Correlation between culture-identified bacteria in the middle nasal meatus and CT score in patients with chronic rhinosinusitis

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1 Journal of Medical Microbiology (2014), 63, DOI /jmm Correlation between culture-identified bacteria in the middle nasal meatus and CT score in patients with chronic rhinosinusitis Barbora Uhliarova, 1,2 Renata Karnisova, 3 Martin Svec 2 and Andrea Calkovska 1 Correspondence Barbora Uhliarova b.uhliarova@gmail.com 1 Department of Physiology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia 2 Department of Otorhinolaryngology, FD Roosevelt Faculty Hospital, Banska Bystrica, Slovakia 3 Department of Clinical Microbiology, FD Roosevelt Faculty Hospital, Banska Bystrica, Slovakia Received 16 September 2013 Accepted 11 October 2013 The aim of this study was to compare bacteriological findings in the middle nasal meatus in patients with chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP) and healthy controls, and to investigate the correlation between the prevalence of culture-identified bacteria and the severity of sinus disease. Bacterial culture was performed using a swab from the middle nasal meatus under endoscopic control in 72 patients with CRSwNP, 25 patients with CRSsNP and 59 healthy controls. Computed tomography (CT) scans were graded for severity using the Lund Mackay scoring system. Patients with more severe forms of CRS with and without nasal polyps had significantly higher rates of pathogenic bacteria in the middle nasal meatus compared with patients with lower CT scores of the paranasal sinuses. There were no significant differences in bacterial species among CRSwNP, CRSsNP and control patients. These results demonstrate, for the first time, that colonization by pathogenic bacteria in patients with CRSwNP and CRSsNP is associated with a more severe form of the disease, as assessed by a pre-operative CT scan of the paranasal sinuses. The results suggest a role for bacterial infection in the pathogenesis of CRS. However, bacteria do not appear to play a role in the development of nasal polyposis in patients with CRS. INTRODUCTION Chronic rhinosinusitis (CRS) is often described as a chronic inflammatory condition of the sinonasal mucosa, but the syndrome is actually defined by its clinical presentation rather than by markers of inflammation. CRS has been defined as persistence of two or more rhinosinusitis-related symptoms (nasal congestion, facial pain/pressure, anterior or posterior nasal drainage and reduced or absent sense of smell) for more than 12 weeks, confirmed by local objective signs (direct visualization or imaging studies) (Fokkens et al., 2012). CRS is divided into two subgroups, CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP), which differ in their immunological patterns. CRSwNP is associated with a T- helper-cell type 2-shifted immune response, which includes a predominance of eosinophils and IL-5 as the dominant cytokine, whereas CRSsNP is a T-helper-cell type 1- dominated disease with predominance of mononuclear Abbreviations: CRS, chronic rhinosinusitis; CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; CT, computed tomography; MRSA, meticillin-resistant Staphylococcus aureus. cells and IFN-c in the nasal tissue (Fokkens et al., 2012; Niederfuhr et al., 2009). CRS affects approximately 5 15 % of the general population both in Europe and the USA. It has a considerable impact on quality of life and causes a high financial burden to society (Fokkens et al., 2012; Kok et al., 2009). An infectious aetiology of CRS has long been postulated. In contrast to microbiology tests performed in patients with acute rhinosinusitis, there are no definitive and consistent data on the distribution of bacteria in patients with CRS and their role in the pathogenesis of the disease. Variability in results from CRS studies is caused by the use of different techniques (e.g. harvesting method), variations in culture methods, prior use of antibiotics and difficulties in distinguishing between the colonizing agents and true pathogens. The aim of this study was to evaluate the relationship between the bacterial microenvironment in the middle nasal meatus and the severity of sinus disease, expressed as a computed tomography (CT) score, in patients with CRSwNP and CRSsNP G 2014 SGM Printed in Great Britain

