Author's response to reviews

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1 Author's response to reviews Title:Clinical and treatment-related risk factors for nosocomial colonisation with extensively drug-resistant Pseudomonas aeruginosa in a haematological patient population: a matched case control study Authors: Matthias Willmann (matthias.willmann@med.uni-tuebingen.de) Anna M Klimek (anna@klimek.de) Wichard Vogel (wichard.vogel@med.uni-tuebingen.de) Jan Liese (jan.liese@med.uni-tuebingen.de) Matthias Marschal (matthias.marschal@med.uni-tuebingen.de) Ingo B Autenrieth (ingo.autenrieth@med.uni-tuebingen.de) Michael Buhl (michael.buhl@med.uni-tuebingen.de) Silke Peter (silke.peter@med.uni-tuebingen.de) Version:2Date:7 November 2014 Author's response to reviews: see over

2 Institut für Medizinische, Elfriede-Aulhorn-Str. 6, Tübingen, Germany Dr. Shirish Huprika Associate Editor Dr. Matthias Willmann, MD, MSc, DTM&H Group leader: Clinical Microbiology Elfriede-Aulhorn-Str. 6, Tübingen Germany Tel / Fax 07071/ Dear Dr. Huprika, The authors of the manuscript entitled Clinical and treatment-related risk factors for nosocomial colonisation with extensively drug-resistant Pseudomonas aeruginosa in a haematological patient population would like to thank for the valuable recommendations and criticism of the associate editor and the reviewers. We took every point into serious consideration in order to improve our manuscript and provided a point-by-point response to the editor and reviewers (see below, responses in blue). Additionally, we had another editorial look at the English syntax and have removed errors as asked by reviewer 1. We would be grateful for your consideration of our revised manuscript for publication in BMC Infectious Diseases. Yours sincerely, Dr. Matthias Willmann Universitätsklinikum Tübingen Anstalt des öffentlichen Rechts Sitz Tübingen Geissweg Tübingen Tel / Steuer-Nr /09402 USt.-ID: DE Aufsichtsrat Hartmut Schrade (Vorsitzender) Vorstand Prof. Dr. Michael Bamberg (Vorsitzender) Gabriele Sonntag (Stellv. Vorsitzende) Prof. Dr. Karl Ulrich Bartz-Schmidt Prof. Dr. Ingo B. Autenrieth Jana Luntz Baden-Württembergische Bank Stuttgart BLZ Konto-Nr IBAN: DE SWIFT-Nr.: SOLADEST Kreissparkasse Tübingen BLZ Konto-Nr IBAN: DE SWIFT-Nr.: SOLADES1TUB Page1 von 15

3 Point-by-point response Editor s comments This is an interesting study and well written manuscript. The reviewers have highlighted some important issues and questions that deserve attention. The major issue that needs attention is the significance of colonization with XDR-PA. Was there a difference in clinical outcome (e.g. 30-day or hospital mortality) between cases and controls? If the authors are able to demonstrate a difference, this will further strengthen the significance of their findings. The editor asked to compare the difference in mortality between cases and controls. We have investigated the in-hospital mortality, and it is 4/31 = 12.9% for cases and 2/93 = 2.2% for controls (chi square test, P = 0.016). Although this seems to strengthen the significance of our finding one need to be careful about the interpretation of this result. We have not directly investigated the impact of XDR-PA colonisation on mortality. To investigate this we would need to include patients with infection (who are excluded in our study when the first detected isolate was recovered from an infectious process and not from a screening swab). Also, confounding factors would need to be investigated to acquire a high degree of internal study validity. We have previously conducted a similar study and found a higher mortality in patients with bloodstream infection due to MDR-PA or MBL-producing PA when compared to patients with bloodstream infections due to a susceptible PA [1]. Appropriate treatment was found to be a protective and independent predictor of mortality. Colonisation with a pathogen is a known risk factor for subsequent infection with the same bacterium [2]. This shows - on the one hand - that the individual patient could benefit if screening would reveal the actual carrier status. An appropriate empirical treatment could then be chosen if necessary. Also, the spread of such resistant pathogens could be reduced due to efficient infection control measures which could Page2 von 15

