Abstract
Respiratory specimens from cystic fibrosis (CF) patients challenge microbiological laboratories with their complexity of pathogens and atypical variants. We evaluated the diagnostic procedures in German laboratories by use of a questionnaire. Although most laboratories followed guidelines, some of them served only a small number of patients, while others did not use the recommended selective agars to culture the particular CF-relevant species.
TEXT
Cystic fibrosis (CF) patients suffer from chronic recurrent airway infections, which cause phenotypical changes (small-colony variants [SCVs]; mucoid phenotypes) of typical CF pathogens and the selection of multiresistant and rare bacteria such as Burkholderia cepacia complex (BCC) or nontuberculous mycobacteria due to repeated antimicrobial therapies and adaptation of the bacteria to this particular niche. To provide optimal microbiological diagnostic special culture conditions, knowledge and experience about the typical CF pathogens and their phenotypical changes are required.
(Part of this work was presented at the Annual Meeting of the German Society for Hygiene and Microbiology, 2012, Hamburg, Germany [CFP01].)
The status of the microbiological diagnostic procedures of laboratories serving CF centers, which participate in the German “Quality assurance cystic fibrosis project” (1), was evaluated by use of a questionnaire. First, contributing CF centers were asked to provide the address of their microbiological laboratories. Second, a questionnaire based on the German microbiological infectious quality standards for airway infections in CF (Mikrobiologisch-infektiologische Qualitätsstandards [MiQ]) (2) which were published by the German Society for Hygiene and Microbiology (3) and on the protocols provided by the consultant laboratories for CF microbiology (Institute of Microbiology, Medical School Hannover, Hannover, Germany; Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Munich, Germany) was sent to the laboratories. A translation of the main recommendations by the MiQ, which are comparable to the guidelines of the UK Cystic Fibrosis Trust (4) is provided in the supplemental material (see Table S1 in the supplemental material).
Seventy-eight of 83 (94%) CF centers provided the address of the microbiological laboratory, and all these laboratories were contacted. Forty-five of 61 (74%) laboratories responsible for the microbiological testing of 4,664 CF patients (56% of CF patients in Germany in 2010) responded to the questionnaire. Eight laboratories (18%) provide microbiological service for less than 25 CF patients. Eighty-nine percent of the laboratories reported that they used guidelines, and most of them used the MiQ (88%).
All laboratories used media to culture the most prevalent CF pathogens such as Haemophilus influenzae, Staphylococcus aureus, and Pseudomonas aeruginosa, supportively and/or selectively (Table 1) (2). Some reported the use of more than one medium. Thirteen laboratories used mannitol salt agar as a selective agar for S. aureus, and eight laboratories employed chromogenic agar (Table 1). Although thymidine auxotroph S. aureus has been reported for the first time to be cultured on mannitol salt agar (5), the study of Kipp et al. (6) showed that the newer chromogenic agar media facilitate a selective and more sensitive growth of S. aureus without further confirmatory testing or subculture for susceptibility testing as well as the identification of S. aureus SCVs (2, 6–8). However, in this study (6), the authors did use only mannitol salt agar provided by one company. Therefore, it is not clear whether the use of mannitol salt agar of other companies may yield better results for SCVs (6). It is important to use special culture conditions for the isolation of SCVs, because these phenotypes are easily overgrown on standard agar by cocolonizing bacteria due to their slow growth and their small colony size and are therefore difficult to isolate. Furthermore, it is of special interest to improve culture conditions for these phenotypes, because they are associated with chronic or persistent infections and with decreased pulmonary function and are more resistant to antibiotics than the normal phenotype (9–14). Whereas all laboratories reported that they informed the clinic about pathogens with mucoid phenotypes, the culture of SCVs was reported by only 70% of investigators.
TABLE 1.
Culture medium | No. of laboratories reporting use of the indicated culture medium (%) |
---|---|
Columbia blood agar | 41 (91) |
Chocolate agara | 45 (100) |
MacConkey agar | 32 (71) |
Selective media for: | |
S. aureusb | 31 (69) |
P. aeruginosac | 19 (42) |
BCCd | 41 (91) |
Chocolate agar alone (n = 18) and chocolate agar with bacitracin (n = 18), oleandomycin (n = 8), or vancomycin (n = 1).
Columbia blood agar with nalidixic acid (n = 1) or aztreonam (n = 1), colistin-nalidixic acid agar (n = 7), mannitol salt agar (n = 13), CHROMagar Staph. aureus (n = 5), S. aureus ID agar (n = 3), and brain heart infusion NaCl agar (n = 1).
Pseudomonas isolation agar (n = 3), cetrimid agar (n = 15), and cetrimid broth (n = 1).
