Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

BMC Microbiology

Geneviève Héry-Arnaud¹  Christopher Payan¹  Gilles Rault⁴  Sophie Vallet¹  Sylvie Boisramé-Gastrin⁵  Stéphanie Gouriou⁵  Jeanne Hardy⁵  Sylvain Rosec²  Rozenn Le Berre³  Florence Le Gall¹ 

[1] Département de Bactériologie-Virologie, Hygiène Hospitalière et Parasitologie-Mycologie, CHRU Brest, Brest F-29200, France;INSERM-CIC 0502, CHRU Brest, Brest F-29200, France;Département de Médecine Interne et Pneumologie, CHRU Brest, Brest F-29200, France;Centre de Perharidy, CRCM, Roscoff F-29680, France;EA 3882-Laboratoire de Biodiversité et d’Ecologie Microbienne (LUBEM), SFR 148 ScInBioS, Faculté de Médecine, Université de Brest, Brest F-29200, France

关键词: Early detection;    qPCR;    Cystic fibrosis;    Pseudomonas aeruginosa;   

Others  :  1143571
DOI  :  10.1186/1471-2180-13-143

 received in 2013-01-16, accepted in 2013-06-13,  发布年份 2013

【 摘 要 】

Background

The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.

Methods

We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.

Results

Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.