4 be delayed if no such a screening is performed. These points were further discussed in the revised version of our manuscript: In the chapter Results : (line ) In-hospital mortality was 12.9% (4/31) for cases compared to 2.2% (2/93) for the controls (P = 0.016). In the chapter Discussion : (line ) We have found a significantly higher in-hospital mortality among case patients compared to controls (12.9% versus 2.2%, P = 0.016). But this results need to be interpreted with caution since we have not directly investigated the impact of XDR-PA colonisation on mortality. However, it strengthens the findings of a previous study in our setting where mortality was significantly different between patients with bloodstream infection due to metallo-β-lactamase (MBL) producing P. aeruginosa or Non-MBL producing P. aeruginosa ( 61% versus 34%) [1]. Additionally, Tacconelli et al have reported that 9% of patients with a new hospital-acquired colonisation with an antibiotic resistant bacterium developed subsequently an infection due to the same pathogen [2]. Thus, identifying the patients carrier status could improve chances to choose an appropriate empirical treatment when necessary, particularly in an institution with a relevant incidence of multidrug-resistant pathogens. Further comments from the editor: 1. The study identifies "cipro administration" as a risk factor. Does this reflect prophylaxis, treatment or both? Was both prophylactic and treatment exposure of other antibiotics also assessed? Page3 von 15

5 Indeed, ciprofloxacin administration reflects both treatment and prophylaxis. Exposure to other antibiotics would reflect treatment since we use exclusively ciprofloxacin for prophylaxis in neutropenic patients at our institution. 2. Since their study identifies cipro as a risk factor, has their institution suspended cipro prophylaxis in neutropenic patients? Would include in discussion. We have not suspended ciprofloxacin prophylaxis in neutropenic patients. According to our national guidelines we use ciprofloxacin for prophylaxis in patients with a neutrophil count < 500 cells/µl ( We do not doubt that these patients benefit from this approach. However, in the light of the recent study results we do consider a switch from ciprofloxacin to cefuroxime prophylaxis due to an expected lower selection pressure. We have added this information to the discussion: (line ) Ciprofloxacin is used in our institution for prophylaxis in neutropenic patients. We have added this option to the conclusion: (line ) Additionally, a switch to alternative regimes for prophylaxis in neutropenic patients is a recommendable option. 3. The choice of control group is an important consideration and a number of articles have been published regarding the choice of controls with negative cultures vs. controls with nonresistant positive cultures. The authors need to weigh the pros and cons of each and why they chose to include both. Page4 von 15

6 Harris et al have excellently discussed this difficult issue [3]. Generally, control groups selection would depend on the question a study should answer. With the present study we wanted to answer the question: What are the risk factors of acquiring an XDR - P. aeruginosa among hospitalized patients? For this purpose we agree with Harris et al. that the control group must be selected from the same source population that gives rise to the case patients. This population is in our case all adult patients hospitalized for 48h who were weekly screened for the presence of P. aeruginosa and who were not colonisied with an XDR-PA at admission. This includes a minority of 16 control patients with a susceptible P. aeruginosa. These patients had a chance of being exposed to an XDR-PA and thus could have become a case patient. Inclusion of such control patients could lead to a selection bias according to Harris et al. [3]. This is due to a potentially less frequent exposure to antibiotics which are active against susceptible strains and would thus inhibit their growth. However, due to the fact that only a minority of these patients are included in the control group we are confident that the selection bias described by Harris et al. would only be a minor issue if at all. Furthermore, a distortion of the observed effect of antibiotics is most likely to be expected if the whole control group would consist of patients with a susceptible pathogen as shown in another study [4]. Our control group selection is also based on a further consideration: Removing patients with susceptible P. aeruginosa from the control group (and thus from the source population) could introduce another selection bias and affect the internal and external validity of our study since we do not only investigate antibiotic exposure but also other risk factors [5]. This selection bias could even have a greater impact on the study results, particularly in multivariate models. For these reasons we have decided to use our actual study design and we are confident that we have chosen an approach as unbiased as possible. Page5 von 15