2 Bacteria affect the severity of chronic rhinosinusitis METHODS Study population. The study was conducted with 156 patients divided into three groups. A total of 72 patients with CRSwNP (mean age 50.2 years, range years) and 25 patients without CRSsNP (mean age 48.8 years, range years) were diagnosed according to the European position paper on rhinosinusitis and nasal polyps (Fokkens et al., 2012) and scheduled for functional endoscopic sinus surgery for persisting symptoms following unsuccessful long-term conservative treatment. Medical treatment included topical and systemic steroids, antihistamines, antibiotics and nasal saline lavage. A total of 59 patients (mean age 37.2 years, range years) admitted for surgical treatment for another reason (i.e. chronic tonsillitis, benign and malignant tumours of the head and neck) were allocated to a control group if there was no history of sinus abnormalities and no signs of mucosal inflammation on nasal endoscopy. Study design and inclusion/exclusion criteria. All patients gave their informed consent and the study was approved by the ethics committees of the Jessenius Medical Faculty, Comenius University, Martin, and the FD Roosevelt Faculty Hospital, Banska Bystrica, Slovakia. The inclusion criteria were: more than 12 weeks of CRS symptoms, and endoscopic and radiological criteria of CRS, as established in the European position paper on rhinosinusitis and nasal polyps (Fokkens et al., 2012). Patients with systemic or local antibiotic treatment within the 4 weeks before enrolment, recent upper respiratory infection, acute exacerbation of CRS, suspected sinusitis of dental origin, increased C-reactive protein levels, cystic fibrosis or unilateral sinus disease were excluded. The prevalence of bronchial asthma in the patients with CRS and differences in colonization by pathogenic bacteria in asthmatic and non-asthmatic subjects were also recorded. CT. Disease severity in CRS patients was established using a preoperative CT scan of the paranasal sinuses and the Lund Mackay scoring scale (Lund & Mackay, 1993). The right or left sinuses were divided into six portions: the maxillary sinus, anterior ethmoid sinuses, posterior ethmoid sinuses, sphenoid sinus, frontal sinus and ostiomeatal complex. The severity of sinus mucosal inflammation or fluid accumulation was scored as 0 (complete lucency), 1 (partial lucency) or 2 (complete opacity). Mild mucosal thickening without fluid collection was scored as 0; mild mucosal thickening with fluid collection causing partial lucency was scored as 1; and moderate or severe mucosal thickening without fluid collection causing partial lucency, but not complete opacity, scored as 1. In addition, the ostiomeatal complex was scored as either 0 (not obstructed) or 2 (obstructed). Points from all 10 locations sinuses and bilateral ostiomeatal complexes were counted to give a total Lund Mackay score that could range from 0 (complete lucency of all sinuses) to 24 (complete opacity of all sinuses). According to the value of the CT score, the patients were divided into two groups: less severe disease (0 15 points) and more severe disease (16 24 points) (Fig. 1). Bacterial culture. Middle nasal meatus swab specimens were obtained under endoscopic control using sterile cotton-wool swabs and transported in Stuart s transport medium to the microbiological laboratory within 2 4 h. The swabs were inoculated onto sheep blood agar (Columbia agar; Bio-Rad), chocolate agar with a bacitracin disc or MacConkey agar (Bio-Rad) and placed into a 7 % CO 2 incubator at 37 uc. The plates were examined after h of incubation. The incubation was further extended to 48 h to detect slow-growing microbes. Identification of colonies to genus or species level was based upon typical colony morphology by subculture, Gram stain, standard rapid tests (catalase, pyrrolidonyl aminopeptidase and oxidase tests), latex agglutination tests and biochemical tests. All Fig. 1. Coronal CT score of the sinuses of a patient with CRSwNP, revealing bilateral obstruction to the ostiomeatal complex and complete opacity of the displayed sinuses (anterior ethmoid sinuses and maxillary sinuses), more severe disease. pathogenic strains were tested for their susceptibility to antimicrobial agents using the agar diffusion method (by EUCAST) with commercial discs (Oxoid). Staphylococcus aureus and coagulase-negative staphylococci were identified on the basis of colony morphology, catalase test and detection of Staphylococcus aureus clumping factor by a commercial latex test (Pastorex Staph Plus; Bio-Rad). Meticillin-resistant Staphylococcus aureus (MRSA) strains were identified after subculture onto chromogenic selective