,10,14 enabled a

more thorough analysis of the risk factors and clinical associations of small-colony variants than was possible in previous studies. We hypothesised that S aureus smallcolony variants commonly infect children with cystic fibrosis in the USA and are independently associated with worse lung disease indicators, including lung function and exacerbation frequency. The goal of this study was to define the burden and consequences of small-colony variant infection in the US paediatric population of patients with cystic fibrosis to better inform ongoing laboratory and therapeutic strategies for the management of cystic fibrosis lung disease.

Methods

Study design and participants In this observational study, children with a documented cystic fibrosis diagnosis were recruited from five cystic fibrosis centres in the USA: Seattle Children’s Hospital, Seattle, WA; Boston Children’s Hospital, Boston, MA; Texas Children’s Hospital, Houston, TX; University of Pittsburgh Children’s Hospital, Pittsburgh, PA; and the University of Alabama Children’s Hospital, Birmingham, AL. Patients were eligible if they were aged 6–16 years and had a minimum of two cystic fibrosis clinic visits and two respiratory cultures in the previous 12 months at enrolment. The minimum age for inclusion was 6 years to increase the likelihood of obtaining reliable spirometric data. Patients were excluded if they had a previous lung transplant or comorbidities that would interfere with data interpretation (eg, premature birth, non-cystic fibrosis immuno­ deficiency, or congenital or structural lung disease). Patients who had participated in our previous single-centre study of small-colony variants15 were excluded from this study. Approval was obtained from each respective site’s institutional review board and patients gave written informed consent or assent before undergoing any study activities. This report conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guide­lines.28

Procedures Participants were observed prospectively for up to 2 years during regularly scheduled, quarterly cystic fibrosis clinic visits (a timeline of visits is provided in the appendix [p 5]). Information was collected on the date of actual visits, but not on the clinic schedule, precluding a comparison between scheduled and actual visit dates. At enrolment, we collected baseline data including height, weight, body-mass index (calculated using the 2006 WHO reference populations for children aged 6 months to <5 years, and 2007 WHO reference population for children older than 5 years),29 race and ethnicity, pancreatic status (determined by faecal elastase or prescription of pancrease supplements), genotype (cystic fibrosis transmembrane conductance regulator [CFTR] variants), lung function (percentage of predicted

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forced expiratory volume at 1 s [FEV1]), and existing medications and treat­ment. Participants recorded their use of antibiotics in either written or online datalogs, from which all antibiotic use for the 3 months preceding each study visit were recorded by research staff for analyses. Lung function measure­ ments and culture results, including detection of S aureus, P aeruginosa, Burkholderia sp, Achromobacter sp, Haemophilus sp, and Stenotrophomonas maltophila, were recorded at each site visit by site research coordinators. Percentage of predicted FEV1 was calculated using the reference equations for spirometry from the Global Lung Function Initiative.30 Respiratory exacerbations were defined as in our previous study,15 as the administration of new inpatient or outpatient antibiotics for respir­atory symptoms while on study. Because this definition required accurate antibiotic treatment data not consist­ ently available for the US cystic fibrosis population, we did not define exacerbation frequency before study commencement. Maintenance antibiotics were permitted during the study. Respiratory tract samples—sputum or oropharyngeal swabs—were taken at baseline and at each visit, stored at 4°C after collection, and shipped overnight on ice to the central laboratory of the Cystic Fibrosis Foundationfunded Therapeutics Development Network Center for Cystic Fibrosis Microbiology at Seattle Children’s Hospital. All respiratory specimens were cultured at this central laboratory within 48 h after collection, as previously described.15 Culture results from the central study laboratory were not provided to treating physicians. Small-colony variants were identified using a specialised method that has been previously described,15 but with the additional analysis of growth on a laboratory medium (ie, Mueller-Hinton agar) on which small-colony variants grow very poorly. Smallcolony variant auxotrophic testing was done on LuriaBertani agar plates with disks impregnated with thymidine (5 μg; Sigma Aldrich, St Louis, MO, USA), haemin (Oxoid), menadione (1·5 μg), or polysorbate 80 (10% solution; Sigma Aldrich). Auxotrophy for carbon dioxide was assessed after growth of small-colony variants on blood agar plates and Luria-Bertani agar in air compared with 5% carbon dioxide after 24 h incubation at 35°C.

Outcomes The main study outcomes were the prevalence of S aureus small-colony variants, including thymidine-dependent, haemin-dependent, menadione-dependent, carbon dioxide-dependent, fatty-acid-dependent, and unknown subtypes, among participants (ie, those who were ever positive for small-colony variants over the 2-year study period). Clinical outcomes were percentage of predicted FEV1, both on average during the 2-year study period and change during the study, and frequency of respiratory exacerbations during the study. 4