7 To address this point in the manuscript we have extended the discussion by the following paragraph: (line ) Moreover, the inclusion of patients with Non-XDR-PA colonisation into the control group might have led to a selection bias according to Harris et al [3, 4]. In the present study, this should have caused only a minor bias if at all due to the low proportion of these patients among the control group (17.2%). Regarding these patients as a part of the source population from which case patients could arise prevents a general selection bias and retains the internal and external validity of our study [5]. 4. Regarding the controls, it is mentioned that 16 controls were colonized with non-xdr-pa. Does this mean that the other 77 controls had negative cultures? If so this should be clarified. And regarding the choice of controls, would the results be different if the controls were restricted to patients with negative cultures? Yes, the remaining 77 control patients had negative cultures during their hospital stay. They were screened weekly. We have added this information in the result section: (line ) The remaining 77 control patients (82.8%) were not colonized with P. aeruginosa. Additionally, the editor asked whether the results would be different if excluding the 16 controls with a Non-XDR-PA. We think that three major uncertainties would arise by such an analysis: 1. Since we have performed a matched case control study with an individual matching, the removal would affect 14 matching groups and thus 14 cases that could not be matched to three controls. The effect on the analysis in an unbalanced conditional regression model is unpredictable. 2. Harris et al made their comparison of control groups with different numbers of Page6 von 15

8 individuals in the control groups [4]. This approach does not take into account that the study power should be equal for an appropriate comparison. Thus, shifts in the odds ratios could be caused by a different estimation within a wider variance. This would be the same in our case. 3. As mentioned above, the potential introduction of a selection bias could affect the impact estimation for other variables, a particular problem in multivariate models. For these reasons we think that a comparison and its interpretation should only be made with caution and do not recommend such an additional analysis. However, if the editor regards this as a critical point we could run the analysis. In this case it must be noted that due to our complex modeling we would probably need more than the 4 weeks usually given for a revision. 5. Were only screening cultures used to define cases and controls or were clinical cultures also utilized? The authors that that active surveillance was performed with rectal and pharyngeal cultures. Yet 8 cases were based on respiratory cultures. Were these clinical cultures? These issues should be clarified. Our study focuses entirely on the colonisation with XDR-PA. Patients with the first detection of an XDR-PA in a clinical sample were not considered eligible for our study. However, case patients could have an infection with an XDR-PA later during their hospital stay. We have considered screening swabs from the pharynx as cultures from the respiratory tract. The misunderstanding has been resolved in the revised manuscript: (line ) Twenty-three case patients (74.2%) developed a rectal and eight case patients (25.8%) a pharyngeal XDR-PA colonisation. Page7 von 15

9 6. Are other carbapenems (ertapenem, imipenem, and doripenem) not used in their hospital? Why weren't exposure to these carbapenems included in the analysis? This should be clarified. Our Department of Haematology uses almost exclusively meropenem. Other carbapenems are rarely used and were thus not investigated. Reviewer 1 comments: Minor Essential Revisions 1. Would include underlying hematologic disease in Table I and in Step I of the conditional logistic regression analysis. The investigation of underlying haematological conditions as a variable with potential impact on the risk of XDR-PA colonisation is a great point. However, due to the variety of different haematological conditions it would be difficult to include them into the logistic regression analysis. The overall number of each condition would be too small to perform a meaningful analysis. Such an approach should be performed in multicenter studies with a sample size that allows for such investigations. By including the standardised SAPSII score into our models we have at least estimated the severity of the underlying diseases. Surprisingly, we have not seen a significant effect. 2. Are fluoroquinolones used for neutropenic prophylaxis at your center? It would be helpful to know whether these were received prior to admission (on an outpatient basis) - ie, did cipro use during the hospitalization reflect continuation Page8 von 15