MRSA medium (ORSAB; Oxoid) and during testing of their susceptibility to antimicrobial agents (cefoxitin 22 mm or MIC oxacillin 2 mg/l by EUCAST). Further confirmation was made using a latex agglutination method that detects penicillin-binding protein 2 (MRSA-Screen; Denka Seiken). Small-bowl colonies with a-haemolysis sensitive to the optochin test (zone inhibition 14 mm) and soluble in bile salt solution enabled rapid identification of Streptococcus pneumoniae strains. Satellitism around colonies of Staphylococcus aureus on blood agar, transparent watercolours colony on chocolate agar, which grew around a bacitracin disc, Gram stain and the requirement of two factors, X (haemin) and V (NAD), for growth were used to differentiate species of Haemophilus influenzae. Streptococcus pyogenes (Lancefield group A) group A Streptococcus was identified by colonial appearance, Gram stain, catalase test and biochemical test (API 20 Strep; biomérieux). A b-lysine-producing Staphylococcus aureus strain was streaked across a plate with a coated swab to identify Streptococcus agalactiae (Lancefield group B) group B Streptococcus. A CAMP test was also used for subculture colonies that had a less distinct zone of b- haemolysis, negative pyrrolidonyl arylamidase test and negative catalase test. Colonies of Moraxella catarrhalis were identified growing as drops of wax glided on plate surface and they were oxidase, catalase and indoxylacetate test positive. The Gram-negative species Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella species, Escherichia coli, Acinetobacter calcoaceticus and Morganella morganii were identified growing as lactose-positive or lactose-negative colonies on MacConkey agar. Further identification 29

3 B. Uhliarova and others was performed by subculturing on TSI agar (LAB M) and using biochemical tests (API; biomérieux). Bacterial strains from the Czech National Collection of Type Cultures and strains from our own collection were used as reference representatives of Gram-positive and Gram-negative bacteria associated with upper respiratory tract infections: Staphylococcus aureus CCM3953, Staphylococcus aureus MRSA CCM4750, Streptococcus pneumoniae CCM4424, Streptococcus pyogenes CCM4425, E. coli CCM3954, P. aeruginosa CCM3955 and H. influenzae NCTC 8468 (Oxoid). Statistical analysis. Frequencies of categorical data were tabulated and evaluated using a x 2 test with Yates s correction. For other data, medians and interquartile ranges were calculated and tested with a Kruskal Wallis or Mann Whitney test. Statistical analysis was performed using STATISTICA CZ 10. All conclusions are based on a significance level of P,0.05. RESULTS Among 156 patients screened for this study, 72 had CRSwNP, 25 had CRSsNP and 59 represented a control group with no signs of CRS. The median CT score was 17.5 in the CRSwNP group and 9.2 in the CRSsNP group. There was a significantly higher rate of more severe forms of disease (CT score 16 points) in the CRSwNP group compared with the CRSsNP group (P,0.0001). Moreover, a tendency to a higher incidence of asthma was found in the CRSwNP group (P50.07) (Table 1). Bacterial growth was present in 63 of the 72 samples (88 %) from patients with CRSwNP, 24 of the 25 samples (96 %) from patients with CRSsNP and 52 of the 59 samples (88 %) from patients in the control group. Polymicrobial growth was recorded in 43, 38 and 21 % of samples in the CRSwNP, CRSsNP and control groups, respectively. Aerobic micro-organisms isolated from the middle nasal meatus of patients are shown in Table 2. There were no significant differences in bacteriological species from the middle nasal meatus among CRSwNP, CRSsNP and control patients. Coagulase-negative Staphylococcus species, Staphylococcus aureus and Corynebacterium species were most commonly identified in all three groups of patients. Coagulase-negative Staphylococcus species, Corynebacterium species, Streptococcus viridans and Neisseria species are considered commensals of the upper respiratory tract; therefore, the distribution of true pathogens in patients was as follows: 31 samples (43 %) in CRSwNP patients, nine (36 %) in CRSsNP patients and 16 (27 %) in the control group, with no significant differences among the three groups (P50.17). Comparing the presence of micro-organisms and the severity of the disease according to the pre-operative CT investigation of paranasal sinuses using the Lund Mackay scoring system, there was a significantly higher rate of pathogenic bacteria in patients with a