Treatment intensity and characteristics of MRSA infection in CF

Journal of Cystic Fibrosis 10 (2011) 201 206 www.elsevier.com/locate/jcf Original Article Treatment intensity and characteristics of MRSA infection in CF Marianne S. Muhlebach a,, Meredith Miller b, Lisa M. LaVange c, Greg Mayhew c, Jennifer S. Goodrich d, Melissa B. Miller e a Department of Pediatrics, University of North Carolina, Chapel Hill, Bioinformatics, NC 27599-7217, USA b University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA c Department of Biostatistics, University of North Carolina at Chapel Hill, 137 East Franklin Str. Chapel Hill, NC 27599-8030, USA d Clinical Microbiology-Immunology Laboratories, University of North Carolina Hospitals, Chapel Hill, NC 27599-7525, USA e Department of Pathology and Laboratory Medicine, University of North Carolina, School of Medicine, Chapel Hill, NC 27599-7525, USA Received 14 September 2010; received in revised form 14 February 2011; accepted 20 February 2011 Abstract Background: Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and interchange of hospital-associated strains carrying the staphylococcal chromosomal cassette mec-ii (SCCmec-II) with those in the community (SCCmec-IV) has increased. This study assesses the impact of MRSA and different MRSA types on clinical outcomes, medication use, and antibiotic sensitivities. Methods: MRSA isolates from CF patients at our center were typed by SCCmec- and pv l status. Patient characteristics, lung function and nutrition are compared between MRSA types and to age, gender and Pseudomonas aeruginosa matched patients with chronic methicillin sensitive S. aureus (MSSA) infection. Results: Seventy-two percent of patients carry pv l negative SCCmec-II isolates. Seventeen percent of all MRSA were SCCmec-IV pv l positive (USA300). These patients were younger and fewer had chronic P. aeruginosa infection, whereas pv l -negative SCCmec-IV isolates show highest antibiotic resistance. Nutritional outcomes and FEV1 percent predicted (75.1 ± 2.7 versus 77.9 ± 2.7) did not differ in patients with MRSA compared to those with MSSA but MRSA patients received more pulmonary maintenance but not oral antibiotic medications. Conclusion: Patients with chronic MRSA are treated more intensely than age, gender and Pseudomonas aeruginosa matched MSSA-positive patients but clinical characteristics within MRSA patients vary depending on MRSA types. 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Methicillin resistant Staphylococcus aureus (MRSA); Medication use; Molecular typing; Outcome 1. Introduction Staphylococcus aureus (S. aureus) is one of the most frequent organisms in CF patients in the US with a prevalence of 45% in infants to children two years of age and peaking around 62% in school age children [1,2]. In recent years there has been a steep increase in the proportion of S. aureus isolates that are methicillin resistant (MRSA) from 2% in 2001 to 21.2% in 2007 per CFF Annual Patient-Registry-Report (http://www. cff.org/). Although prevalence in most European countries is Corresponding author. Tel.: +1 919 966 1055; fax: +1 919 966 6179. E-mail address: Marianne_Muhlebach@med.unc.edu (M.S. Muhlebach). much lower there has been an increase in MRSA during this time frame. MRSA, initially described in the 1960s only in hospital settings, has spread in frequency within and outside the healthcare environment. Infection with MRSA in previously healthy subjects i.e., community-associated MRSA (CA- MRSA) was originally described in the 1980s as causing complicated skin infections and necrotizing pneumonia. In the US one particular strain, USA300 has been especially successful [3]. So-called healthcare-associated MRSA (HA- MRSA) and CA-MRSA differ in several aspects other than time of contact between the patient and the healthcare system (http:// cdc.gov/ncidod/dhqp/ar_mrsa_ca.html). Methicillin resistance is mediated by production of low-affinity penicillin binding 1569-1993/$ - see front matter 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2011.02.004

202 M.S. Muhlebach et al. / Journal of Cystic Fibrosis 10 (2011) 201 206 protein 2a that is encoded by the meca gene, which is located on the staphylococcal chromosomal cassette mec (SCCmec). To date five different SCCmec types have been described, and of these, SCCmec types I, II, and III are typically healthcareassociated strains in the US. These SCCmec strains carry additional resistance genes making them resistant to many classes of antibiotics. Conversely, CA-MRSA typically harbor the smaller SCCmec types IV or V carrying only mecamediated resistance resulting in susceptibility to a wider variety of antibiotic classes. Studies performed prior to the recent spike in MRSA showed either no or controversial impact of MRSA on clinical outcomes in CF [4,5]. More recent epidemiologic studies show varying effects of MRSA on clinical outcomes. A cross-sectional study including CF patients with S. aureus as the sole organism showed that those with MRSA had lower lung function than those with MSSA [6]. Using the same database Sawicki et al. found that longitudinal decline in lung function was worse in patients with