10 of outpatient prophylaxis? As mentioned in the response to the editor, we give ciprofloxacin for prophylaxis in neutropenic patients. Usually, medication is started in the hospital but in a few cases patients would have received ciprofloxacin before admission. Since our hospital software system does not provide us with reliable documentation about outpatient medication we have not included this in the analysis but do not expect a severe bias due to the low frequency of this event. 3. Would include the number of control patients with negative surveillance cultures and the number with non-xdr-pa surveillance cultures in the text. These numbers are now included in the revised manuscript (see response to editor). 4. The definition of XDR-PA was slightly confusing on the initial read. It appears that the definition of XDR-PA was based upon the paper by Magiorakos et al. However, starting on line 97, however, it says that "XDR-PA was defined as resistant to meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin..." (no initial mention of aztreonam and aminoglycosides). I am assuming this means that meropenem was the carbapenem tested at your institution, etc. If the XDR-PA definition (defined by Magiorakos et al) was utilized, would consider adjusting the wording to indicate which XDR-PA definition was used and which antimicrobials were tested at your center. Resonse Page9 von 15

11 The reviewer is correct in that this sentence would mislead the reader. We used the CDC/ECDC criteria and have modified the revised manuscript to make this clear. We deleted the sentence and have replaced it with: (line ) XDR-PA was defined according to the CDC/ECDC criteria. The following antimicrobials were tested at our center: gentamicin, tobramycin, amikacin, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, ciprofloxacin, levofloxacin, meropenem, aztreonam, fosfomycin, and colistin. The former sentence should express that all XDR-PA in our setting were resistant to the four bactericidal standard antibiotics that can be administered in monotherapy (meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin), saying that we think that these are the clinically most relevant strains. Since this is also mentioned in the result section we do not want to mislead the reader here. 5. Would also address the limitation of using a control group comprised of those with negative screening cultures or from whom a non-xdr-pa was isolated (versus a control group with negative screening cultures only or non-xdr-pa screening cultures only). The potential selection bias is mentioned in the revised manuscript and comprehensively discussed in our replies to the editor s comments. Discretionary Revisions Page10 von 15

12 1. If the information is available, would include data on whether the cases and controls were ever previously colonized with Pseudomonas (XDR vs not). All cases included in the analysis have not been known to be a carrier of an XDR-PA previously. Unfortunately, we do not have reliable data from other hospitals where some patients were treated before they were admitted to our institution. To our knowledge we are the only institution in our region which performs a comprehensive screening for P. aeruginosa colonisation. However, we have performed a screening at admission where actual carriers would have been identified and excluded from the study. Reviewer 2 comments: This is an interesting manuscript and the study is sound. The authors evaluated risk factors to acquire multi-resistant Pseudomonas aeruginosa in a Haematology Unit and, based on their results, developed a clinical risk score. They proposed in the discussion that this score may determine patients at high risk, directing and rationalizing the use of surveillance cultures. There are few points that I believe require clarifying. 1- The authors did not describe the characterisitics of the Haematology department in which the study was conducted. For example, how many beds were there? What type of patients were hospitalised in the Unit? We have provided the reader with further information about our Haematology department in the Patients and Methods chapter: (line 82-87) Page11 von 15

13 The study was performed on the wards of the Department of Haematology in a 1500-bed tertiary teaching hospital in Tübingen, Germany. There are 80 beds at the department for the treatment of patients having various haematological-oncological conditions, such as leukaemia, lymphoma and multiple myeloma. Stem cell transplantations are regularly performed. One ward is an intensive care unit with single rooms. 2- The authors did not describe how surveillance was conducted. It seems that cultures were collected on admission of the patients to the unit, but this is not clearly stated. Also, it is not clear what was the frequency of the culturing during hospitalisation. We have already described this in the original manuscript in the Patients and Methods chapter: (line in the revised manuscript) Routine screening for P. aeruginosa was carried out at admission and weekly thereafter. The screening involved rectal and pharyngeal swabs. 3- In results (page 9, line185) the authors mention that all isolates of the case patients were resistant to aztreonam. However they only investigated genes that encode metallobetalactamases, that do not usually hydrolyse aztreonam. The authors should discuss this issue and the mechanisms that may be present among the isolates. Although this is a very interesting point we unfortunately do not have a solid base for such a discussion. Resistance to monobactams can be mediated by enzymatic activity (ESBLs, OXA, Page12 von 15

