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Journal of Clinical Microbiology, January 2000, p. 32-39, Vol. 38, No. 1
Departments of Medical
Microbiology,1 Clinical
Microbiology,2 Medical
Biochemistry,3
Pediatrics,4 Obstetrics and
Gynecology,5
Surgery,6 and
Medicine,7 University of Turku, Turku,
Finland
Received 6 July 1999/Returned for modification 28 August
1999/Accepted 22 September 1999
A broad-range bacterial PCR targeting rRNA genes (rDNAs) was used
to directly analyze 536 clinical samples obtained from 459 hospitalized
patients during a 4-year study period. The molecular diagnosis based on
DNA sequencing of the PCR product was compared to that obtained by
bacterial culture. The bacteriological diagnosis was concordant for 447 (83%) specimens. Broad-range rDNA PCR was the only method that yielded
an etiologic diagnosis for 11 (2.4%) of 459 patients. Compared to
culture and clinical assessment, the sensitivity of the PCR method
combined with sequencing was 74.2%, and the specificity was between
98.7 and 99.6%. At present, the described molecular approach proved
superior to bacterial culture in two clinical situations: infections
caused by bacteria with unusual growth requirements and specimens taken
during antimicrobial treatment of the patient.
The phylogenetic classification of
microbes based on rRNA gene (rDNA) sequences (4, 34) offers
a well-defined framework which can be used to develop molecular tools
for microbial identification (1, 29). The rRNA molecules
typically consist of highly conserved sequences interspersed with
regions of more variable sequences (7, 17). Together with
the development of the PCR technique (21), this knowledge
has provided an approach for recognition of bacterial pathogens. It is
possible to rationally design probes covering a desired phylogenetic
group of bacteria, whether this group is a certain division, genus, or species.
When PCR primers are targeted at the most conserved rDNA gene
sequences, amplification of the genes of virtually any bacterial species is possible. On the basis of the DNA sequence of the
amplification product, the bacterium can then be located in the
phylogenetic tree (32). During recent years, this approach
has proven its usefulness for the identification of bacterial isolates
that are difficult to classify by their phenotypic properties. Even
more distinguishing and fascinating has been the application of this method to the identification of rare pathogens with unusual growth requirements and, indeed, of those which can be visualized in tissue
but not cultured. The recognition of Bartonella henselae as
the cause of bacillary angiomatosis (26) and that of
Tropheryma whippelii as the uncultured bacillus associated
with Whipple's disease (27, 33) were among the first
demonstrations of the power of this method. Subsequently, broad-range
bacterial PCR has been used to amplify bacterial genes directly from
clinical samples, e.g., biopsy specimens from diseased tissue, with the aim of establishing etiologic diagnoses of various bacterial diseases in a clinical setting. These earlier studies have involved a limited number of specimens and have focused on one defined bacterial disease
only, e.g., endocarditis (6, 11), intra-amniotic infection
(8, 12), meningitis (16), bacteremia
(18), and keratitis (15).
Since the beginning of 1994, our laboratory has applied the PCR
technique to the amplification of the 23S or 16S rDNA genes and DNA
sequencing of the PCR products for the diagnosis of disease in
hospitalized patients. We report here on our 4-year laboratory experience with the use of this molecular approach as a diagnostic tool
in a clinical setting and delineate the potential advantages and
disadvantages associated with the clinical application of this method.
Samples.
From the beginning of 1994 through the end of 1997, a total of 536 clinical specimens from 459 patients were analyzed at
the Department of Medical Microbiology, Turku University, Turku,
Finland, by the broad-range bacterial PCR assay. These included 415 specimens of body fluids, 63 biopsy specimens from tissues, and 58 pus
samples from various abscesses (Table 1).
All samples were sent from the Turku University Central Hospital.
During this study, the PCR assays were requested by the attending
medical doctors for clinical purposes.
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Direct Amplification of rRNA Genes in Diagnosis of
Bacterial Infections
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Specimens analyzed by the 23S rDNA PCR and culture as
well as the number and percentage of positive results for various
sample types
DNA purification. DNA was extracted from the fresh tissue samples after proteinase K (0.1 mg/ml) digestion (56°C, 2 to 17 h) with two phenol-chloroform-isoamyl alcohol extractions followed by one ether wash as described earlier (11). Synovial, pleural, and amniotic fluids as well as pus samples were concentrated by centrifugation, and organic DNA extraction was performed as described above (12). Extraction of DNA from cells of bone marrow was performed similarly, after gradient centrifugation (Ficoll, Pharmacia, Sweden) and proteinase K treatment. Cerebrospinal fluid and small volumes (0.5 to 1 ml) of pericardial fluid were heated (94°C, 10 min) and treated with proteinase K (16), followed by heat inactivation of the enzyme.
