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Evaluation of the Gram stain of sputa from cystic fibrosis patients to predict the presence of Staphylococcus aureus
Diagnostic Microbiology and Infectious Disease 77 (2013) 99–100
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Evaluation of the Gram stain of sputa from cystic fibrosis patients to predict the presence of Staphylococcus aureus Leandro Reus Rodrigues Perez a,⁎, Cícero Armídio Gomes Dias a, b a b
Microbiology unit, Hospital Mãe de Deus, Porto Alegre – RS, 90880-480, Brazil Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 7 January 2013 Received in revised form 12 June 2013 Accepted 14 June 2013 Available online 23 July 2013
a b s t r a c t We evaluated the presence of Gram-positive cocci in cluster, seen in Gram-stained smears of cystic fibrosis (CF) sputa versus the microbiological culture for the prediction of the presence of S. aureus. Gram stain provided low accuracy (69.2%; odds ratio, 4.8; 95% confidence interval, 61.3–76.1) for predicting S. aureus in CF sputum. © 2013 Elsevier Inc. All rights reserved.
Keywords: Gram stain Cystic fibrosis Staphylococcus aureus
The Gram stain is the most commonly used tool in microbiology laboratories for evaluating morphology and arrangement of bacterial cells. Furthermore, the Gram stain allows a careful review of the clinical specimen with respect to its microscopic quality, mainly on clinical specimens that are necessarily obtained from a tract colonized by commensal microorganisms, such as sputa (Sharp, 2003). Although a specimen with high degree of difficulty for processing and interpretation, sputa have been largely used for analysis in patients with suspect of having chronic obstructive pulmonary disease (Roche et al., 2007), community-acquired pneumonia (Miyashita et al., 2008), and cystic fibrosis (CF) patients (Burns et al., 1998; Nair et al., 2002; Sadeghi et al., 1994). Cultures using selective media are recommended to obtain a higher yield of isolation of the main microorganisms involved with a respiratory disorder in CF patients—Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and Burkholderia cepacia (Cystic Fibrosis Foundation, 1994). However, cultures using selective media usually require a longer period of evaluation compared the conventional cultures, and for these cases, results of Gram stain examination could guide the antimicrobial choice for initial empirical treatment (Heineman et al., 1977). The objective of this study was to evaluate the presence of a particular morphotype, Gram-positive cocci in cluster (GPCC), seen in Gram-stained smears of sputa collected from CF patients versus the microbiological culture for the prediction of the presence of S. aureus. This study is a substudy in the evaluation of the use of a selective chromogenic agar to isolate methicillin-resistant S. aureus (MRSA) (Perez et al., 2012). Specimens collected from CF patients admitted at ⁎ Corresponding author. Tel.: +55-5193690440. E-mail address: [email protected] (L.R.R. Perez). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.06.022
Hospital de Clínicas de Porto Alegre, the main care center for CF patients in the south of Brazil, between January and April 2011, were included. Smears were prepared and stained by Gram stain according to standard procedure (Chapin and Murray, 1999). Quality of samples was inspected according to microscopic analysis and summarized in good quality (GQ) or poor quality (PQ) based upon presence of epithelial cells (b10 and ≥10, respectively) in low-power field (LPF). Additionally, sputa were cultured on conventional media (5% sheep blood agar and mannitol salt agar) and the MRSA ID (bioMérieux, Marcy l’Etoile, France), a chromogenic selective agar to allow the growth of S. aureus. For both smear preparation and culture plate inoculation, a purulent portion of sputum was selected. After 24 to 48 h of incubation at 35 °C, plates were examined. Results from microscopy and culture were obtained independently and carried out by double-blind analysis. Statistical analyses were carried out using SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA). Prevalence ratios, sensitivity, specificity, positive and negative predictive values, and odds ratio (OR) and 95% confidence intervals (CIs) were calculated. The measurement of observer