MRSA, however both, before and after acquisition of this pathogen [7]. Using the CFF Registry Dasenbrook et al. showed a more rapid decline in lung function in adolescents who acquired MRSA compared to those who remained MRSA negative in a 10-year cohort study; however no effect was seen in adults acquiring MRSA [8]. Most recently a higher mortality risk in patients with chronic MRSA infection was shown [9]. While there is increasing concern for a negative impact of MRSA none of these studies examined the potential impact of different MRSA strains, the effect and intensity of therapy that patients receive, and if different SCCmec types affect antibiotic sensitivities. In this study we hypothesize that infection with CA-MRSA versus HA-MRSA is associated with different patient characteristics in CF and antibiotic susceptibilities. Further we compare treatment intensity and outcomes in patients with MRSA to an age, gender and P. aeruginosa status matched methicillin sensitive S. aureus (MSSA) patient group. 2. Methods 2.1. Patients Patients with MRSA were selected based on prospective collection of all MRSA positive respiratory secretions obtained from CF patients seen at the UNC Pediatric and Adult CF clinics between October 2005 and October 2007 [10]. One isolate per patient was included but previous and subsequent microbiological data were tracked to confirm that the patient had chronic MRSA carriage ( 3 MRSA positive cultures/year). Patients with chronic MSSA infection were selected to be the same gender, age within 0.5 years, and have the same Pseudomonas status (see Clinical data) as the matched MRSA subject. Diagnosis of CF had been confirmed per standard recommendations [11]. Approval to perform this research using a waiver to obtain consent was obtained from our Institutional Review Board. 2.2. Clinical data P. aeruginosa infection status was classified into three categories (never, intermittent and chronic) based on Leeds criteria, with the modification of including mucoid phenotype in the definition given the negative impact of mucoid P. aeruginosa on lung function [12,13]. Pulmonary function was measured at each clinic visit per standard clinical protocol using Sensormedics spirometer and was expressed as percent of predicted normal values [14,15]. For children b 8 years old, preschool specific equations were used [16,17]. Use of oral antibiotics, routine medications and number of exacerbations (IV and orally treated) were extracted from clinic notes and in some cases from PortCF. For longitudinal data analyses lung function results were downloaded from the CF Foundation Patient Registry data at our center (PortCF). 2.3. Microbiology methods Respiratory samples were processed per routine methodologies for CF specimens [18]. Detection of MRSA was based on growth on mannitol salt agar followed by a positive tube coagulase test or a positive BactiStaph latex agglutination test (Remel, Lenexa, KS). Screening tests were performed using oxacillin screening agar and full susceptibility testing was done by Kirby Bauer disk diffusion following CLSI guidelines [19]. Molecular methods used to determine PVL status and SCCmec type were performed as previously described [10]. 2.4. Statistical methods Statistical analyses were performed using SAS Version 9.2 (SAS, Cary, NC). Cross-sectional comparisons were performed using t-tests for continuous variables and chi-squared tests for categorical variables. For longitudinal analyses mixed linear models were fit to repeated measurements of FEV 1 and BMI that appropriately accounted for randomly missing observations and correlations among the repeated measurements on a given patient [20]. Age at time of infection and gender were included as model covariates. Two sets of mixed linear models were fit to address two different hypotheses. One set included separate intercepts and slopes for MRSA and MSSA patients and assumed a constant linear trend across the two-year period surrounding the time of infection. The second set of models allowed for the slopes to differ between the months prior to infection and the months following infection. This second set of models allowed testing for an effect for change in FEV 1 at the time of infection. 3. Results 3.1. MRSA isolates and molecular characterization One-hundred and twenty MRSA isolates from distinct patients were collected over a 2 year period, which is representative of our