more severe form of the disease (CT score 16 points) within both the CRSwNP group (P50.02) and the CRSsNP group (P50.02) (Table 3). No significant difference between colonization by pathogenic bacteria and the presence of bronchial asthma was found in patients with CRS. In the CRSwNP group, eight asthmatic patients had positive culture for pathogenic bacteria and 14 patients had a negative culture (P50.4). In the CRSsNP group, pathogenic bacteria were found in two patients with bronchial asthma and in one patient the culture was negative (P50.2). DISCUSSION Despite the high prevalence of CRS, its pathogenesis and the reasons behind the development of nasal polyps in some patients are still unclear (Fokkens et al., 2012). In this study, we investigated bacteriological colonization of the middle nasal meatus in a well-defined study population of patients with CRS with and without polyps and controls, and the relationship of bacteriological colonization with the severity of sinus disease as expressed by CT scan. There is general agreement in the literature that bacteria are present within the paranasal sinuses in most patients with CRS. The maxillary antrum and middle meatus are generally considered sterile in normal populations (Hamilos, 2000; Klossek et al., 1998). In patients with clinical and radiographic evidence of CRS, % demonstrate recoverable bacteria from the ethmoid or maxillary sinuses (Bhattacharyya, 2002). In a review of 174 patients who underwent surgery for chronic maxillary sinusitis, Biel et al. (1998) identified bacteria in 94 % of patients, with 19.5 % of patients having polymicrobial infection. Table 1. Demographic and disease characteristics of the study population Characteristic CRSwNP (n572) CRSsNP (n525) Controls (n559) Age [years (range)] 50.2 (22 76) 48.8 (23 79) 37.2 (18 66) Male/female (n) 45/27 14/11 31/28 CT score [median (range)] 17.5 (10 24) 9.2 (8 18) NA CT score 16 points [n (%)] 53 (74) 5 (20) NA Asthma bronchiale [n (%)] 22 (31) 3 (12) 0 NA, Not applicable. 30 Journal of Medical Microbiology 63

4 Bacteria affect the severity of chronic rhinosinusitis Table 2. Aerobic bacteria found in the three patient groups with positive culture Bacteria [n (%)] CRSwNP (n563) CRSsNP (n524) Controls (n552) Gram-positive Coagulase-negative Staphylococcus 40 (63) 16 (67) 39 (75) Corynebacterium species 16 (25) 4 (17) 9 (17) Staphylococcus aureus 15 (24) 6 (25) 12 (23) Streptococcus viridans 5 (8) 4 (17) 1 (2) Streptococcus pneumoniae 5 (8) 0 0 Streptococcus agalactiae group B Streptococcus 4 (6) 0 0 Streptococcus pyogenes group A Streptococcus 1 (2) 0 0 b-haemolytic streptococci 1 (2) 0 0 MRSA Gram-negative Neisseria species 3 (5) 1 (4) 1 (2) Pseudomonas aeruginosa 1 (2) 0 1 (2) Proteus mirabilis 0 1 (4) 2 (4) Klebsiella species 2 (3) 0 1 (2) Haemophilus influenzae 1 (2) 2 (8) 0 Escherichia coli 2 (3) 1 (4) 0 Acinetobacter calcoaceticus 1 (2) 1 (4) 0 Morganella morganii 1 (2) 0 0 Moraxella catarrhalis (2) Polymicrobial growth 27 (43) 9 (38) 11 (21) More recently, a trend towards Gram-negative bacteria involvement in CRS has been identified. Mantovani et al. (2010) found a 58 % prevalence of Gram-negative bacteria and a 28 % prevalence of Pseudomonas species in patients with CRS. Similar findings have been reported by other authors (Bolger, 1994; Nadel et al., 1998; Zurak et al., 2009). It is clear from most of these studies that bacteria are present within the ethmoid and maxillary sinuses in patients with a history of CRS. However, interpretation of these results is difficult. Methodological differences lead to variability in the results from CRS studies. Several studies (Jiang et al., 1993, 2002; Ozcan et al., 2002) have suggested that culture after endoscopic harvesting of secretions from the middle meatus is a feasible alternative to anthral punction, being effective for identifying pathogens and a non-invasive method for the aetiological diagnosis of CRS. Moreover, the middle meatus drains the anterior ethmoid, frontal and maxillary sinuses, and thus the bacteriology of this area better reflects the microbiology of the paranasal sinuses when compared with material from the maxillary punction (Jiang et al., 2002). For the reasons mentioned above, we decided on the simple, non-invasive but effective method of taking a mucosal swab from middle nasal meatus under endoscopic control. Large numbers of positive cultures of Gram-positive bacteria were found in all three groups of patients in this study. The most frequently isolated agent was coagulasenegative