Statistical analysis We recorded baseline demographic, clinical, and laboratory characteristics for all participants; we compared the characteristics of participants who ever had small-colony variant S aureus with those of patients who were never positive for small-colony variant S aureus during the study period. We summarised continuous variables using means and SDs and made comparisons using the two-sample t test if normally distributed; otherwise, we summarised variables using medians and IQRs and made comparisons using the Wilcoxon–Mann–Whitney test. Categorical variables were summ­arised using counts and proportions, and compared between study groups using Pearson’s χ² test or Fisher’s exact tests as appropriate. We considered the timing of visits as a continuous variable (follow-up time in months since baseline) in our initial analyses, and found time to have no effect on the results; therefore, time was treated as a discrete variable in all analyses thereafter. Potential sources of bias in this study included selection (eg, centre and participant), informational (eg, recall), and confounding biases that can lead to misinterpretation of data. We adjusted for confounding bias using multivariate analyses. We chose a sample size of 250 children with cystic fibrosis a priori using data from previous studies for estimates; the sample size was calculated, assuming a cumulative prevalence of small-colony variants of 25%, to provide sufficient power to explore clinical associations with disease severity, including 80% power to detect significant differences in lung function. We determined the prevalence of S aureus small-colony variants and used univariate and multivariate logistic regression models to assess the correlates of smallcolony variant detection among participants with cystic fibrosis. In univariate and multivariate analyses, we assessed unadjusted associations between being ever infected with small-colony variant S aureus and several predefined predictors—ie, culture results at baseline, patient demographics (age, sex, race), CFTR genotype, pancreatic status, sweat chloride values, medications, and antibiotics. To identify independent predictors of small-colony variant detection, we adjusted for the following a priori selected confounders in the multivariate logistic regression: age, sex, race, CFTR genotype, and treatment with a CFTR modulator. Our multivariate analyses included correlates that had strong, plausible theoretical and clinical links with small-colony variant S aureus, even if they were not significant in univariate analysis. We did our multivariate analyses using a graded approach, first adjusting for visit number, demographics, genotype, and treatment with CFTR modulators; then adding P aeruginosa culture status; and finally baseline lung function measures, to provide estimates of the individual contributions of P aeruginosa status and baseline lung function separately. We calculated means (SD) of the percentage of predicted FEV1 at each visit on the basis of the available data, and tabulated the data for participants for whom

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small-colony variants were never detected and for whom small-colony variants were ever detected during the study. Visual inspection of longitudinal percentage of predicted FEV1 data using spaghetti plots suggested that, for this dataset, a random intercept-only linear mixedeffects regression model was more plausible than a model with both random intercept and slope. Therefore, we assessed associations between small-colony variants with differences and longitudinal changes in lung function, as measured by percentage of predicted FEV1, using linear mixed-effects regression models with random intercept, with an exchangeable correlation matrix and a robust variance estimator. We assessed the association between infection with small-colony variants and occurrence of respiratory exacerbations using generalised estimating equation logistic regression models, with an exchangeable correlation matrix and a robust variance estimator. We constructed multivariate regres­ sion models on the basis of carefully selected potential cofactors and confounders, including age, CFTR genotype, sweat chloride values, medications, other pathogens, sex, race, and baseline lung function, on the basis of the known and conceivable associations between these characteristics, study outcomes, and cystic fibrosis microbiology. We analysed the association between small-colony variants and recorded exposure to antibiotics in the quarter before each culture under consideration. We did all analyses both comparing patients with no small-colony variants with those with small-colony variants, and separately, those with thymidine-dependent or nonthymidine-dependent subtypes of small-colony variants, to identify associations both with subsequent clinical outcomes and also pre-existing clinical risk factors (eg, antibiotics) which are known to select for small-colony variants.8,9 Because initial analyses indicated associations between small-colony variants selected by sulfonamides and poor outcomes, we individually investigated the association between sulfonamides and those outcomes to distinguish the clinical effects of this antibiotic from the small-colony variant it selects. In this analysis, we also quantified the use of this and other antibiotics and we quantified the prevalence of small-colony variants at each study site to determine the generalisability of our findings. For all analyses, we used two-sided tests and a 5% level of significance. Because participants had different numbers of study visits during their 2-year follow-up period, resulting in fewer having data for visit 8, all analyses were restricted to data from visits 1–7. We did analyses using Stata 15.1.

Role of the funding source The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