agreement for categorical date (Kappa coefficient) was determined, and the results were classified according to previously described (Landis and Koch, 1977). A total of 146 sputa from CF patients were evaluated. Of these, 128 (87.7%; 95% CI, 81.3–92.1) samples presented a number of epithelial cells less than 10 per LPF and 18 (12.3%; 95% CI, 7.9–18.6) presented more than 10 epithelial cells per LPF, being considered sputa of GQ and PQ, respectively. GPCCs were observed in 77/128 (60.2%; 95% CI, 51.5–68.2) samples with GQ and, for these, 56 (72.7%; 95% CI, 61.9 to 81.4) cultures showed grown of S. aureus. Among samples with PQ, GPCC were present in 13/18 (72.2%; 95% CI, 49.1 to 87.5) and in 8/13 (61.5%;
100
L.R.R. Perez, C.A.G. Dias / Diagnostic Microbiology and Infectious Disease 77 (2013) 99–100
Table 1 Sensitivity, specificity, positive and negative predictive values, accuracy, and OR of Gram stain findings for determining the culture results presenting S. aureus in sputum specimens from CF patients Variable
Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Accuracy (%) Odds ratio a b c
Gram stain findings presenting Gram-positive cocci in clusters b10/LPFa (95% CI, range in %)
≥10/LPFb (95% CI, range in %)
Totalc (95% CI, range in %)
76.7 61.8 72.7 66.7 70.3 5.3
80 (49–94.3) 37.5 (13.7–69.4) 61.5 (35.5–82.3) 60 (23.1–88.2) 61.1 (38.6–79.7) 2.4 (2.9–19.8)
77.1 (67–84.8) 58.7 (46.4–70) 71.1 (61–79.4) 66.1 (53–77.1) 69.2 (61.3–76.1) 4.8 (2.3–9.8)
(65.8–84.9) (48.6–73.5) (61.9–81.4) (53–78) (61.9–77.5) (2.5–11.5)
N = 128; cases including only Gram stain presenting b10 epithelial cells per LPF. N = 18; cases including only Gram stain presenting 10 or more epithelial cells per LPF. N = 146; considering (a + b) situation.
95% CI, 35.5 to 82.3) S. aureus were isolated in culture. Sensitivity of microscopic examination was 77.1% (64/83), being 76.7% (56/73) and 80% (8/10) for GQ and PQ, respectively. Total agreements (accuracy) were of 90 (70.3% of observations) and 11 (61.1% of observations) for the group of samples of GQ and PQ, respectively. Overall, only 69.2% (101/146) accuracy was achieved. According to our results, the measurement of agreement between the presence of GPCC and the growth of S. aureus was classified as “fair” agreement (kappa = 0.36; 95% CI, 0.21–0.57). Moreover, OR for recovering S. aureus on culture when GPCC is visualized in GQ samples Gram stained was 2 times superior than PQ samples (OR = 5.3 versus OR = 2.4). Several studies had discussed the utility of microbiological findings, to predict lower respiratory tract infection, and the role of sputum Gram stain remains unclear. Theerthakarai et al. (2001) indicated that sputum Gram stain, for patients with non-severe community-acquired pneumonia without co-morbid factors, does not provide diagnostically useful information and does not help in guiding initial therapy. Garcia-Vázquez et al. (2004) found that Gram stain of sputum samples was useful in guiding microbiological diagnosis of community-acquired pneumonia in only 14.4% of the hospitalized patients evaluated in the study. For CF patients, Nair et al. (2002) showed that a subjective evaluation of sputum samples was superior to the Gram stain in identifying adequacy for culture. Besides this, these authors showed a poor correlation between culture results and Gram stain for gram-negative pathogens. Sadeghi et al. (1994) showed a positive predictive value of the Gram stain for growth from acceptable sputum samples of 86.3% for S. aureus, superior to the value obtained for us in this study (Table 1); however, they also showed a very poor sensitivity for microscopic examination. There is a number of potential explanations for this lack of association between the Gram stain and culture results. These include lack of homogeneity and differences in bacterial densities throughout the sample. Besides this, influence of previous antimicrobial therapy (a variable that was not controlled in this and in the studies of Sadeghi et al.) may influence results. Respiratory secretions of CF patients may be polymicrobial, making important the microscopic examination to recognize different morphotypes and to predict the presence of co-colonization, a condition related to lung function decline (Hudson et al., 1993). Another special issue in CF patients is the recommendation by some authors (Heineman et al., 1977; Medici et al., 1988) that even “inadequate” (≥10 epithelial cells in LPF) must be accepted for culture. Nair et al. (2002) showed that, in 90% of the samples that