PCR. The primers used in the 23S and 16S rDNA PCRs have been described earlier (16). To screen for the presence of bacteria in a sample, a PCR with primers MS 37 and MS 38 (which amplify an approximately 850-bp sequence of the 23S rDNA) was performed (16). On the basis of sequence analysis of the 23S rDNA and experimental data, these primers cover several bacterial subdivisions, as described previously (16).
The bacterial DNA present in a 23S rDNA PCR-positive sample was identified by sequencing the 16S and/or 23S rDNA. The 23S rDNA PCR assay was used for the initial screening of the samples because of its higher sensitivity compared to that of the previously described 16S rDNA PCR method. 16S rDNA was preferably used for sequencing, since (in 1997) our database contained 6,800 bacterial 16S rDNA sequences (3,400 of which were complete) but only 204 bacterial 23S rDNA sequences (131 of which were complete).Analytic sensitivity of the assay. When serial dilutions of Escherichia coli ATCC 25922 (American Type Culture Collection [ATCC], Manassas, Va.) cells were used as the template, the analytical sensitivity of the 23S rDNA PCR was about 10 CFU/reaction, and this was also the case in the presence of 50,000 Ficoll-isolated human mononuclear blood cells per reaction when the cells were mixed with the bacteria prior to DNA isolation. However, on the basis of the intensity of the band it was estimated that at least 100 CFU would be needed for a successful sequencing reaction. When Streptococcus pyogenes ATCC 8184 was used as a target, the sensitivity of the 23S rDNA PCR assay was 2,000 CFU/reaction.
Precautions to avoid false-positive and false-negative PCR results. Precautions taken to avoid laboratory contamination included physical separation of the pre-PCR areas used for sample preparation, DNA extraction, and preparation of the reaction mixtures from the post-PCR areas (22). The staff working in the post-PCR areas did not participate in handling of the samples or PCR reagents before amplification. Biosafety hoods and positive-displacement pipettes or barrier tips were used when handling specimens and reagents to avoid carryover and amplicon contamination.
Two negative controls were included in each PCR series: a sample preparation control with sterile water that went through the same DNA extraction steps as the clinical samples and a reagent control with sterile water as a template. As a positive control, 50 ng of DNA from Neisseria meningitidis group B strain 10026 (National Collection of Type Cultures, London, United Kingdom) was used. To avoid amplification of possible bacterial DNA contaminants in the reagents, reaction mixtures were irradiated with UV light for 3 to 3.5 min before addition of the target DNA. Since April 1997, all samples have also been examined for inhibition of PCR and/or degradation of DNA by amplifying a 429-bp fragment of the human growth hormone gene (24) in a separate reaction by using the same parameters used for the 23S rDNA PCR. When tested by serial dilution experiments with human mononuclear cells, the detection limit of this PCR was about 1,000 cells/reaction. All human samples that were originally PCR negative were further analyzed for inhibition by spiking 90 ng of human DNA (about 10-fold the minimal amount required) in the reaction tube before retesting. Samples found to be strongly inhibitory were excluded from further analyses.Sequencing. The sequencing reactions were performed as described earlier, either manually (11) or semiautomatedly (16). The practice of sequencing of the amplified PCR product varied during the study period from sequencing of selected samples after a special request by the clinician in 1994 to routine sequencing of all PCR-positive samples in 1997.
Comparative sequence analyses and databases. The 16S rDNA sequences that were obtained were compared with sequences in a database made up of sequences from GenBank (2), EMBL (30), and the ribosomal database project (19) by using an in-house algorithm (16). At the beginning of the study period in 1994 the database contained about 3,000 bacterial sequences, and at the end of the study period in 1997 it contained about 6,800 bacterial sequences. For comparison of the 23S rDNA sequences, the FastA program (25) was used. The interpretation of the sequencing results was based on the guidelines given by Stackebrandt and Goebel (28). Below, the best matches and sequence homologies are reported according to the original results; that is, the sequences that were originally analyzed were not reanalyzed by comparison with the sequences in the expanded database that was available at the end of the study. Most often the result of the sequence analysis was reported at the genus level. Some bacterial 16S rDNA or 23S rDNA sequences in the database were distinct enough to allow identification to the species level (6).
Conventional microbiological methods. In addition to being referred for PCR analysis, the clinical samples were also sent to the microbiology laboratory of the Turku University Central Hospital for bacterial or mycobacterial cultures by conventional methods. The routine culture methods for most samples included aerobic culture on blood agar and chocolate agar plates and, when appropriate, anaerobic culture on a fastidious anaerobic agar plate. Prior to cultivation, liquid samples were concentrated by centrifugation and biopsy specimens were homogenized in brain heart infusion broth (Gibco BRL, Life Technologies, Paisley, Scotland). Identification of the colonies isolated was based on routine microbiological methods (10). Gram staining was performed with the specimen if requested by the attending clinician. Detection of mycobacteria was based on routine culture methods with the use of the BACTEC 460 TB Hood system (Becton Dickinson Diagnostic Instrument Systems, Cockeysville, Md.), followed by identification with AccuProbe (GenProbe, San Diego, Calif.).
