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* From the Department of Pediatrics and Communicable Diseases (Mss. McMenamin and Zaccone, and Dr. LiPuma), University of Michigan Medical School, Ann Arbor, MI; and the Laboratorium voor Microbiologie (Drs. Coenye and Vandamme), Universiteit Gent, Ghent, Belgium.
Correspondence to: John J. LiPuma, MD, Department of Pediatrics, University of Michigan, 1150 W. Medical Center Drive, 8323 MSRB III, Box 0646, Ann Arbor, MI 48109-0646; e-mail: jlipuma{at}umich.edu
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Study objectives: To determine the rate of misidentification of B cepacia recently recovered from CF sputum culture of persons receiving care in US treatment centers.
Design: Bacterial isolates cultured from CF sputum and putatively identified as B cepacia or other related nonlactose-fermenting Gram-negative species were referred from participating treatment centers. Isolates underwent polyphasic analyses employing phenotypic (selective media and biochemical testing) and genotypic (polymerase chain reaction) assays to determine species identification. Taxonomic evaluations were performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified-fragment length polymorphism analysis.
Measurements and results: A total of 1,051 isolates recovered from 608 patients were received from 115 treatment centers in 91 US cities. Among the isolates identified as B cepacia by referring laboratories, 11% could not be confirmed as B cepacia by polyphasic analyses. In addition, 36% of isolates not specifically identified by the referring laboratory or identified as a species other than B cepacia were, in fact, found to be members of the B cepacia complex.
Conclusions: Rates of misidentification of B cepacia remain unacceptably high among US treatment centers. These data suggest the need for increased awareness of this problem among CF centers and their affiliated laboratories, better adherence to recommended protocols for evaluation of CF sputum, and greater use of reference laboratories equipped to provide advanced analyses.
Key Words: acquisition • Burkholderia cepacia • colonization • cystic fibrosis • identification
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The taxonomic complexity of B cepacia also contributes to unreliable identification. At least six distinct species of closely related bacteria comprise what is now referred to as the B cepacia complex.5 In addition, CF sputum isolates that defy identification based on conventional phenotypic analyses may represent novel bacterial taxa, such as the recently named genus, Pandoraea.6
Since the spring of 1997, the CF Foundation Burkholderia cepacia Research Laboratory and Repository has accepted CF sputum isolates for analysis. A polyphasic approach that employs both phenotypic and genotypic analyses has been used to confirm species identification. The results of these analyses demonstrate that the rate of misidentification of B cepacia and related bacteria remains high among US treatment centers.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 1,051 isolates cultured from 608 persons were referred from 115 CF treatment centers (108 affiliated microbiology laboratories) in 91 cities in the United States. Ninety-four percent of isolates were recovered from patients between June 1997 and March 1999. Isolates were assigned to one of two groups. The first group consisted of 770 isolates presumptively identified as B cepacia by the referring laboratory. This group also included isolates designated as "? B cepacia" or "possible B cepacia." The second group was comprised of 281 isolates that were either identified by the referring laboratory as another nonfermenting Gram-negative species, or were not specifically identified to the species level. This group included 40 isolates designated as "Burkholderia species" or "possible Burkholderia species."
Phenotypic Analysis
Isolates were recovered from agar slant or transport media and
subcultured onto both Mueller Hinton (MH) agar and B
cepacia-selective agar (BCSA), prepared as described
previously.7
Growth on both media was recorded after
incubation at 34°C in an ambient atmosphere for at least 48 h.
Isolates were stored in sterile skim milk at - 80°C.
For conventional biochemical testing, bacteria from either a fresh (24 to 48 h) MH agar plate culture or from frozen stock that had been subcultured twice onto MH agar were inoculated into the test reagent. Prior to inoculation, MH plates were inspected for purity, and oxidase testing of each isolate was performed with 1% tetramethyl p-phenylenediamine dihydrochloride. Lysine decarboxylase, o-nitrophenyl-ß-D-galactoside (ONPG), and oxidation-fermentation sugars (sucrose and lactose) were obtained from Remel (Lenexa, KS). Biochemical tests were incubated at 37°C in an ambient atmosphere, and were examined daily for 7 days, except ONPG, which was examined at 24 h and at 48 h. Phenotypic identification of B cepacia complex bacteria was based on weakly positive oxidase reaction, positive ONPG and lysine decarboxylase reactions, and acid production from sucrose and lactose.8 The exception was Burkholderia multivorans (B cepacia complex genomovar II), which was identified by a variable lysine decarboxylase reaction and by failure to produce acid from sucrose.9
Commercial identification systems were used as an adjunct to identify species (eg, Burkholderia gladioli and Stenotrophomonas maltophilia) not specifically identified by conventional biochemical and polymerase chain reaction (PCR) testing (below). The systems were used according to the recommendations of the manufacturers, and included RapID NF Plus (Innovative Diagnostic Systems; Norcross, GA) and Uni-N/F and N/F Screen (Remel).