14 class A carbapenemases) and efflux systems such as MexAB-OprM [6]. From the laboratory point of view we have investigated the reason for meropenem resistance rather than the mechanism for the clinically less relevant aztreonam non-susceptibility (Aztreonam was not administered to patients during the study period, supplementary Table S2). This leaves us with no more than pure speculation for the discussion concerning that question. We would like to avoid this and would rather focus on the major topic of our work. However, this could be an interesting topic for future, laboratory-based studies. 4- The authors evaluated the odds ratio using the score and the cutt-off proposed by them. However it would be expected that the score perfom well when evaluating the sample from which it originated. It is necessary that this scoring system be evaluated using another group of patients and this should be clearly stated. The reviewer mentions two points. It would be expected that the score performs well when derived from odds ratios that were retrieved from the results of the actual study population. We do not agree with this statement since a score must still have a decent discriminatory power to perform well and this is not necessarily the case even when derived from the investigated population. However, we do certainly agree with the second point which says that the score should be evaluated on another patient population before giving a final statement about its performance. We have included this information in our revised manuscript: (line ) We are aware that our study has several limitations. The CRS is derived from the odds ratios of significant risk factors in our study population. Its true performance would need to be evaluated on another patient cohort in the same setting, preferably with a prospective design. Page13 von 15

15 5- The authors should mention that this study was conducted in a specific unit (Haematolgy) and that its results may not apply to other settings. This point was already discussed among the study limitations in our original manuscript: (line in the revised manuscript) Also, this is a single-center study and results are usually not transferable to other settings. However, it must be stated that even results of multi-center studies as well as recommendations from generally accepted guidelines can turn out as inappropriate. An example are the results from a research group in eastern China who found the American Thoracic Society (AST) guidelines criteria not reliable for the prediction of multidrug-resistant organisms in their hospital [7]. References 1. Willmann M, Kuebart I, Marschal M, Schroppel K, Vogel W, Flesch I, Markert U, Autenrieth IB, Holzl F, Peter S: Effect of metallo-beta-lactamase production and multidrug resistance on clinical outcomes in patients with Pseudomonas aeruginosa bloodstream infection: a retrospective cohort study. BMC infectious diseases 2013, 13: Tacconelli E, De Angelis G, Cataldo MA, Mantengoli E, Spanu T, Pan A, Corti G, Radice A, Stolzuoli L, Antinori S et al: Antibiotic usage and risk of colonization and infection with antibiotic-resistant bacteria: a hospital population-based study. Antimicrob Agents Chemother 2009, 53(10): Page14 von 15

16 3. Harris AD, Karchmer TB, Carmeli Y, Samore MH: Methodological principles of casecontrol studies that analyzed risk factors for antibiotic resistance: a systematic review. Clin Infect Dis 2001, 32(7): Harris AD, Samore MH, Lipsitch M, Kaye KS, Perencevich E, Carmeli Y: Control-group selection importance in studies of antimicrobial resistance: examples applied to Pseudomonas aeruginosa, Enterococci, and Escherichia coli. Clin Infect Dis 2002, 34(12): Elwood M: Critical Appraisal of Epidemiological Studies and Clinical Trials. Oxford: Oxford University Press; 2007: Poole K: Pseudomonas aeruginosa: resistance to the max. Frontiers in microbiology 2011, 2: Xie J, Ma X, Huang Y, Mo M, Guo F, Yang Y, Qiu H: Value of American Thoracic Society guidelines in predicting infection or colonization with multidrug-resistant organisms in critically ill patients. PLoS One 2014, 9(3):e Page15 von 15