Analysis of the microbiological methods. An infectious diseases specialist (P.K. or O.R.) examined the hospital records of all patients with positive PCR and/or culture results with the aim of evaluating the clinical importance of these findings.
Statistics. Statistical analyses were performed by Fisher's exact test with the use of the SAS System for Windows (release 6.12; SAS Institute Inc., Cary, N.C.).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR and sequencing. Of all 536 samples analyzed, 73 (14%) were positive by the 23S rDNA PCR (Table 1), and sequencing was attempted for 59 of them. The microbe was recognized to the genus level in 45 (76%) samples, among which the recognition was based on the 16S rDNA sequence for 35 samples and on the 23S rDNA sequence for 10 samples. In one sample from which only the 23S rDNA sequence was available, no matching sequence was found in the database. The sequence was later found to be identical to that of the Propionibacterium acnes strain isolated by bacterial culture from the same sample. In six samples, the presence of several species was suggested on the basis of the typical electropherogram produced by the sequencer. In seven samples, sequencing was not considered possible even after reamplification or the reaction did not produce an interpretable sequence.
Comparison of molecular methods and bacterial culture. An overall presentation of the results obtained by PCR and sequencing compared to the results of routine culture after resolution of the clinical importance of the findings is shown in Fig. 1.
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, negative result. 
, sum.
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Effect of antimicrobial treatment on recovery of microbes. A tendency for a specimen to be culture negative but PCR positive was observed if the patient was receiving antimicrobial treatment at the time that the specimen was taken. A comparison of the antimicrobial status (effective antimicrobial agents versus no antimicrobial treatment) of the patients for whom results are presented in Tables 2 and 3 showed that if the patient was receiving effective antimicrobial treatment, the infective agent was significantly more likely to be detected by rRNA amplification only (Table 3) than by both conventional and molecular methods (Table 2) (P = 0.000016; Fisher's exact test). Data for the specimens that were taken during ineffective antimicrobial treatment and those that yielded fastidious microbes were excluded from the statistical analysis.
Sensitivities and specificities of the methods. For evaluation of sensitivity and specificity, samples from groups 1.1, 2.1, and 2.2 in Fig. 1 were designated true PCR positive, samples from group 3.1 were designated false PCR negative, and samples from groups 3.2 and 4 were designated true PCR negative. The sample from patient 4 (Table 2) as well as a second sample with evident PCR contamination found in the clinical evaluation (a synovial fluid sample belonging to group 2.3) was designated as false PCR positive. Data for the remaining samples were excluded from the analysis because of the difficulty in judging the significance of the finding in the absence of sequence data.
As resolved by the culture result and clinical evaluation as described above, the sensitivity of the PCR assay combined with sequencing was 74.2%, and the specificity was 99.6%, while the positive and negative predictive values of this method were 95.8 and 96.5%, respectively. If all five samples with sequencing failures in group 2.3 were designated contaminations, the specificity of the method would be 98.7%, with a positive predictive value of 88.5%. The performance of the PCR with various sample types is shown in Table 5.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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We assess in this study the usefulness and practicability of molecular methods in a clinical setting on the basis of an analysis of 536 patient samples examined by a broad-range bacterial PCR assay. Our results show that the molecular findings were in good agreement with those obtained by conventional microbiological methods. The results were identical for 447 (83%) samples, including 418 culture-negative and PCR-negative samples and 29 culture-positive samples from which significant pathogens were sequenced, as defined by clinical assessment.
An important finding was that for 17 samples, the PCR assay combined with sequencing succeeded in recognizing an etiologically significant bacterial pathogen that did not grow in conventional bacterial culture, although the species identified was routinely cultivatable from 12 samples. Clinical evaluation of these culture-negative patients revealed that all of them had been receiving antimicrobial treatment at the time that the specimen for PCR and culture was taken. This finding supports our previous hypothesis that in patients receiving empiric antimicrobial treatment, molecular methods might be superior to bacterial culture in providing the etiologic diagnosis (16). In the present study, this hypothesis could be statistically verified.