Only 91% (41/45) of laboratories indicated the application of a selective agar to culture BCC (Table 1) (2). In contrast, Zhou et al. reported the use of such an agar in the United States in 99% (136/138) of laboratories (15). Since four of the German laboratories did not use a selective agar, which is recommended by the MiQ as well as by the United Kingdom guidelines as one of the basic culture media, it might be the case that these laboratories will miss BCC isolates (2, 4). As shown in the study of Gilligan et al., in cultures of sputum samples from 169 CF patients, BCC isolates were cultured on BCC selective agar from 35 patients, but when they were cultured on MacConkey agar, BCC isolates were found for only 21 patients, thereby missing 40% of BCC isolates (16). Because of the slow growth of some BCC strains, they can easily be overgrown by other microorganisms, thereby making their isolation very difficult (17). Shreve et al. showed that the prevalence of typical CF pathogens and their epidemiology were influenced by the frequency of the microbiological testing, by the employment of selective media for S. aureus, P. aeruginosa, and BCC as well as adequate culture conditions (18). The correct isolation of pathogens is important not only to treat CF patients appropriately but also to allow conclusions about the impact of special bacteria on disease progression (18).
In the case of culture of BCC isolates, only 60% of laboratories contacted their CF center directly to initiate special hygiene measures. Considering that patient-to-patient transmission is reported for BCC isolates (19), it becomes clear how important it is to detect this pathogen correctly and to provide rapid information of positive cultures (20). Moreover, infection with BCC strains is associated with higher mortality and high resistance to antibiotics (21–23).
All laboratories reported the identification of Gram-negative nonfermenting bacteria, including P. aeruginosa and BCC by using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (n = 29) and/or API 20 NE (n = 26). Some laboratories indicated the use of multiple systems. In general, the laboratories used MALDI-TOF mass spectrometry (n = 28), an API system (n = 28), and/or Vitek 2 (n = 24) for bacterial identification.
Although 93% of the laboratories indicated that they performed confirmatory tests for special pathogens, only 56% used such tests to confirm BCC (Table 2). Seven laboratories specified that they used sequencing or PCR of the recA gene as the method of choice (2, 4). It has been shown that phylogenetic analysis of recA can be used to confirm BCC and that PCR-restriction fragment length polymorphism (RFLP) analysis of recA is superior to that of 16S rRNA gene with regard to discrimination of BCC species (20, 24).
TABLE 2.
Pathogen | No. of laboratories using the following confirmatory testa: |
|||
---|---|---|---|---|
PCR | 16S sequencing | Probe | Others | |
MRSAb (n = 36) | 24 | 0 | 1 | 17 |
BCC (n = 25) | 9 | 18 | 0 | 10 |
Some laboratories reported the use of more than one confirmatory test, 7 for MRSA and 8 for BCC.
MRSA, methicillin-resistant S. aureus.
For susceptibility testing, 89% of laboratories reported that they utilized more than one method. Agar diffusion (n = 34) and/or Etest (n = 31) were employed most frequently, followed by Vitek 2 (n = 27). In the EuroCareCF quality assessment (25), most laboratories used disc diffusion and/or Etest without any major errors. Earlier studies showed that automated systems do not always produce reliable results for susceptibility testing of P. aeruginosa and other nonfermenting Gram-negative bacteria, especially if adapted phenotypes, such as mucoid isolates or SCVs, were cultured and are therefore not recommended (26, 27). There were efforts made by Otto-Karg et al. to improve the automated susceptibility testing of Gram-negative nonfermenters with Vitek 2, providing good results for bacteria, which were clearly categorized resistant or susceptible (28). However, the results were poorer for strains with MICs near the breakpoints (28).
To conclude, most, but not all, consulted laboratories in Germany are using quality standards specifically elaborated for the microbiological diagnostic of CF respiratory specimens (2). In our questionnaire, we identified a lack of use of selective media especially for such important pathogens as BCC and special phenotypes such as S. aureus SCVs. To overcome uncertainties concerning culture media, including selective and new media and their specific culture conditions, further instructions for microbiological laboratories serving CF centers are necessary. Moreover, the culture of CF specimens should be performed by laboratories serving a certain number of patients to be able to provide special culture methods and experience in identifying unusual phenotypes and species.
Supplementary Material
ACKNOWLEDGMENTS
We thank all CF centers for revealing the respective laboratories and all laboratories for answering the questionnaire and providing information about their microbiological diagnostic procedures.
Footnotes
Published ahead of print 3 January 2014
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02866-13.