Results Between July 1, 2014, and May 26, 2015, 230 from the five centres were enrolled (number of participants by site is provided in the appendix [p 6]). One centre (Seattle Children’s Hospital) had fewer participants than the other sites because children who participated in a previous single-centre study15 of small-colony variants were excluded from the current study. Participants were followed-up for 2 years, with a mean of 6·4 visits (SD 1·14) per participant (range 2–9 visits; figure 1). The mean interval between visits was 3·94 months (SD 1·77) and the median interval was 3·45 months (IQR 2·99–4·44). Demographic and clinical character­ istics of participants at baseline and a comparison of participants who were never and ever positive for S aureus small-colony variants are provided in table 1. The mean age of participants was 11·7 years (SD 3·1), and 125 (54%) participants were homozygous for the Phe508del mutation in the CFTR protein (CFTR variants in the study population are listed in the appendix [p 4]). 126 (55%) of 230 patients received maintenance anti­ biotics during the study: inhaled tobramycin, aztreonam, or azithromycin. Of 1444 analysable respiratory samples from study participants, 237 (16%) were sputum and 1207 (84%) were oropharyngeal swabs. This distribution was similar among study sites. During the study and after enrolment, 64 (28%) of 230 participants were positive for small-colony variants (appendix pp 6–7, 9), as defined by poor growth of isolated S aureus on indicator media. 55 (86%) of these participants were negative for small-colony variants at enrolment (based on baseline cultures) but became culture positive at least once during the study, and nine (14%) were positive for small-colony variants at enrolment and positive at least once during the study (appendix p 9). The overall prevalence of small-colony variants during the study (28%) was similar to the prevalence at baseline of canonical cystic fibrosis pathogens P aeruginosa (66 [29%] of 230) and MRSA (57 [25%] of 230) in the study population (appendix p 8). The overall prevalence of MSSA, MRSA, and small-colony variants of each S aureus category (MSSA small-colony variant and MRSA small-colony variant) during the study is shown in the appendix (p 7). Prevalence of small-colony variants varied among the five study sites between 14% (seven of 50, Texas Children’s Hospital) and 35% (18 of 51, Boston Children’s Hospital; appendix p 6). Participants with S aureus small-colony variants ever detected during the study were older, more likely to be using dornase alfa at baseline, and less likely to be using hypertonic saline at baseline. Use of the CFTR modulator ivacaftor was uncommon in this paediatric population, being used exclusively among participants who were never positive for small-colony variants (table 1). Baseline lung function, measured as mean percentage of predicted FEV1, was significantly lower among those who were positive for small-colony variants during the study (p=0·0145; table 1). Participants who were positive for small-colony variants

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230 enrolled in study and completed baseline assessment 51 Boston Children’s Hospital 50 Texas Children’s Hospital 50 University of Pittsburgh Children’s Hospital 29 Seattle Children’s Hospital 50 University of Alabama Children’s Hospital 0 did not attend quarterly visit 230 attended first quarterly visit 226 provided respiratory tract sample 221 completed spirometry 230 provided treatment history for assessment of respiratory exacerbations 4 did not attend quarterly visit 226 attended second quarterly visit 225 provided respiratory tract sample 222 completed spirometry 230 provided treatment history for assessment of respiratory exacerbations 9 did not attend quarterly visit 221 attended third quarterly visit 221 provided respiratory tract sample 217 completed spirometry 226 provided treatment history for assessment of respiratory exacerbations 20 did not attend quarterly visit 210 attended fourth quarterly visit 205 provided respiratory tract sample 208 completed spirometry 221 provided treatment history for assessment of respiratory exacerbations 47 did not attend quarterly visit 183 attended fifth quarterly visit 176 provided respiratory tract sample 181 completed spirometry 210 provided treatment history for assessment of respiratory exacerbations 104 did not attend quarterly visit 126 attended sixth quarterly visit 115 provided respiratory tract sample 121 completed spirometry 183 provided treatment history for assessment of respiratory exacerbations 185 did not attend quarterly visit 45 attended seventh quarterly visit 41 provided respiratory tract sample 45 completed spirometry 126 provided treatment history for assessment of respiratory exacerbations 224 did not attend quarterly visit 6 attended eighth quarterly visit 5 provided respiratory tract sample 6 completed spirometry 45 provided treatment history for assessment of respiratory exacerbations

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during the study were also more likely to have baseline respiratory cultures that were positive for MSSA, MRSA, or MSSA small-colony variants, or a combination of these (appendix p 8). A similar number of participants had repeated detection of small-colony variants (more than one positive culture during study—ie, persistent infection; n=30) and only one detection (ie, intermittent infection; n=34) during the study (appendix pp 9–10), with no difference in age, sex, or genotype between these groups (appendix p 10). Most participants with either persistent (29 [97%] of 30) or intermittent (31 [91%] of 34) smallcolony variant infections were using dornase alfa, suggesting that persistence of small-colony variants was not attributable to this medication. To determine the association between S aureus smallcolony variants and lung function, we first compared mean percentage of predicted FEV1 measures at each of the study visits for participants with and without small-colony variants detected during the study. Participants with smallcolony variants had consistently lower lung function than did those without small-colony variants at all study visits with sufficient data (figure 2; appendix p 11–12). Smallcolony variants were associated with significantly lower percentage of predicted FEV1 across visits 1–7 in univariate analyses (regression coefficient –7·07, 95% CI –12·20 to –1·95; p=0·0068) and multivariate analyses after adjusting for both patient demographics and P aeruginosa culture status (–5·50, –10·51 to –0·48; p=0·0316; appendix p 12). Regression analyses of the association between detection of small-colony variants and rate of change in percentage of predicted FEV1 did not show a significant interaction between detection of small-colony variants and follow-up visit number in either univariate or multivariate analyses (appendix p 13), indicating that participants with and without small-colony variants had similar rates of decrease in percentage of predicted FEV1 during the study. Percentage of predicted FEV1 was lower for participants with either intermittent or persistent small-colony variant infection compared with those who were negative for small-colony variants, but results were only statistically significant for participants with inter­ mittent infections (appendix p 14). The prevalence of respiratory exacerbations among participants who were ever positive and never positive for small-colony variants is shown in figure 3 and the appendix (pp 15–16). Participants with small-colony variants had significantly more exacerbations during the study across visists 1–7 than did those without small-colony variants for visits 1–7 (appendix p 16). Detection of smallcolony variants was significantly associated with increased odds of respiratory exacerbation in both the univariate

Figure 1: Study profile Treatment histories were recorded as written or online datalogs, therefore some discrepancies exist between the number who attended visits and those who provided treatment history data for each quarterly timepoint.