Conclusion

Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

【 授权许可】

   
2013 Le Gall et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Ballmann M, Rabsch P, von der Hardt H: Long-term follow up of changes in FEV1 and treatment intensity during Pseudomonas aeruginosa colonisation in patients with cystic fibrosis. Thorax 1998, 53(9):732-737.
• [2]Ciofu O, Riis B, Pressler T, Poulsen HE, Hoiby N: Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation. Antimicrob Agents Chemother 2005, 49(6):2276-2282.
• [3]Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, Robertson CF, Grimwood K: Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis. J Pediatr 2001, 138(5):699-704.
• [4]Oliver A, Mena A: Bacterial hypermutation in cystic fibrosis, not only for antibiotic resistance. Clin Microbiol Infect 2010, 16(7):798-808.
• [5]Stuart B: Early eradication of pseudomonas aeruginosa in patients with cystic fibrosis. Paediatr Respi Rev 2010, 11(3):177-184.
• [6]Gibson RL, Burns JL, Ramsey BW: Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 2003, 168(8):918-951.
• [7]Hoiby N, Frederiksen B, Pressler T: Eradication of early Pseudomonas aeruginosa infection. J Cyst Fibros 2005, 4(Suppl 2):49-54.
• [8]Valerius NH, Koch C, Hoiby N: Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment. Lancet 1991, 338(8769):725-726.
• [9]Oliver A, Canton R, Campo P, Baquero F, Blazquez J: High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 2000, 288(5469):1251-1254.
• [10]Pierre M, Le Berre R, Tiesset H, Faure K, Guery B, Desseyn JL, Galabert C, Beghin L, Beermann C, Gottrand F, et al.: Kinetics of Pseudomonas aeruginosa virulence gene expression during chronic lung infection in the murine model. Med Mal Infect 2008, 38(6):318-323.
• [11]Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP: Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 2000, 407(6805):762-764.
• [12]Stewart PS, Franklin MJ: Physiological heterogeneity in biofilms. Nat Rev Microbiol 2008, 6(3):199-210.
• [13]O’May CY, Reid DW, Kirov SM: Anaerobic culture conditions favor biofilm-like phenotypes in Pseudomonas aeruginosa isolates from patients with cystic fibrosis. FEMS Immunol Med Microbiol 2006, 48(3):373-380.
• [14]Anuj SN, Whiley DM, Kidd TJ, Bell SC, Wainwright CE, Nissen MD, Sloots TP: Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes. Diagn Microbiol Infect Dis 2009, 63(2):127-131.
• [15]Kidd TJ, Ramsay KA, Hu H, Bye PT, Elkins MR, Grimwood K, Harbour C, Marks GB, Nissen MD, Robinson PJ, et al.: Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients. J Clin Microbiol 2009, 47(5):1503-1509.
• [16]Wellinghausen N, Kothe J, Wirths B, Sigge A, Poppert S: Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from patients with cystic fibrosis. J Clin Microbiol 2005, 43(8):4070-4075.
• [17]Spilker T, Coenye T, Vandamme P, LiPuma JJ: PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 2004, 42(5):2074-2079.
• [18]Ferroni A, Sermet-Gaudelus I, Abachin E, Quesnes G, Lenoir G, Berche P, Gaillard JL: Phenotypic and genotypic characteristics of non fermenting atypical strains recovered from cystic fibrosis patients. Pathol Biol (Paris) 2003, 51(7):405-411.
• [19]Qin X, Emerson J, Stapp J, Stapp L, Abe P, Burns JL: Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis. J Clin Microbiol 2003, 41(9):4312-4317.
• [20]Xu J, Moore JE, Murphy PG, Millar BC, Elborn JS: Early detection of Pseudomonas aeruginosa–comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF). Ann Clin Microbiol Antimicrob 2004, 3:21. BioMed Central Full Text
• [21]Deschaght P, Schelstraete P, Lopes dos Santos Santiago G, Van Simaey L, Haerynck F, Van Daele S, De Wachter E, Malfroot A, Lebecque P, Knoop C, et al.: Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients. BMC Microbiol 2010, 10:245. BioMed Central Full Text
• [22]Billard-Pomares T, Herwegh S, Wizla-Derambure N, Turck D, Courcol R, Husson MO: Application of quantitative PCR to the diagnosis and monitoring of Pseudomonas aeruginosa colonization in 5-18-year-old cystic fibrosis patients. J Med Microbiol 2011, 60(Pt 2):157-161.
• [23]Logan C, Habington A, Lennon G, Cronin F, O’Sullivan N: Evaluation of the efficacy of real-time polymerase chain reaction for the routine early detection of Pseudomonas aeruginosa in cystic fibrosis sputum and throat swab specimens. Diagn Microbiol Infect Dis 2010, 68(4):358-365.
• [24]McCulloch E, Lucas C, Ramage G, Williams C: Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population. J Cyst Fibros 2011, 10(1):21-24.
• [25]Hoboth C, Hoffmann R, Eichner A, Henke C, Schmoldt S, Imhof A, Heesemann J, Hogardt M: Dynamics of adaptive microevolution of hypermutable Pseudomonas aeruginosa during chronic pulmonary infection in patients with cystic fibrosis. J Infect Dis 2009, 200(1):118-130.
• [26]Mena A, Smith EE, Burns JL, Speert DP, Moskowitz SM, Perez JL, Oliver A: Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation. J Bacteriol 2008, 190(24):7910-7917.
• [27]Finnan S, Morrissey JP, O’Gara F, Boyd EF: Genome diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients and the hospital environment. J Clin Microbiol 2004, 42(12):5783-5792.
• [28]Mathee K, Narasimhan G, Valdes C, Qiu X, Matewish JM, Koehrsen M, Rokas A, Yandava CN, Engels R, Zeng E, et al.: Dynamics of Pseudomonas aeruginosa genome evolution. Proc Natl Acad Sci USA 2008, 105(8):3100-3105.
• [29]Whiley DM, Lambert SB, Bialasiewicz S, Goire N, Nissen MD, Sloots TP: False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation? Crit Rev Microbiol 2008, 34(2):71-76.
• [30]Joly B, Pierre M, Auvin S, Colin F, Gottrand F, Guery B, Husson MO: Relative expression of Pseudomonas aeruginosa virulence genes analyzed by a real time RT-PCR method during lung infection in rats. FEMS Microbiol Lett 2005, 243(1):271-278.
• [31]Moissenet D, Bingen E, Arlet G, Vu-Thien H: Use of 16S rRNA gene sequencing for identification of “Pseudomonas-like” isolates from sputum of patients with cystic fibrosis. Pathol Biol (Paris) 2005, 53(8-9):500-502.
• [32]Lee TW, Brownlee KG, Conway SP, Denton M, Littlewood JM: Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros 2003, 2(1):29-34.
• [33]Degand N, Carbonnelle E, Dauphin B, Beretti JL, Le Bourgeois M, Sermet-Gaudelus I, Segonds C, Berche P, Nassif X, Ferroni A: Matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis patients. J Clin Microbiol 2008, 46(10):3361-3367.
• [34]Desai AP, Stanley T, Atuan M, McKey J, Lipuma JJ, Rogers B, Jerris R: Use of matrix assisted laser desorption ionisation-time of flight mass spectrometry in a paediatric clinical laboratory for identification of bacteria commonly isolated from cystic fibrosis patients. J Clin Pathol 2012, 65(9):835-838.
• [35]Deschaght P, Van Daele S, De Baets F, Vaneechoutte M: PCR and the detection of Pseudomonas aeruginosa in respiratory samples of CF patients. A literature review. J Cyst Fibros 2011, 10(5):293-297.
• [36]Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, Sindelka R, Sjoback R, Sjogreen B, Strombom L, et al.: The real-time polymerase chain reaction. Mol Aspects Med 2006, 27(2–3):95-125.
• [37]Anonyme: Recommandations pour l’analyse bactériologique des prélèvements d’expectoration chez les patients atteints de mucoviscidose. In REMIC - Référentiel en microbiologie médicale. 2nd edition. Edited by Société Française de Microbiologie. Paris; 2010:99-104.
• [38]Davison J: Genetic exchange between bacteria in the environment. Plasmid 1999, 42(2):73-91.
• [39]Aparna MS, Yadav S: Biofilms: microbes and disease. Braz J Infect Dis 2008, 12(6):526-530.
• [40]Masters CI, Shallcross JA, Mackey BM: Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction. J Appl Bacteriol 1994, 77(1):73-79.
• [41]Deschaght P, De Baere T, Van Simaey L, Van Daele S, De Baets F, De Vos D, Pirnay JP, Vaneechoutte M: Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients. BMC Microbiol 2009, 9:244. BioMed Central Full Text