M.S. Muhlebach et al. / Journal of Cystic Fibrosis 10 (2011) 201 206 203 CF population. Repeat MRSA isolates obtained from 26 patients 2 years later showed persistence of the same molecular strain within a given patient, consistent with our prior findings [10]. After exclusion of patient samples with insufficient information (n = 11), transplant recipients (n = 11), or variable appearance of MRSA and MSSA (n = 16) eighty-two pediatric and adult patients were included in further analyses. Bacterial isolates of 58 patients were positive for SCCmec-II and one patient for SCCmec-III; thus, SCCmec types usually associated with healthcare-associated infections in the US were present in ~72% (n=59) of patients and all were pvl negative. Isolates from 23 patients (28%) carried the SCCmec-IV MRSA; of these 14 isolates were also pvl positive; thus 17% of all MRSA were SCCmec-IV pvl positive, and further characterization showed these to be USA300 strains [10]. Clinical characteristics of all MRSA positive patients and MRSA types are summarized in Table 1. Patients with pvlpositive SCCmec-IV MRSA strains were younger than those with SCCmec-II (p = 0.039), but not those with pvl negative SCCmec- IV (p=0.063). The younger age of patients with pvl positive SCCmec-IV strains is likely a result of the increased spread of USA300 strain in recent years. The time of initial positive MRSA culture could be determined for 62 patients. Duration of MRSA infection was longer for patients harboring SCCmec-II strains (5.0±0.8 years, n=42) compared to the seven patients with pvlpositive SCCmec-IV strains (1.8±0.4 years, p=0.005) but not the 13 patients with pvl-negative SCCmec-IV strains (2.1 ± 0.5 years, p=0.075). Prevalence and type of P. aeruginosa were significantly higher in patients with SCCmec-II strains compared to patients with SCCmec-IV (86.4% versus 52.2%, p=0.001) when classification was based on SCCmec type only. Comparison with inclusion of pvl status showed a higher proportion of chronic P. aeruginosa infection in patients with SCCmec-II isolates compared to pvl-positive SCCmec-IV (p = 0.004) but not to pvl-negative SCCmec-IV (p = 0.133); however sample sizes are very small for the last comparison. Table 1 Background characteristics of MRSA positive patients overall and by SCCmec status. MRSA (n=82) SCCmec-II SCCmec-IV, SCCmec-IV, All (n=59) pvl neg (n=9) pvl pos (n=14) Number (%) male 39 (47.6) 29 (50.8) 5 (55.5) 5 (35.7) Age (years) a : 17.6±1.0 18.3±1.1 21.2±3.8 12.8±2.4 mean±se N (%) N18 years 34 (41.5) 26 (44.1) 5 (55.5) 4 (28.6) P. aeruginosa 2005 2007: n (%) Never 10 (12.2) 3 (5.1) 2 (22.2) 5 (35.7) Intermittent 9 (11.0) 5 (8.5) 1 (11.1) 3 (21.4) Chronic/mucoid 63 (76.8) 51(86.4) 6 (66.7) 6 (42.9) a The date used for calculation of age was Aug. 1, 2007. For further comparison of P. aeruginosa infection a group of MSSA infected patients was used with same mean age, similar gender distribution (51.2% male) and 13.4% never, 13.4% intermittent, and 73.6% chronic P. aeruginosa infection. 3.2. Antimicrobial susceptibilities Resistance to trimethoprim sulfamethoxasole (TMP SMX) was low, both for initial isolates and those collected in 2007: 5.3% and 2.2% for SCCmec-II (n=52), 11.11% and 12.5% among pvl-negative SCCmec-IV (n = 8), and no resistance among pvl-negative SCCmec-IV isolates (n = 11). Clindamycin resistance or inducible resistance was present in 98% of the initial and 100% of subsequent SCCmec-II isolates. Again, pvlnegative versus pvl-positive SCCmec-IV isolates differed: 62.5% of pvl-negative versus 7.1% and 33.3% of pvl-positive isolates were clindamyin resistant at initial isolation and in 2007, respectively. Most MRSA isolates, regardless of molecular type, were resistant to erythromycin (75 93%). Resistance to gentamicin was low (0 11%) for all MRSA types, except pvl-negative SCCmec-IV isolates 25% (2/8). No isolates were reported indeterminate or resistant to vancomycin. Linezolid susceptibilities were not routinely evaluated. 3.3. Treatment intensity and outcomes of patients with MRSA compared to MSSA As the different patient characteristics between MRSA types may affect outcomes we compared the MRSA patients to an age, gender, and P. aeruginosa matched cohort with MSSA to test for differences in outcomes and treatments. Cross-sectional comparisons of lung function using the best measures in 2007, showed that mean FEV 1 percent predicted was 75.1 ± 2.7% among MRSA patients compared to 77.9 ± 2.7% among MSSA patients (p=0.486). Mean FEF 25 75 percent predicted was 60.9±4.3% versus 64.1±4.5% in MRSA and MSSA patients, respectively (p = 0.600). Similarly there was no difference in nutritional status in 2007 as measured by BMI percentile (41.3±3.4% for MRSA versus 41.0±3.1% for MSSA; p=0.946). Patients with MRSA versus MSSA infection had a similar number of exacerbations (2.4 ± 0.2 versus 2.1 ± 0.2; p=0.186) and number of clinic visits (4.8±2.0 versus 4.9±0.3, p=0.871) in 2006. Pancreatic sufficiency (96% of MRSA and 90% in MSSA p = 0.212) and insulin dependent diabetes (6/82 in MSSA compared to 12/82 MRSA (p = 0.212) were similar. Chronic pulmonary medications included use of tobramycin solution for inhalation (TSI e.g. TOBI ), mucolytics (DNase and hypertonic saline), inhaled steroids, and chronic oral azithromycin. As shown in Table 2 there was a trend for higher use of chronic medications in the MRSA patients for each of those medications, reaching significance for inhaled steroids and tobramycin (p=0.003 and 0.008, respectively). Analysis of use of any of these five medications showed that a higher proportion of patients with MRSA were prescribed at least one of the five pulmonary maintenance medications than patients with MSSA (p=0.001). In the year prior to these measures patients with MRSA received 72.2±6.1 days of oral and IV antibiotics for exacerbations versus 61.2 ±4.8 in the MSSA group; p=0.157; oral antibiotics only were: 52.2±5.4 versus 41.7±3.5; p=0.091.