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unadjusted analysis (OR 1·73, 95% CI 1·19–2·52; p=0·0045) and multivariate analysis adjusted for demographic variables and P aeruginosa culture status (OR 1·49, 1·04–2·15; p=0·031) but no longer reached significance when adjusting for lung function at enrolment (appendix p 16). Interstingly, P aeruginosa culture positive status was associated with lower odds of exacer­bation in this model (OR 0·55, 95% CI 0·38–0·79; p=0·0014; appendix p 15). Participants with small-colony variants detected in multiple cultures (ie, persistent infection) had significantly higher odds of an exacerbation than those with small-colony variants detected once (ie, intermittent infection; appendix p 17). We investigated whether different small-colony variant subtypes had different associated clinical outcomes (ie, lung function and respiratory exacerbations). Thymidine-dependent small-colony variants appeared to account for most of the clinical associations observed for all small-colony variants in this population. For example, participants with thymidine-dependent small-colony variants had significantly lower percentage of predicted FEV1 in univariate analysis (regression coefficient –13·10, 95% CI –20·09 to –6·10; p=0·0003) and in multivariate analysis (–10·49, –17·25 to –3·73; p=0·0024) while participants with the other subtypes did not (figure 2; appendix p 20). Participants with thymidine-dependent small-colony variants also had a higher risk of respiratory exacerbations (OR 2·81, 95% CI 1·69 to 4·67; p=0·0001; figure 3, table 2)—even after adjusting for patient demo­ graphics, P aeruginosa culture status, and baseline predicted FEV11 (OR 2·17, 1·33 to 3·57; p=0·0021; table 3)—than did those with all other small-colony variant types. In multivariate models including small-colony variants, MRSA status, and P aeruginosa culture status, only MRSA small-colony variants, which were predominantly thymidine dependent, were associated with both decreased lung function (–8·44, 95% CI –16·15 to –0·72; p=0·00318) and increased odds of exacerbations (2·15, 1·24 to 3·71; p=0·0061; appendix pp 21022). MSSA smallcolony variants were also associated with lower lung function in multivariate models but not with respiratory exacerbations. Normal colony MRSA were not associated with lung function (–5·12, –10·74 to 0·51; p=0·08) or respiratory exacerbations (1·36, 0·91 to 2·03; p=0·13) in these mutlivariate analyses. Similarly, another cystic fibrosis pathogen, P aeruginosa, was not associated with lung function (–4·77, –10·36 to 0·83; p=0·10) in the multivariate model (appendix p 21) but was independently associated with reduced odds of exacerbations (0·54, 0·36 to 0·81; p=0·0028; appendix p 22). When assessing the association between detection of small-colony variants and previous exposure to antibiotics, only treatment with sulfonamides in the 3 months before a visit was significantly associated with detection of smallcolony variants by univariate analysis (OR 2·22, 95% CI 1·06–4·66; p=0·0338; table 4). Multivariate analyses

Total population (n=230)

Never positive for small-colony variant (n=166)

Ever positive for small-colony variant (n=64)

p value

Demographic Age, years

11·7 (3·1)

11·5 (3·1)

12·5 (3·0)

0·0302

Weight, kg

40·5 (14·4)

40·6 (14·8)

40·1 (13·5)

0·82

Height, cm

145·4 (17·1)

144·9 (17·7)

146·6 (15·5)

0·51

18·5 (3·0)

18·6 (3·1)

18·1 (3·0)

Body-mass index, kg/m²* Sex

··

··

··

Male

109 (47%)

79 (48%)

30 (47%)

Female

121 (53%)

87 (52%)

34 (53%)

Race White

··

··

··

0·22 0·92 ·· ·· 0·78

219 (95%)

158 (95%)

61 (95%)

··

Black or African American

6 (3%)

4 (2%)

2 (3%)

··

Asian or Pacific Islander

2 (1%)

2 (1%)

0

··

Native American or Alaskan Native

1 (<1%)

1 (1%)

0

··

Unknown

2 (1%)

1 (1%)

1 (2%)

··

··

··

Hispanic race Hispanic Non-Hispanic Unknown

·· 0·70

10 (4%)

8 (5%)

2 (3%)

··

219 (95%)

157 (95%)

62 (97%)

··

1 (<1%)

1 (1%)

0

··

··

0·20

Clinical Pancreatic status Pancreatic sufficient Pancreatic insufficient Genotype

··

··

15 (7%)

13 (8%)

2 (3%)

··

215 (94%)

153 (92%)

62 (97%)

··

··

··

··

0·41

CFTR Phe508del homozygous

125 (54%)

87 (52%)

38 (59%)

··

CFTR Phe508del heterozygous

80 (35%)

62 (37%)

18 (28%)

··

Other Sweat chloride value Baseline FEV1 % predicted

25 (11%)

17 (10%)

8 (13%)

··

101·5 (15·3)

100·7 (17·0)

103·6 (9·4)

0·27

90·4 (18·9)

92·4 (18·6)