Staphylococcus (68 % of positive cultures). Gramnegative bacteria growth was seen in 18 % of cultures. The difference between the recovery rate and rates of Grampositive, Gram-negative and polymicrobial growth between the groups of patients with CRSwNP and CRSsNP was not significant. This is in accordance with the study of Brook and Frazier (2005), which suggested that the microbiology of the maxillary sinus of patients with chronic sinusitis with polyposis is not different from that of patients who Table 3. Presence of pathogenic bacteria and their relationship to severity of disease n (%) CT score 16 points [n (%)] CT score,16 points [n (%)] P value CRSwNP (74) 19 (26) Pathogenic micro-organisms 31 (43) 27 (87) 4 (13) 0.02 CRSsNP 25 5 (20) 20 (80) Pathogenic micro-organisms 9 (36) 5 (56) 4 (44) 0.02 Controls 59 NA NA Pathogenic micro-organisms 16 (27) NA NA NA, Not applicable. 31

5 B. Uhliarova and others develop chronic sinusitis without polyposis. The recovery rate for aerobic bacteria in our study was also similar to that found in the literature (Bhattacharyya, 2002; Zurak et al., 2009). Staphylococcus aureus is a frequent colonizer of the nasal cavity in white subjects, with an average persistent carrier rate of % of adults (Van Crombruggen et al., 2011). An increased colonization rate of Staphylococcus aureus has previously been demonstrated in patients with CRSwNP (63.6 %) but not in patients with CRSsNP (27.3 %) versus control subjects (Van Zele et al., 2004). In our study, there were no significant differences between the groups in the detection of Staphylococcus aureus in the middle nasal meatus. Therefore, these bacteria are unlikely to be the main reason for nasal polyp formation in patients with CRSwNP. Moreover, in accordance with Niederfuhr et al. (2009), we did not find significant differences in the bacteriological features of mucosal swabs from the middle nasal meatus among CRSwNP, CRSsNP and control patients. Therefore, a bacteriological pathogenesis of the polyps in CRSwNP patients seems unlikely and the general use of antibiotics in patients with CRS is questionable. Interestingly, in both groups of patients with CRS with and without nasal polyps, we found a significant correlation between the presence of pathogenic bacteria and disease severity, as expressed by CT score. Patients with more severe forms of CRS with and without nasal polyps (CT score 16 points) had significantly higher rates of pathogenic bacteria in the middle nasal meatus than patients with lower CT score of the paranasal sinuses. Similar results were found by Bhattacharyya et al. (2001), where the degree of inflammatory cellular infiltrate into the mucosa in CRS correlated with the radiographic extent of disease. It is probable that bacterial infection results in immunological changes within the sinonasal mucosa, with subsequent thickening and CRS. However, bacterial infection alone is insufficient to produce CRS in most patients, and other host factors are also important. Asthma and CRS are frequently associated in some patients, but their interrelationship is not clearly understood (Bousquet et al., 2001). The prevalence of asthma has been reported to be higher in patients with nasal polyps, and 7 % of asthma patients have nasal polyps compared with a lower percentage in the non-asthma population (Settipane & Chafee, 1977). In addition, nasal polyps are diagnosed more frequently (10 15 %) in patients with non-atopic and late-onset asthma. In our study, the rate of bronchial asthma was higher in the CRSwNP group (31 %) than the CRSsNP group (12 %), but the difference was not significant (P50.07). Colonization by pathogenic bacteria did not differ in our groups of patients and there were no significant differences between asthmatic and non-asthmatic patients. Zurak et al. (2009) confirmed the hypothesis that bacterial colonization in chronically inflamed sinuses may have an impact on neutrophil granulocyte activation in patients with bronchial asthma, which was not confirmed for patients with CRS without asthma. Moreover, a study by Ikeda et al. (2011) demonstrated that bacterial infection induces exacerbation of asthma secondary to worsening of sinonasal disease. In conclusion, the results of this study illustrate, for the first time, that colonization by pathogenic bacteria in CRSwNP and CRSsNP patients is associated with a more severe form of the disease, as assessed by pre-operative CT scanning of the paranasal sinuses. 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