would have been rejected by their quality score, CF pathogens were isolated on culture media. In counterpoint to this, in our study, a loss of specificity was noted when we include PQ samples (37.5% versus 61.8%). On the other hand, the low number of PQ samples (N = 18) included is a limitation of this study, and investigations including a larger number of samples with this characteristics are required. Our results indicate that microscopic examination presents low accuracy to predict the presence of S. aureus in sputum cultures of CF patients. References Burns JL, Emerson J, Stapp JR, Yim DL, Krzewinski J, Louden L, et al. Microbiology of sputum from patients at cystic fibrosis centers in the United States. Clin Infect Dis 1998;27:158–63. Chapin KC, Murray PR. Stains. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th edn. Washington, DC: American Society for Microbiology; 1999. p. 1677–9. Cystic Fibrosis Foundation. Microbiology and infectious disease in cystic fibrosis. Consensus Conference, Concepts in Care, vol. V. Consensus Conference. Bethesda, Md: Cystic Fibrosis Foundation; 1994. p. 1–26. Garcia-Vázquez E, Marcos MA, Mensa J, de Roux A, Puiq J, Font C, et al. Assessment of the usefulness of sputum culture for diagnosis of community-acquired pneumonia using the PORT predictive scoring system. Arch Intern Med 2004;164:1807–11. Heineman HS, Chawla JK, Lofton WM. Misinformation from sputum cultures without microscopic examination. J Clin Microbiol 1977;6:518–27. Hudson VL, Wielinski CL, Regelmann WE. Prognostic implications of initial oropharyngeal bacterial flora in patients with cystic fibrosis diagnosed before the age of two years. J Pediatr 1993;122:854–60. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159–74. Medici TC, von Graevenitz A, Shang H, Bohni E, Wall M. Gram stain and culture of morning and 24 h sputum in the diagnosis of bacterial exacerbation of chronic bronchitis: a dogma disputed. Eur Respir J 1988;1:923–8. Miyashita N, Shimizu H, Ouchi K, Kawasaki K, Kawai Y, Obase Y, et al. Assessment of the usefulness of sputum Gram stain and culture for diagnosis of community-acquired pneumonia requiring hospitalization. Med Sci Monit 2008;14:171–6. Nair B, Stapp J, Stapp L, Buqni L, Van Dalfsen J, Burns JL. Utility of Gram staining for evaluation of the quality of cystic fibrosis sputum samples. J Clin Microbiol 2002;40:2791–4. Perez LR, Antunes AL, Bonfanti JW, Pinto JB, Roesch EW, Rodrigues D, et al. Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar. J Clin Microbiol 2012;50: 2506–8. Roche N, Kouassi B, Rabbat A, Mounedji A, Lorut C, Huchon G. Yield of sputum microbiological examination in patients hospitalized for exacerbations of chronic obstructive pulmonary disease with purulent sputum. Respiration 2007;74:19–25. Sadeghi E, Matlow A, MacLusky I, Karmali MA. Utility of Gram stain in evaluation of sputa from patients with cystic fibrosis. J Clin Microbiol 1994;32:54–8. Sharp SE. Cumitech 7B, Lower respiratory tract infections. In: Sharp SE, editor. Coordinating. Washington, DC: American Society for Microbiology; 2003. Theerthakarai R, El-Halees W, Ismail M, Solis RA, Khan MA. Nonvalue of the initial microbiological studies in the management of nonsevere community-acquired pneumonia. Chest 2001;119:181–4.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Evaluation of the Gram stain of sputa from cystic fibrosis patients to predict the presence of Staphylococcus aureus Leandro Reus Rodrigues Perez a,⁎, Cícero Armídio Gomes Dias a, b a b
Microbiology unit, Hospital Mãe de Deus, Porto Alegre – RS, 90880-480, Brazil Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 7 January 2013 Received in revised form 12 June 2013 Accepted 14 June 2013 Available online 23 July 2013
a b s t r a c t We evaluated the presence of Gram-positive cocci in cluster, seen in Gram-stained smears of cystic fibrosis (CF) sputa versus the microbiological culture for the prediction of the presence of S. aureus. Gram stain provided low accuracy (69.2%; odds ratio, 4.8; 95% confidence interval, 61.3–76.1) for predicting S. aureus in CF sputum. © 2013 Elsevier Inc. All rights reserved.