85·5 (19)

0·0145

Medications and treatments† Bronchodilator

208 (90%)

150 (90%)

58 (91%)

0·95

Pancreatic enzyme supplement

213 (93%)

151 (91%)

62 (97%)

0·12

Inhaled steroid

122 (53%)

83 (50%)

39 (61%)

0·14

11 (5%)

6 (4%)

5 (8%)

0·18

Hypertonic saline

161 (70%)

123 (74%)

38 (59%)

0·0290

Dornase alfa

193 (84%)

133 (80%)

60 (94%)

0·0117

17 (7%)

17 (10%)

Oral steroid

CFTR modulator (ivacaftor)

0

0·0078

Data are mean (SD) or n (%) and p values. *Calculated using the 2006 WHO reference populations for children aged 6 months to <5 years and 2007 WHO reference population for children ≥5 years. †Medications or treatments participants were given at enrolment.

Table 1: Demographic and clinical characteristics of participants at enrolment

confirmed this association for all small-colony variant subtypes (OR 1·89, 1·03–3·49; p=0·0407; appendix p 18) and the association was even stronger for thymidinedependent small-colony variants in multivariate analyses (OR 10·88, 3·83–30·88, p<0·0001; appendix p 27). Participants who used dornase alfa also had significantly

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100 90 80 FEV1 % predicted

70 60 50 40 30 20 10 0 Baseline Number of participants Never positive for 164 small-colony variant Ever positive for 64 small-colony variant Ever positive for 35 non-thymidine-dependent small-colony variant Ever positive for 29 thymidine-dependent small-colony variant

Never positive for small-colony variant Ever positive for non-thymidine-dependent small-colony variant Ever positive for small-colony variant Ever positive for thymidine-dependent small-colony variant 1

2

3 4 Visit number

5

6

7

157

159

154

147

129

86

30

64

63

63

61

52

35

15

35

34

35

34

27

19

9

29

29

28

27

25

16

6

Figure 2: Lung function measures at each study visit Mean percentage of predicted FEV1 is shown for quarterly visits 1–7 for patients who were ever positive for Staphylococcus aureus small-colony variants (thymidine-dependent subtype, non-thymidine-dependent subtypes, and subtypes combined, non-exclusively, as indicated) and those who were never positive for small-colony variants (SDs for each mean value are in the appendix [p 10]). Data for visit 8, which relatively few participants attended, are omitted for clarity. FEV1=forced expiratory volume in 1 s.

Proportion of participants with exacerbations (%)

100 90 80 70 60 50 40 30 Never positive for small-colony variant Ever positive for non-thymidine-dependent small-colony variant Ever positive for small-colony variant Ever positive for thymidine-dependent small-colony variant

20 10 0

0

1

Number of participants Never positive for 166 small-colony variant Ever positive for 64 small-colony variant Ever positive for 35 non-thymidine-dependent small-colony variant Ever positive for 29 thymidine-dependent small-colony variant

2

3

4 Visit number

5

6

7

166

162

158

149

131

91

64

64

63

61

52

35

35

35

35

34

27

19

29

29

28

27

25

16

Figure 3: Prevalence of respiratory exacerbations since the previous study visit Proportion of patients with one or more exacerbations since the previous study visit, or since baseline in the case of visit 1, as assessed from spirometry at each clinic visit (absolute numbers of participants are shown in the appendix [p 14]). Visit 8, which relatively few participants attended, is omitted for clarity.

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increased odds of detection of small-colony variants, whereas treatment with hypertonic saline was associated with lower odds in both univariate (table 4) and multi­ variate analyses (appendix p 18). We categorised the biochemical subtype of each smallcolony variant isolate to further investigate their likely selective pressures and antibiotic resistance patterns. 185 small-colony variants were isolated from 64 participants, and each possible auxotrophic type was detected in this population with some patients having multiple auxotrophic types of isolates, including fattyacid-dependent small-colony variants (27 [15%] of 185), which, to our knowledge, have not previously been described in cystic fibrosis respiratory infections (appendix p 19). Most (103 [56%] of 185) of the smallcolony variants detected were thymidine dependent; a higher proportion of MRSA small-colony variants (68 [81%] of 84) than MSSA small-colony variants (35 [35%] of 101) were thymidine dependent. Participants with persistent small-colony variant infections were more likely to have thymidine-dependent small-colony variants than any other small-colony variant subtype (appendix p 10). Because detection of thymidine-dependent smallcolony variants could have been simply a marker of sulfonamide treatment, we analysed whether sulfonamide treatment itself was associated with worse clinical out­ comes. Sulfonamide treatment was not associated with lung function (difference –2·96, 95% CI –7·81 to 1·88; p=0·23; appendix p 23). Sulfonamides were associated with higher odds of respiratory exacerbations (OR 3·12, 95% CI 2·34–4·15; p<0·0001), as were all antibiotics used in this study apart from maintenance macrolides (appendix p 24), most likely reflecting their standard use in exacerbation treatment and the study definition of exacerbation. Sulfonamides were only the fifth most commonly prescribed antibiotic class in this study (appendix p 25). Therefore, the high prevalence of thymidine-dependent small-colony variants could not be attributed to disproportionate use of this antibiotic. However, significantly more participants with MRSA were treated with sulfonamides during the study than were those with only MSSA (50 [60%] of 84 treated with sulfonamides vs 34 [41%] of 84; p=0·0079; appendix p 26), probably explaining the higher frequency of thymidinedependent MRSA-small-colony variants than thymidinedependent MSSA-small-colony variants (appendix p 19). To further investigate the association between medic­ ations and small-colony variants, we compared reported medication use and the frequency of detection of smallcolony variants among the five study sites. Sites with the highest prevalences of small-colony variant generally reported higher mean frequencies of sulfonamide treat­ ment than did those with lower prevalences of smallcolony variants. For example, the sites with the highest and lowest frequencies of detection of small-colony variant (appendix p 6) reported the correspondingly highest and lowest mean frequency of use of sulfo­