204 M.S. Muhlebach et al. / Journal of Cystic Fibrosis 10 (2011) 201 206 Table 2 Use of medications by MRSA and MSSA patients. Medication use a MSSA n (% of patients using med) MRSA n (% of patients using med) 3.4. Longitudinal analyses of lung function and BMI P-value c AZM 40 (48.8) 49 (59.8) 0.210 TSI 19 (23.2) 36 (43.9) 0.008 Hypertonic 56 (68.3) 57 (69.5) 0.999 saline DNase 29 (35.3) 41 (50.0) 0.083 Inhaled steroids 29 (35.3) 49 (59.8) 0.003 Combined 173 232 0.001 meds. b Enzymes 74 (90.2) 79 (96.3) 0.212 Diabetes (insulin) 6 (7.3) 12 (14.6) 0.212 AZM=Azithromycin, TSI=tobramycin solution for inhalation. a Use is categorized as Yes or No, and number and % of patients in each group on the medication are counted. b Combined medications represent the sum of five possible chronic pulmonary medications used in each group. Maximum value would be 410. c Chi-square for comparison of proportions; t-test for combined medications. As patients with MRSA were prescribed a more intense medication regimen at the current time point but had similar lung function we evaluated if either of these outcomes was different prior to and after S. aureus infection. The linear rates of change in FEV 1 from one year prior to one year after infection were 1.98% (±2.90%) per year and 2.81% (± 1.70%) for MSSA (n = 43) and MRSA patients (n = 62), respectively. The difference in slopes between the two patient groups was not statistically significant (p-value = 0.805 after adjusting for gender and age at infection) (Fig. 1). Similarly there were no significant differences in linear rates of change in BMI% across the two-year period surrounding infection for patients with MRSA versus those with MSSA (p-value = 0.310). Age at initial staphylococcal infection was significantly younger for MSSA than for MRSA patients (7.8 ± 1.1 years versus 13.3 ± 1.1 years; p-value b 0.001). Rates of decline before and after S. aureus infection are provided in Fig. 2A (MSSA) and B (MRSA) for FEV 1 %predicted.the models include an additional effect for change in FEV 1 at the time of infection, depicted as a solid dot on the graphs. For MRSA patients, FEV 1 is fairly flat during the months both before and after infection but appears to drop notably at the time of infection; however, this drop was not statistically significant. No difference in slopes for FEV 1 before and after infection was detected (p-value for difference in slopes = 0.874). For MSSA patients, FEV 1 appears to decline both before and after infection but increase somewhat at the time of infection. This increase, however, was not statistically significant, and no significant differences between the slopes before and after infection were noted (p-value for difference in slopes = 0.754). 4. Discussion The current study characterizing MRSA in CF by molecular typing of MRSA shows that demographic and clinical characteristics differ among patients with SCCmec-II or SCCmec-IV isolates. As described in the literature not all SCCmec-IV isolates were pvl positive and these isolates were associated with different clinical characteristics. Further treatment details and outcomes of this population were compared to a matched group of patients with MSSA. Fig. 1. Longitudinal rate of change in FEV1% during the 2 years surrounding the infection with either MRSA (red) or MSSA (blue). Measurements obtained at 3- month intervals were compared for one year pre-infection to one year postinfection centered at the time of initial infection. Fig. 2. Longitudinal change in FEV 1 for MSSA (A) and MRSA (B) before and after acquisition of S. aureus acquisition. Analysis focuses on differences in rates of decline before and after S. aureus infection with an additional effect for change at the time of initial acquisition.