Keywords: Gram stain Cystic fibrosis Staphylococcus aureus
The Gram stain is the most commonly used tool in microbiology laboratories for evaluating morphology and arrangement of bacterial cells. Furthermore, the Gram stain allows a careful review of the clinical specimen with respect to its microscopic quality, mainly on clinical specimens that are necessarily obtained from a tract colonized by commensal microorganisms, such as sputa (Sharp, 2003). Although a specimen with high degree of difficulty for processing and interpretation, sputa have been largely used for analysis in patients with suspect of having chronic obstructive pulmonary disease (Roche et al., 2007), community-acquired pneumonia (Miyashita et al., 2008), and cystic fibrosis (CF) patients (Burns et al., 1998; Nair et al., 2002; Sadeghi et al., 1994). Cultures using selective media are recommended to obtain a higher yield of isolation of the main microorganisms involved with a respiratory disorder in CF patients—Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and Burkholderia cepacia (Cystic Fibrosis Foundation, 1994). However, cultures using selective media usually require a longer period of evaluation compared the conventional cultures, and for these cases, results of Gram stain examination could guide the antimicrobial choice for initial empirical treatment (Heineman et al., 1977). The objective of this study was to evaluate the presence of a particular morphotype, Gram-positive cocci in cluster (GPCC), seen in Gram-stained smears of sputa collected from CF patients versus the microbiological culture for the prediction of the presence of S. aureus. This study is a substudy in the evaluation of the use of a selective chromogenic agar to isolate methicillin-resistant S. aureus (MRSA) (Perez et al., 2012). Specimens collected from CF patients admitted at ⁎ Corresponding author. Tel.: +55-5193690440. E-mail address: [email protected] (L.R.R. Perez). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.06.022
Hospital de Clínicas de Porto Alegre, the main care center for CF patients in the south of Brazil, between January and April 2011, were included. Smears were prepared and stained by Gram stain according to standard procedure (Chapin and Murray, 1999). Quality of samples was inspected according to microscopic analysis and summarized in good quality (GQ) or poor quality (PQ) based upon presence of epithelial cells (b10 and ≥10, respectively) in low-power field (LPF). Additionally, sputa were cultured on conventional media (5% sheep blood agar and mannitol salt agar) and the MRSA ID (bioMérieux, Marcy l’Etoile, France), a chromogenic selective agar to allow the growth of S. aureus. For both smear preparation and culture plate inoculation, a purulent portion of sputum was selected. After 24 to 48 h of incubation at 35 °C, plates were examined. Results from microscopy and culture were obtained independently and carried out by double-blind analysis. Statistical analyses were carried out using SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA). Prevalence ratios, sensitivity, specificity, positive and negative predictive values, and odds ratio (OR) and 95% confidence intervals (CIs) were calculated. The measurement of observer agreement for categorical date (Kappa coefficient) was determined, and the results were classified according to previously described (Landis and Koch, 1977). A total of 146 sputa from CF patients were evaluated. Of these, 128 (87.7%; 95% CI, 81.3–92.1) samples presented a number of epithelial cells less than 10 per LPF and 18 (12.3%; 95% CI, 7.9–18.6) presented more than 10 epithelial cells per LPF, being considered sputa of GQ and PQ, respectively. GPCCs were observed in 77/128 (60.2%; 95% CI, 51.5–68.2) samples with GQ and, for these, 56 (72.7%; 95% CI, 61.9 to 81.4) cultures showed grown of S. aureus. Among samples with PQ, GPCC were present in 13/18 (72.2%; 95% CI, 49.1 to 87.5) and in 8/13 (61.5%;