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namides during the study (25 [51%] of 49 participants at Boston Children’s Hospital, and 11 [23%] of 48 at Texas Children’s Hospital (appendix p 28). Interestingly, the site with the lowest prevalence of small-colony variants (Texas Children’s Hospital) also reported significantly more hypertonic saline use (48 [96%] of 50 participants) than the other sites (ranging 60% to 69%; p≤0·0008 per pairwise site comparison; appendix p 29). Odds ratio

p value

Small-colony variant culture status Never positive for small-colony variant*

1 (ref)

Ever positive for non-thymidinedependent small-colony variant

1·08 (0·68–1·71)

0·76

Ever positive for thymidinedependent small-colony variant

2·81 (1·69–4·67)

0·0001

0·89 (0·83–0·95)

0·0006

Visit number

··

Data in parentheses are 95% CIs. Small-colony variant categories are mutually exclusive. *Baseline category.

Table 2: Association between small-colony variant subtypes and odds of respiratory exacerbations, univariate analyses

Discussion In this US-based multicentre, observational study, S aureus small-colony variants were detected among approximately 28% of children with cystic fibrosis when using sensitive culture methods that are rarely used in clinical laboratories. Small-colony variants were associated with measures of worse lung disease during the study—ie, lower lung function (percentage of predicted FEV1) and higher exacerbation frequency— even in multivariate models that adjusted for lung function at baseline. By comparison, P aeruginosa culture status did not have consistent associations with these poor outcomes in models that included small-colony variants, and associations between MRSA and poor outcomes were apparently attributable to MRSA smallcolony variants in this population. Risk factors for smallcolony variants included age, treatment with dornase alfa at enrolment, and sulfonamide treatment during the 3 months before detection. More detailed analysis showed that these observations were attributable primarily to thymidine-dependent small-colony variants, which were the most common small-colony variant subtype in this study and are known to be selected by sulfonamide

Multivariate analysis 1, adjusted

Multivariate analysis 2, adjusted

Multivariate analysis 3, adjusted

Odds ratio

Odds ratio

Odds ratio

p value

p value

p value

Small-colony variant culture status Never positive for small-colony variant*

1 (ref)

Ever positive for non-thymidinedependent small-colony variant

1·04 (0·65–1·65)

0·88

··

1·01 (0·64–1·58)

1 (ref)

0·97

··

1·01 (0·65–1·57)

1 (ref)

0·97

··

Ever positive for thymidine-dependent small-colony variant

2·50 (1·51–4·12)

0·0003

2·39 (1·46–3·89)

0·0005

2·17 (1·33–3·57)

0·0021

Visit number

0·90 (0·84–0·96)

0·0007

0·90 (0·84–0·96)

0·0007

0·90 (0·84–0·95)

0·0005

Age at enrolment

1·08 (1·02–1·14)

0·0060

1·09 (1·04–1·15)

0·0010

1·08 (1·03–1·14)

0·0042

Sex Male*

1 (ref)

Female

1·42 (1·02–1·97)

·· 0·0373

1 (ref) 1·49 (1·08–2·05)

·· 0·0155

1 (ref) 1·44 (1·05–1·98)

·· 0·0258

Hispanic race Hispanic*

1 (ref)

Non-Hispanic

0·85 (0·38–1·93)

·· 0·70

1 (ref) 0·74 (0·33–1·65)

·· 0·47

1 (ref) 0·77 (0·35–1·67)

·· 0·50

Genotype CFTR Phe508del homozygous*

1 (ref)

CFTR Phe508del heterozygous

1·09 (0·76–1·56)

0·63

··

1·03 (0·73–1·47)

1 (ref)

0·86

··

1·02 (0·72–1·44)

1 (ref)

0·92

··

Other

0·77 (0·44–1·35)

0·36

0·77 (0·44–1·33)

0·34

0·73 (0·42–1·26)

0·25

CFTR modulator No*

1 (ref)

Yes

0·55 (0·28–1·07)

·· 0·08

1 (ref) 0·53 (0·28–1·02)

·· 0·06

1 (ref) 0·57 (0·30–1·08)

·· 0·08

Pseudomonas aeruginosa culture status Never positive*

··

··

1 (ref)

Ever positive

··

··

0·55 (0·38–0·79)

··

··

Baseline FEV1 % predicted

··

·· 0·0014 ··

1 (ref)

··

0·52 (0·36–0·75)

0·0005

0·99 (0·98–1·00)

0·10

Data in parentheses are 95% CIs. Small-colony variant categories are mutually exclusive. Adjusted multivariate analysis 1 is adjusted for patient demographics genotype, and treatment with CFTR modulator, adjusted multivariate analysis 2 also includes Pseudomonas aeruginosa culture status during the study, and adjusted multivariate analysis 3 also includes baseline function. This graded analysis is presented to indicate the incremental association of these key potential confounders with outcomes. CFTR=cystic fibrosis transmembrane conductance regulator. *Baseline category.