Genotypic Analysis
For PCR testing, genomic DNA was prepared from the initial
isolate from each patient and tested by using a series of primer pairs
as described.10
These primers target ribosomal RNA
gene sequences and include pairs specific for the genera
Burkholderia/Ralstonia, as well as pairs specific for species within
the B cepacia complex. A primer pair that amplifies a
conserved 313-base pair 16S ribosomal RNA gene segment from all
bacteria11
was used as a positive control; negative
control PCRs consisted of reactions with all mixture components except
template DNA. For testing of subsequent isolates from an individual
patient in whom a prior isolate had been confirmed as B
cepacia complex, bacteria were lysed by boiling (10 min at
100°C) in sterile ultraviolet-irradiated water and used as template
in species-specific PCR.
Taxonomic Analysis
Isolates for which species identification remained equivocal
after the above analyses underwent additional taxonomic evaluation
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
whole-cell proteins, as well as amplified-fragment length polymorphism
(AFLP) fingerprinting, as described elsewhere.5
12
In
brief, after an incubation period of 48 h, whole-cell protein
extracts were prepared and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed. The densitometric analysis,
normalization and interpolation of the protein profiles, and numerical
analysis were performed by using the GelCompar software package version
4.2 (Applied Maths; Kortrijk, Belgium). For AFLP fingerprinting, DNA
was prepared as described by Pitcher et al.13
The
preparation of template DNA, amplification by PCR, separation of the
fragments (using an ABI Prism 377 automated DNA sequencer; PE
Biosystems; Foster City, CA), and numerical analysis were
performed as described previously.12
In previous studies,
whole-cell protein and AFLP analysis have provided excellent taxonomic
resolution, and the results obtained are generally in agreement with
DNA-DNA hybridization.14
Species Identification
Initial bacterial isolates from an individual patient were
identified as B cepacia complex if they grew on BCSA, fit
the above criteria for biochemical reactivity, and were positive in
both genus- and species-specific PCRs. Subsequent isolates from
patients in whom a previous isolate was confirmed as B
cepacia complex, were identified as B cepacia complex
based on growth on BCSA, weakly positive oxidase test, and positive
species-specific PCR test. All other species were identified by use of
a commercial identification system and/or taxonomic analysis as above.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overall, 189 bacterial isolates from 149 patients were misidentified. In 74 patients, a sputum culture isolate was mistakenly identified as B cepacia, when in fact it was not a member of the B cepacia complex. In 77 patients, the presence of B cepacia in sputum was missed by the evaluation of the referring laboratory. (In two patients, both types of misidentification occurred.)
Fifty-five of the 108 participating clinical microbiology laboratories (51%), submitted at least one isolate that was misidentified. Participating laboratories each specified one of seven different commercial systems as their primary method of identification, and only 35 laboratories reported the use of conventional biochemical tests to augment commercial test systems.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current study expands these observations with a comprehensive analysis of a large number of CF sputum isolates recently obtained from geographically diverse laboratories in the United States. We demonstrate that misidentification occurs in two ways. Many isolates identified as B cepacia are, in reality, other species. Conversely, many B cepacia isolates are not identified as such and are thus "missed" by routine laboratory testing.
We found that among isolates submitted for analysis, > 1 in 10 identified as B cepacia by referring laboratories are, in fact, not members of the B cepacia complex. Most of these isolates (70%) were identified as other related nonfermenting Gram-negative species that also colonize the CF respiratory tract; among these, B gladioli was most frequently misidentified as B cepacia. A significant proportion (30%) of isolates misidentified as B cepacia by referring laboratories could not definitively be placed in a known bacterial species, although taxonomic analysis confirmed that they were not members of the B cepacia complex.