100
L.R.R. Perez, C.A.G. Dias / Diagnostic Microbiology and Infectious Disease 77 (2013) 99–100
Table 1 Sensitivity, specificity, positive and negative predictive values, accuracy, and OR of Gram stain findings for determining the culture results presenting S. aureus in sputum specimens from CF patients Variable
Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Accuracy (%) Odds ratio a b c
Gram stain findings presenting Gram-positive cocci in clusters b10/LPFa (95% CI, range in %)
≥10/LPFb (95% CI, range in %)
Totalc (95% CI, range in %)
76.7 61.8 72.7 66.7 70.3 5.3
80 (49–94.3) 37.5 (13.7–69.4) 61.5 (35.5–82.3) 60 (23.1–88.2) 61.1 (38.6–79.7) 2.4 (2.9–19.8)
77.1 (67–84.8) 58.7 (46.4–70) 71.1 (61–79.4) 66.1 (53–77.1) 69.2 (61.3–76.1) 4.8 (2.3–9.8)
(65.8–84.9) (48.6–73.5) (61.9–81.4) (53–78) (61.9–77.5) (2.5–11.5)
N = 128; cases including only Gram stain presenting b10 epithelial cells per LPF. N = 18; cases including only Gram stain presenting 10 or more epithelial cells per LPF. N = 146; considering (a + b) situation.
95% CI, 35.5 to 82.3) S. aureus were isolated in culture. Sensitivity of microscopic examination was 77.1% (64/83), being 76.7% (56/73) and 80% (8/10) for GQ and PQ, respectively. Total agreements (accuracy) were of 90 (70.3% of observations) and 11 (61.1% of observations) for the group of samples of GQ and PQ, respectively. Overall, only 69.2% (101/146) accuracy was achieved. According to our results, the measurement of agreement between the presence of GPCC and the growth of S. aureus was classified as “fair” agreement (kappa = 0.36; 95% CI, 0.21–0.57). Moreover, OR for recovering S. aureus on culture when GPCC is visualized in GQ samples Gram stained was 2 times superior than PQ samples (OR = 5.3 versus OR = 2.4). Several studies had discussed the utility of microbiological findings, to predict lower respiratory tract infection, and the role of sputum Gram stain remains unclear. Theerthakarai et al. (2001) indicated that sputum Gram stain, for patients with non-severe community-acquired pneumonia without co-morbid factors, does not provide diagnostically useful information and does not help in guiding initial therapy. Garcia-Vázquez et al. (2004) found that Gram stain of sputum samples was useful in guiding microbiological diagnosis of community-acquired pneumonia in only 14.4% of the hospitalized patients evaluated in the study. For CF patients, Nair et al. (2002) showed that a subjective evaluation of sputum samples was superior to the Gram stain in identifying adequacy for culture. Besides this, these authors showed a poor correlation between culture results and Gram stain for gram-negative pathogens. Sadeghi et al. (1994) showed a positive predictive value of the Gram stain for growth from acceptable sputum samples of 86.3% for S. aureus, superior to the value obtained for us in this study (Table 1); however, they also showed a very poor sensitivity for microscopic examination. There is a number of potential explanations for this lack of association between the Gram stain and culture results. These include lack of homogeneity and differences in bacterial densities throughout the sample. Besides this, influence of previous antimicrobial therapy (a variable that was not controlled in this and in the studies of Sadeghi et al.) may influence results. Respiratory secretions of CF patients may be polymicrobial, making important the microscopic examination to recognize different morphotypes and to predict the presence of co-colonization, a condition related to lung function decline (Hudson et al., 1993). Another special issue in CF patients is the recommendation by some authors (Heineman et al., 1977; Medici et al., 1988) that even “inadequate” (≥10 epithelial cells in LPF) must be accepted for culture. Nair et al. (2002) showed that, in 90% of the samples that