Table 3: Association between small-colony variant subtypes and odds of pulmonary exacerbations, multivariate analyses

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Odds ratio

p value

Aminoglycoside No*

1 (ref)

Yes

1·28 (0·64–2·56)

·· 0·48

Beta-lactam No*

1 (ref)

Yes

1·03 (0·52–2·04)

·· 0·94

Fluoroquinolone No*

1 (ref)

Yes

1·11 (0·51–2·38)

·· 0·79

Glycopeptide No*

1 (ref)

Yes

1·22 (0·44–3·35)

·· 0·70

Lincosamide No*

1 (ref)

Yes

0·86 (0·23–3·28)

·· 0·82

Macrolide No*

1 (ref)

Yes

1·38 (0·77–2·45)

·· 0·28

Polymyxin No*

1

Yes

3·43 (0·89–13·22)

·· 0·07

Rifamycin No*

1 (ref)

Yes

0·36 (0·04–2·99)

·· 0·35

Sulfonamide No*

1 (ref)

Yes

2·22 (1·06–4·66)

·· 0·0338

Tetracycline No*

1 (ref)

Yes

1·74 (0·69–0·24)

·· 0·24

Antifungal treatment No*

1 (ref)

Yes

5·32 (0·47–59·75)

·· 0·18

Hypertonic saline No*

1 (ref)

Yes

0·51 (0·28–0·94)

0·0304

Inhaled steroid No*

1 (ref)

Yes

1·56 (0·87–2·81)

·· 0·14

Dornase alfa No*

1 (ref)

Yes

3·72 (1·26–10·98)

·· 0·0172

Data in parentheses are 95% CIs. Treatment exposures were assessed for the study quarter before the culture under consideration. *Baseline category.

Table 4: Association between treatment and subsequent small-colony variant detection in univariate analysis

treatment;11 however, sulfonamide treat­ment was not a risk factor for worse outcomes in the absence of smallcolony variants. Thymidine-dependent small-colony variants were particularly common among patients wth MRSA, probably because sulfonamide treatment was more common 10

among patients who were positive for MRSA in cultures than among other participants in this study; notably, trimethoprim-sulfamethoxazole is recommended as firstline therapy for MRSA—but not MSSA—cystic fibrosis infections in the USA.31,32 Prevalence of MRSA in patients with cystic fibrosis in the USA has also been shown to be higher than in many other countries;33 therefore, sulfonamides might be expected to be used particularly often in the USA; however, these antibiotics are commonly used in some countries continuously as antistaphylococcal prophylaxis or treatment for patients with cystic fibrosis, neither of which are common practices in the USA.34 Considering our findings, the role of trimethoprimsulfamethoxazole in the management of S aureus respiratory infection in cystic fibrosis merits discussion. We propose that this drug be avoided upon detection of small-colony variants; however, given our results, and because S aureus cystic fibrosis isolates tend to be sulfonamide susceptible,35,36 we do not recommend removing this antibiotic from the cystic fibrosis anti­ staphylococcal armamentarium. Our observations also identified a new plausible candidate mechanism for smallcolony variant selection: treatment with dornase alfa. This enzyme cleaves free DNA near pyrimidines and might release free thymidine, which supports the growth of thymidine-dependent small-colony variants. The import­ ance of this mechanism in small-colony variant selection requires further study. The associations we identified with worse measures of lung disease are consistent with observations from a single-centre US study15 and from European studies that were either small,19,21–23 single centre,19–22 or did not focus on small-colony variants or their associations with clinical outcomes.23 In the US single-centre study,15 small-colony variants were associated with a faster decrease in lung function over time that was not observed in this multicentre study, which might be attributed to the difference in participant population between the single-centre and multicentre studies. The single-centre study population was younger on average (mean age at enrolment 9 years vs 11·7 years in this study), and so the current study might have been less likely to capture initial infection, and less able to determine whether the trajectory of lung function changes after this event. By comparison with small-colony variants, the canonical cystic fibrosis pathogen P aeruginosa was associated with fewer exacer­bations in multivariate analyses in this paediatric cystic fibrosis population, and in models that adjusted for MRSA small-colony variants, P aeruginosa was not significantly associated with reduced lung function. Because small-colony variants have rarely been considered in cystic fibrosis studies or clinical care, these results raise questions about the associations between P aeruginosa, MRSA, and cystic fibrosis clinical outcomes identified in previous studies. The causal relationship between detection of smallcolony variants and clinical outcomes cannot be established in this observational study. Small-colony