REFERENCES
- 1.Sens B, Stern M. 2011. Berichtsband Qualitätssicherung Mukoviszidose 2010 Hippocampus, Bad Honnef, Germany [Google Scholar]
- 2.Hogardt M, Häußler S, Balke B, Kahl BC, Schmoldt S, Leitritz L, Jäger G, Kappler M, Suerbaum S, Heesemann J. 2006. Mikrobiologisch-infektiologische Qualitätsstandards (MiQ). Atemwegsinfektionen bei Mukoviszidose; Urban & Fischer, Munich, Germany [Google Scholar]
- 3.Balke B, Schmoldt S, Häußler S, Suerbaum S, Heesemann J, Hogardt M. 2008. A German external quality survey of diagnostic microbiology of respiratory tract infections in patients with cystic fibrosis. J. Cyst. Fibros. 7:7–14. 10.1016/j.jcf.2007.02.007 [DOI] [PubMed] [Google Scholar]
- 4.Denton M, Doherty C, Foweraker J, Govan J, Hall M, Isalska B, Jones A, Kenna D, Larsen A, Pitt T, Wareham D. 2010. Laboratory standards for processing microbiological samples from people with cystic fibrosis. Report of the UK Cystic Fibrosis Trust Microbiology Laboratory Standards Working Group; Cystic Fibrosis Trust, Kent, United Kingdom [Google Scholar]
- 5.Gilligan PH, Gage PA, Welch DF, Muszynski MJ, Wait KR. 1987. Prevalence of thymidine-dependent Staphylococcus aureus in patients with cystic fibrosis. J. Clin. Microbiol. 25:1258–1261 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kipp F, Kahl BC, Becker K, Baron EJ, Proctor RA, Peters G, von Eiff C. 2005. Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants. J. Clin. Microbiol. 43:1956–1959. 10.1128/JCM.43.4.1956-1959.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Gaillot O, Wetsch M, Fortineau N, Berche P. 2000. Evaluation of CHROMagar Staph. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. J. Clin. Microbiol. 38:1587–1591 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Perry JD, Rennison C, Butterworth LA, Hopley ALJ, Gould FK. 2003. Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus. J. Clin. Microbiol. 41:5695–5698. 10.1128/JCM.41.12.5695-5698.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Besier S, Smaczny C, von Mallinckrodt C, Krahl A, Ackermann H, Brade V, Wichelhaus TA. 2007. Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J. Clin. Microbiol. 45:168–172. 10.1128/JCM.01510-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kahl BC, Herrmann M, Everding AS, Koch HG, Becker K, Harms E, Proctor RA, Peters G. 1998. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J. Infect. Dis. 177:1023–1029. 10.1086/515238 [DOI] [PubMed] [Google Scholar]
- 11.Kahl BC, Duebbers A, Lubritz G, Haeberle J, Koch HG, Ritzerfeld B, Reilly M, Harms E, Proctor RA, Herrmann M, Peters G. 2003. Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study. J. Clin. Microbiol. 41:4424–4427. 10.1128/JCM.41.9.4424-4427.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Häußler S, Tümmler B, Weißbrodt H, Rohde M, Steinmetz I. 1999. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin. Infect. Dis. 29:621–625. 10.1086/598644 [DOI] [PubMed] [Google Scholar]
- 13.Schneider M, Mühlemann K, Droz S, Couzinet S, Casaulta C, Zimmerli S. 2008. Clinical characteristics associated with isolation of small-colony variants of Staphylococcus aureus and Pseudomonas aeruginosa from respiratory secretions of patients with cystic fibrosis. J. Clin. Microbiol. 46:1832–1834. 10.1128/JCM.00361-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wolter DJ, Emerson JC, McNamara S, Buccat AM, Qin X, Cochrane E, Houston LS, Rogers GB, Marsh P, Prehar K, Pope CE, Blackledge M, Déziel E, Bruce KD, Ramsey BW, Gibson RL, Burns JL, Hoffman LR. 2013. Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis. Clin. Infect. Dis. 57:384–391. 10.1093/cid/cit270 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Zhou J, Garber E, Desai M, Saiman L. 2006. Compliance of clinical microbiology laboratories in the United States with current recommendations for processing respiratory tract specimens from patients with cystic fibrosis. J. Clin. Microbiol. 44:1547–1549. 10.1128/JCM.44.4.1547-1549.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Gilligan PH, Gage PA, Bradshaw LM, Schidlow DV, DeCicco BT. 1985. Isolation medium for the recovery of Pseudomonas cepacia from respiratory secretions of patients with cystic fibrosis. J. Clin. Microbiol. 