M.S. Muhlebach et al. / Journal of Cystic Fibrosis 10 (2011) 201 206 205 This study was done after widespread occurrence of MRSA strain USA300 in the community. In our patient population we find this MRSA strain in pediatric and adult patients. Differentiation of MRSA types is relevant given the different characteristics of community- versus hospital-associated MRSA in non-cf patients. Differentiation based on epidemiologic criteria is becoming difficult given the increasing number of HA-MRSA patients with chronic diseases in the community and outbreaks of CA-MRSA in the hospital [21,22]. Mutations and genetic interchange between MRSA strains further complicate molecular classification. Classification as CAversus HA-MRSA in fact may depend if epidemiologic, SCCmec type, PVL status, or sensitivity to clindamycin is used [23]. In this study we use primarily SCCmec typing but also determine pvl status as USA300, the most frequent CA-MRSA in the US, is pvl positive. Within SCCmec type IV six subtypes have been described but not all of these are pvl positive, thus PVL cannot be used as the sole criterion for definition as CA- MRSA [24 26]. Our data show clinically relevant differences between SCCmec types but also within SCCmec-IV depending on pvl status in regard to demographic and clinical parameters, and antimicrobial sensitivities. SCCmec-IV that were pvl negative show the highest rate of resistance, e.g. an 11% resistance to TMP SMX in this group compared to 3 5% in all other patients. This could indicate that these strains belong to different SCCmec-IV subtypes or reflect the higher proportion of adults with more use of antibiotics in this group. Only two prior studies evaluated MRSA SCCmec types in patients with CF [27]. These showed that approximately onethird of MRSA strains recovered from CF patients were the SCCmec type IV or V. Of these 29% were pvl positive, with a higher rate among patients with recent MRSA acquisition [28]. None of our patients had suppurative lung disease as has been described in cases of non-cf [29] and CF pneumonia [30]. A comparison of MRSA-positive patients to patients with MSSA at our center matched for three main risk factors of worse outcomes (age, gender, and P. aeruginosa) showed nodifference in cross-sectional comparison of lung function and nutrition parameters. Use of chronic maintenance medications was higher in MRSA-positive patients despite having comparable disease severity. This could indicate that MRSA-positive patients require more intensive therapy to maintain the same lung function as MSSA-positive patients, a finding that would be consistent with worse outcomes with MRSA as described by Ren et al. [6]. Neither use of antibiotics (either oral or all types of antibiotics) nor frequency of exacerbations was different. Conceivably, MRSApositive patients may be sicker prior to the first isolation of this organism. In fact we see a trend of having the lowest FEV 1 at the time of first MRSA. This explanation is consistent with the more rapid decline prior to and after acquisition of MRSA versus MSSA reported by Sawicki et al. [7].InthisscenarioMRSAmay be a marker rather than a cause of worse disease. As MRSA patients were older at the time of initial S. aureus infection than those with MSSA, comparison of antibiotic use during the year preceding infection is biased by other factors and was not done in this study. Conceivably, medications may have been started prior to onset of MRSA infection and actually be risk factors for development of resistance. The higher rate of nebulizer use or inhaled steroids might be considered such potential risk factors. Use of inhaled tobramycin as recommended for chronic P. aeruginosa infection was higher among patients with MRSA despite the same P. aeruginosa infection status, a finding similar to a study evaluating impact of MRSA in patients without P. aeruginosa where ~30% of patients with MSSA and ~ 60% with MRSA were on inhaled aminoglycosides [6,31]. We can only speculate on explanations for increased use of inhaled aminoglycosides in MRSA positive patients; one, patients with resistant organisms are considered sicker leading to use of additional medications; two, that these patients had prior P. aeruginosa and were continued on inhaled tobramycin. Determination of the presence of P. aeruginosa prior to onset of MRSA infection in the currently studied patients did not reveal a systematic difference in frequency of P. aeruginosa infection at the time of MRSA or MSSA onset (data not shown); three, that high dose inhaled tobramycin is used in hopes of eradicating MRSA, especially as N90% of strains are reported as gentamicin susceptible in vitro. This approach would not be appropriate as systemic aminoglycoside monotherapy is not recommended for MRSA infection due to lack of efficacy. Thus, inhaled tobramycin is unlikely to be effective for MRSA as evident by the high number of patients who use inhaled high dose tobramycin but continue to have MRSA in their sputum. Our analyses of MRSA types are limited by the small sample size, especially when evaluating pvl positive versus negative SCCmec-IV types separately and did not allow for a longitudinal study assessing therapies and concomitant infections. 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