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variants are known to be selected by antibiotics, which are used more frequently in people with more severe symptoms. Therefore, the associations established here might indicate that small-colony variants worsen disease or that they are simply a marker of worse pre-existing disease. Both these explanations might prove to be true. Similar controversy exists for clinical associations identified for MRSA,6,7 multiresistant P aeruginosa,37 and the gradual decrease in microbial diversity in cystic fibrosis sputum observed with advancing age and disease.38 Three observations in our study could be interpreted as supporting the marker hypothesis. First, small-colony variants were not associated with faster decrease in lung function; second, small-colony variants were detected less often in patients treated with ivacaftor, which is predicted to decrease the need for antibiotics; and finally, single detection (ie, intermittent infection), but not repeated detection (persistent infection), was associated with worse lung function. Alternatively, two observations support the possibility that small-colony variants might be pathogenic in cystic fibrosis. First, sulfonamide treatment, which can select small-colony variants, was not independently associated with worse lung function; therefore, small-colony variants do not simply indicate sulfonamide exposure as a proxy of severity. Second, the regression models that identified associations between small-colony variants and both exacerbation risk and worse lung function were adjusted for percentage of predicted FEV1 at enrolment, a measure of pre-existing disease severity. Future studies of small-colony variant preventive or treatment interventions might be able to address this question. At least one previous study, undertaken in Australia, did not identify small-colony variants among bronchoalveolar lavage samples from children with cystic fibrosis.39 Here, most samples taken from participants were oropharyngeal swabs. Although small-colony variants could primarily infect the upper airways rather than lower airways, the prevalence of S aureus cystic fibrosis respiratory infection is substantially lower in the USA than in Australia, where anti­ staphylococcal antibiotic prophylaxis and treatment on detection are routine practices.2,40 Therefore, smallcolony variants might be less common and their clinical impact might be lower in populations where anti­ staphylococcal strategies are used (although small-colony variants have been detected often in European countries where such strategies are common20). We previously identified small-colony variants in the sputum of paediatric patients with cystic fibrosis,15 which might more accurately reflect lower airway microbiology than do oropharyngeal swabs. Another limitation of using oropharyngeal swabs in this study was the inability to measure inflammatory markers, some of which have been associated with both specific bacteria and outcomes in cystic fibrosis—eg, neutrophil elastase.41,42 A future study using sputum or bronchoalveolar lavage samples might be able to address these questions.

This study was also limited by the quarterly respiratory sampling regimen and by the fact that each culture identified only the most abundant morphologically distinct isolates, under-sampling the diversity of infecting S aureus cell populations. Small-colony variants are known to revert to a normal colony phenotype during subculture, which can lead to an incorrect categorisation. The study was also limited by relatively few observations at visits 7 and 8, and lack of respiratory exacerbation data at baseline. While the effects of potential confounders were adjusted for in multivariate analyses, selection (ie, centre and participant selection) and informational (eg, recall) biases might reduce the generalisability and accuracy of the findings. Small-colony variants are not routinely identified by most clinical laboratories, including many of those specifically dedicated to cystic fibrosis microbiology, and susceptibility testing is not usually possible due to their slow growth. However, methods have been developed to identify these variants and test their antibiotic suscept­ ibilities that are not yet in widespread use.35 The high prevalence of small-colony variants and their clinical associations identified in this and previous studies of other cystic fibrosis populations15,19–22 indicate that consideration should be given to the routine adoption of these methods by clinical microbiology laboratories, particularly for samples from chronic infections in which small-colony variants are often present, including those of the airway, skin and skin structure, bone, and medical devices.8,9 We also recommend that small-colony variant epidemiological and susceptibility data be recorded in registries, such as the US Cystic Fibrosis Patient Registry, which currently tracks P aeruginosa, MRSA, and other socalled standard cystic fibrosis pathogens.2 Contributors DJW, FMO, and LRH did the literature search, analysed and interpreted the data, prepared figures and tables, and wrote the manuscript. JLB and LRH conceived the study. JE, JLB, SM, MB, DJW, and LRH designed the study. SM, LN, AU, DMO, MM, WH, and RLG enrolled participants. All authors, except JE, JLB, FMO, and LRH, contributed to data collection. All authors critically assessed the manuscript for content and approved the final version. Declaration of interests DJW, RLG, AU, JLB, and LRH report grants from Cystic Fibrosis Foundation during the conduct of the study. RLG reports grants from the National Institutes of Health (NIH) and other support from Vertex Pharmaceuticals outside of the submitted work. JLB reports grants from Seattle Children’s Research Institute during the conduct of the study. LRH reports grants from the NIH during the conduct of the study, and he is the director of the Center for Cystic Fibrosis Microbiology (Seattle, WA, USA), a national resource centre supported by the Cystic Fibrosis Foundation. In this role, LRH provides consultation to both private and public bodies doing research in cystic fibrosis microbiology but does not accept direct compensation for this role; all financial arrangements are made with LRH’s institution. All other authors declared no competing interests. Acknowledgments This study was supported by grants from the Cystic Fibrosis Foundation (BURNS03Y2, HOFFMA14A0, SINGH15R0) and the National Institutes of Health (K24HL141669, P30DK089507). We thank the children and their families who participated in this study.

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www.thelancet.com/respiratory Published online November 11, 2019 https://doi.org/10.1016/S2213-2600(19)30365-0