Among the group of isolates identified by referring laboratories as a species other than B cepacia (or not identified to the species level), our analysis revealed a considerable proportion (36%) to be B cepacia complex species. By design, we also included in this group isolates for which the analysis of the referring laboratory indicated the genus Burkholderia, but did not identify a specific species. These isolates, in fact, accounted for many of the missed B cepacia in this study. However, a greater proportion (nearly one half) of the isolates later identified as B cepacia complex had been submitted to us as "unknown" or "nonfermenting Gram negative."
It is likely that the rates of misidentification detected in this study are in part attributable to selection bias; although referring laboratories were invited to submit all Burkholderia recovered from CF sputum, some may have been more likely to submit difficult-to-identify isolates. Nevertheless, it seems apparent that B cepacia and related bacteria are frequently misidentified. There are likely several reasons for this. Recent work indicates that clinical microbiology laboratories vary in the degree to which they adhere to recommended protocols for evaluation of CF sputum.4 In the current study, only one third of laboratories reported use of conventional biochemical tests to augment commercial test systems.
Microbiology laboratories also vary with respect to the types of selective growth media and commercial systems used for bacterial identification. The selective media (oxidation-fermentation polymyxin bacitracin lactose agar, and Pseudomonas cepacia agar) used by many laboratories support growth of species other than B cepacia. Henry et al17 recently demonstrated that BCSA, the selective media used in our study, is superior to other media in suppressing non-Burkholderia species while supporting growth of B cepacia. At least seven different commercial systems were used as the primary identification method by laboratories participating in this study. It is clear from our work (data not shown) that not all systems perform equally in identification of B cepacia complex and related bacteria. Kiska et al18 and van Pelt et al19 found considerable differences in the accuracy of commonly used commercial identification systems and recommend the use of reference laboratories and PCR-based analysis to confirm identity of Burkholderia species.
The complex taxonomy of B cepacia and related species that colonize the CF respiratory tract also contributes to difficulties with accurate identification. Certain species of the B cepacia complex have phenotypes that would be considered atypical based on conventional parameters for "B cepacia."8 9 B multivorans (B cepacia genomovar II) in particular may be misidentified, owing to its failure to oxidize sucrose and its variable lysine decarboxylase activity. Strains of some other species, most notably B gladioli, may be easily confused with B cepacia, as this study demonstrates. The difficulty in distinguishing these species is also illustrated by a study reporting apparent interpatient transmission of B gladioli.20 Further analysis by us (data not shown) and others21 indicates that the isolates described are actually B cepacia.
Many bacteria that colonize the CF pulmonary tract defy definitive species assignment. Our study identified many isolates that could not be placed in a known bacterial species. Several of these share phenotypic and genotypic features and are likely to represent novel species. In fact, comprehensive taxonomic analysis, including DNA-DNA hybridization studies, of such isolates has recently identified another species (genomovar VI) within the B cepacia complex,22 as well as a new bacterial genus designated Pandoraea.6 Further taxonomic study will most likely define additional species with the capacity to colonize the CF respiratory tract. The clinical significance of these species is unknown and will require ongoing investigation.
In summary, our study demonstrates relatively high rates of misidentification of B cepacia among CF treatment centers in the United States. Such diagnostic uncertainty is widespread; one half of the laboratories participating in this study submitted at least one isolate that had been misidentified. The lack of uniform methods of identification and the taxonomic diversity of B cepacia complex and related species contributes to this problem. These observations suggest the need for increased awareness of this problem among CF centers and their affiliated laboratories, better adherence to recommended protocols for evaluation of CF sputum, and greater use of reference laboratories equipped to provide more complete analyses, including genotypic methods that are not yet commercially available.
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Acknowledgements |
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Footnotes |
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This work was supported by a grant (to Dr. LiPuma) from the Cystic Fibrosis Foundation (United States). Dr. Vandamme is indebted to the Fund for Scientific Research-Flanders (Belgium) for a position as postdoctoral fellow. Dr. Coenye acknowledges the support received from the Vlaams Instituut voor Bevordering van Wetenschappelijk-technologisch Onderzoek in de Industrie (Belgium) in the form of a bursary for advanced study. Drs. Vandamme and Coenye acknowledge the financial support by the Cystic Fibrosis Trust (United Kingdom; grant RS15).
Received for publication October 1, 1999. Accepted for publication January 12, 2000.
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