would have been rejected by their quality score, CF pathogens were isolated on culture media. In counterpoint to this, in our study, a loss of specificity was noted when we include PQ samples (37.5% versus 61.8%). On the other hand, the low number of PQ samples (N = 18) included is a limitation of this study, and investigations including a larger number of samples with this characteristics are required. Our results indicate that microscopic examination presents low accuracy to predict the presence of S. aureus in sputum cultures of CF patients. References Burns JL, Emerson J, Stapp JR, Yim DL, Krzewinski J, Louden L, et al. Microbiology of sputum from patients at cystic fibrosis centers in the United States. Clin Infect Dis 1998;27:158–63. Chapin KC, Murray PR. Stains. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th edn. Washington, DC: American Society for Microbiology; 1999. p. 1677–9. Cystic Fibrosis Foundation. Microbiology and infectious disease in cystic fibrosis. Consensus Conference, Concepts in Care, vol. V. Consensus Conference. Bethesda, Md: Cystic Fibrosis Foundation; 1994. p. 1–26. Garcia-Vázquez E, Marcos MA, Mensa J, de Roux A, Puiq J, Font C, et al. Assessment of the usefulness of sputum culture for diagnosis of community-acquired pneumonia using the PORT predictive scoring system. Arch Intern Med 2004;164:1807–11. Heineman HS, Chawla JK, Lofton WM. Misinformation from sputum cultures without microscopic examination. J Clin Microbiol 1977;6:518–27. Hudson VL, Wielinski CL, Regelmann WE. Prognostic implications of initial oropharyngeal bacterial flora in patients with cystic fibrosis diagnosed before the age of two years. J Pediatr 1993;122:854–60. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159–74. Medici TC, von Graevenitz A, Shang H, Bohni E, Wall M. Gram stain and culture of morning and 24 h sputum in the diagnosis of bacterial exacerbation of chronic bronchitis: a dogma disputed. Eur Respir J 1988;1:923–8. Miyashita N, Shimizu H, Ouchi K, Kawasaki K, Kawai Y, Obase Y, et al. Assessment of the usefulness of sputum Gram stain and culture for diagnosis of community-acquired pneumonia requiring hospitalization. Med Sci Monit 2008;14:171–6. Nair B, Stapp J, Stapp L, Buqni L, Van Dalfsen J, Burns JL. Utility of Gram staining for evaluation of the quality of cystic fibrosis sputum samples. J Clin Microbiol 2002;40:2791–4. Perez LR, Antunes AL, Bonfanti JW, Pinto JB, Roesch EW, Rodrigues D, et al. Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar. J Clin Microbiol 2012;50: 2506–8. Roche N, Kouassi B, Rabbat A, Mounedji A, Lorut C, Huchon G. Yield of sputum microbiological examination in patients hospitalized for exacerbations of chronic obstructive pulmonary disease with purulent sputum. Respiration 2007;74:19–25. Sadeghi E, Matlow A, MacLusky I, Karmali MA. Utility of Gram stain in evaluation of sputa from patients with cystic fibrosis. J Clin Microbiol 1994;32:54–8. Sharp SE. Cumitech 7B, Lower respiratory tract infections. In: Sharp SE, editor. Coordinating. Washington, DC: American Society for Microbiology; 2003. Theerthakarai R, El-Halees W, Ismail M, Solis RA, Khan MA. Nonvalue of the initial microbiological studies in the management of nonsevere community-acquired pneumonia. Chest 2001;119:181–4.