22:5–8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Tablan OC, Carson LA, Cusick LB, Bland LA, Martone WJ, Jarvis WR. 1987. Laboratory proficiency test results on use of selective media for isolating Pseudomonas cepacia from simulated sputum specimens of patients with cystic fibrosis. J. Clin. Microbiol. 25:485–487 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Shreve MR, Butler S, Kaplowitz HJ, Rabin HR, Stokes D, Light M, Regelmann WE. 1999. Impact of microbiology practice on cumulative prevalence of respiratory tract bacteria in patients with cystic fibrosis. J. Clin. Microbiol. 37:753–757 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Saiman L, Siegel J. 2004. Infection control in cystic fibrosis. Clin. Microbiol. Rev. 17:57–71. 10.1128/CMR.17.1.57-71.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Mahenthiralingam E, Baldwin A, Dowson CG. 2008. Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J. Appl. Microbiol. 104:1539–1551. 10.1111/j.1365-2672.2007.03706.x [DOI] [PubMed] [Google Scholar]
- 21.Isles A, Maclusky I, Corey M, Gold R, Prober C, Fleming P, Levison H. 1984. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J. Pediatr. 104:206–210. 10.1016/S0022-3476(84)80993-2 [DOI] [PubMed] [Google Scholar]
- 22.Frangolias DD, Mahenthiralingam E, Rae S, Raboud JM, Davidson AG, Wittmann R, Wilcox PG. 1999. Burkholderia cepacia in cystic fibrosis. Variable disease course. Am. J. Respir. Crit. Care Med. 160:1572–1577. 10.1164/ajrccm.160.5.9805046 [DOI] [PubMed] [Google Scholar]
- 23.Fauroux B, Hart N, Belfar S, Boulé M, Tillous-Borde I, Bonnet D, Bingen E, Clément A. 2004. Burkholderia cepacia is associated with pulmonary hypertension and increased mortality among cystic fibrosis patients. J. Clin. Microbiol. 42:5537–5541. 10.1128/JCM.42.12.5537-5541.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Mahenthiralingam E, Bischof J, Byrne SK, Radomski C, Davies JE, Av-Gay Y, Vandamme P. 2000. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J. Clin. Microbiol. 38:3165–3173 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Hogardt M, Ulrich J, Riehn-Kopp H, Tümmler B. 2009. EuroCareCF quality assessment of diagnostic microbiology of cystic fibrosis isolates. J. Clin. Microbiol. 47:3435–3438. 10.1128/JCM.01182-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Burns JL, Saiman L, Whittier S, Krzewinski J, Liu Z, Larone D, Marshall SA, Jones RN. 2001. Comparison of two commercial systems (Vitek and MicroScan-WalkAway) for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients. Diagn. Microbiol. Infect. Dis. 39:257–260. 10.1016/S0732-8893(01)00234-6 [DOI] [PubMed] [Google Scholar]
- 27.Sader HS, Fritsche TR, Jones RN. 2006. Accuracy of three automated systems (MicroScan WalkAway, VITEK, and VITEK 2) for susceptibility testing of Pseudomonas aeruginosa against five broad-spectrum beta-lactam agents. J. Clin. Microbiol. 44:1101–1104. 10.1128/JCM.44.3.1101-1104.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Otto-Karg I, Jandl S, Müller T, Stirzel B, Frosch M, Hebestreit H, Abele-Horn M. 2009. Validation of Vitek 2 nonfermenting Gram-negative cards and Vitek 2 version 4.02 software for identification and antimicrobial susceptibility testing of nonfermenting Gram-negative rods from patients with cystic fibrosis. J. Clin. Microbiol. 47:3283–3288. 10.1128/JCM.00505-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Henry DA, Campbell ME, LiPuma JJ, Speert DP. 1997. Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J. Clin. Microbiol. 35:614–619 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Becton Dickinson. 2003. BDTM Cepacia Medium • BDTM OFPBL Agar. Instructions for use–ready-to-use plated media. PA-254481.02. BD Diagnostic Systems, Heidelberg, Germany: https://www.bd.com/europe/regulatory/Assets/IFU/HB/CE/PA/PA-254481.pdf [Google Scholar]
- 31.Oxoid Limited 2013. Burkholderia cepacia selective supplement. Code SR0189. Oxoid Limited, Thermo Fisher Scientific Inc., Basingstoke, United Kingdom: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0995&org=16&c=UK&lang=EN [Google Scholar]
- 32.Welch DF, Muszynski MJ, Pai CH, Marcon MJ, Hribar MM, Gilligan PH, Matsen JM, Ahlin PA, Hilman BC, Chartrand SA. 1987. Selective and differential medium for recovery of Pseudomonas cepacia from the respiratory tracts of patients with cystic fibrosis. J. Clin. Microbiol. 25:1730–1734 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.