Another advantage of bacterial PCR is connected to its broad-range nature: rare or unexpected pathogens can be found. Patient 8 (Table 3) was the first patient in Finland to receive a diagnosis of bartonella endocarditis; until then, bartonella infections had been unknown in our country (11). Both Bartonella and the other fastidious pathogen found by molecular methods in the present study, Ureaplasma urealyticum, can be cultured with relative ease, with the precondition that they are regarded as potential etiologic agents of infection and specific growth conditions are provided. Considering the numerous unknown rDNA sequences found in environmental samples (9), it is noteworthy that no novel bacterial species were detected in this study with a large set of clinical material.
A comparison to culture results revealed difficulties in detecting gram-positive species and mycobacteria by PCR. On the basis of sequence analysis, there is no systematic discrimination of gram-positive species (with the exception of the genus Bifidobacterium) with the 16S rDNA-specific primers (14). Also, the 23S rDNA-specific primers have successfully amplified several gram-positive bacteria, including the genera most often missed in the present study, Streptococcus and Staphylococcus, as well as mycobacteria. Thus, the problems are due to difficulties in breaking the cell walls of these organisms during sample preparation, resulting in a failure in the DNA extraction process. Microscopically, an abundance of S. pyogenes cells remains intact after boiling and incubation of the sample with proteinase K (14). Therefore, finding a more effective method of breaking the cell walls would be of vital importance but may be difficult, since the addition of enzymes to the reaction tubes inevitably increases the risk of contamination with bacterial DNA in the reagents. According to our preliminary experiments, mechanical disruption of bacteria could be a more optimal solution.
In any PCR laboratory, strenuous efforts should be taken to avoid contamination of the reagents and samples by amplicons and high-copy-number DNA in other samples. The risk of contamination is still higher for broad-range bacterial PCR than for specific assays, since traces of environmental bacterial fragments from reagents, vessels, etc., will be amplified. Thus, the goal is to determine a level at which the sensitivity of the PCR assay is sufficient for finding the possible pathogens in the samples while simultaneously avoiding the detection of occasional bacterial DNA in the reagents and/or the environment, leading to false-positive results. In the present material, PCR contaminations appeared to be less of a problem than expected, because in comparison to culture results, only one sample with unequivocal contamination and one sample with probable contamination were detected.
Application of rRNA phylogeny to the identification of bacteria, whether the rDNA is amplified directly from clinical samples or is isolated from bacteria in pure cultures, always allows a given (single) sequence to be located somewhere in the bacterial phylogeny (13). Thus, the identity of no isolate, even a novel one, remains totally unknown. At present, a 16S rDNA-based system for identification of bacterial strains is also available commercially (MicroSeq; Applied Biosystems, Foster City, Calif.). However, phylogenic identification based only on ribosomal sequences does have its limitations. Several examples show that information on rRNA sequences (16S or 23S) is not in agreement with DNA-DNA relatedness or the phenotypic properties of a bacterial species. Clearly separated species can have completely identical (within the limits of sequencing errors) 16S rRNA molecule sequences (5, 20, 28), but, on the other hand, intrastrain variations in the 16S rRNA sequences are common (3). In general, it is obvious that the 16S rRNA (or 23S rRNA) identity does not guarantee the species identity (5, 28). This is especially true if only partial 16S or 23S rRNA sequences are used for species identification. In every case, the decision must be made individually on the basis of similarity values obtained from the sequence comparisons, with careful consideration of the quality of the database.
The clinical shortcomings related to the use of broad-range bacterial PCR and sequencing are also of concern. Often only the bacterial genus can be determined, while the probable species remains undefined. This may cause difficulties, for example, regarding various staphylococcal species, as the therapeutic guidelines for S. aureus and coagulase-negative staphylococcal infections are different. In addition, the antimicrobial susceptibility of the causative agent remains unknown. Polymicrobial infections are also problematic due to the inability of the current procedure to identify microorganisms when several are present in the same specimen. Furthermore, sequence homology to species commonly present in water or the environment (31) but of little pathogenic potential should always raise concern about contamination and warrants a careful clinical evaluation.
On account of financial and professional requirements, the performance of the kind of a molecular assay described here is restricted to only a few highly specialized laboratories. The median turnaround time for PCR was 2 days from the time of arrival of the sample at the laboratory, and the results of the sequence analysis were obtained from 2 to 14 days after retrieval of the positive PCR result. Therefore, the approach reported here is rather slow and is too labor-intensive to be used in clinical microbiology. However, the development of automated DNA amplification techniques together with rapid and affordable sequence detection systems will contribute to the usefulness of the direct molecular approach in the near future. The introduction of novel high-density DNA microarrays may also revolutionize pathogen detection based on molecular techniques (23).
In conclusion, we report here on a comparison of direct broad-range PCR and sequencing and routine culture in bacteriological diagnostics for 536 samples obtained from 459 hospitalized patients. Although not currently competitive with conventional methods, the direct molecular approach appears to be a valuable complementary technique for the diagnosis of infectious diseases. At present, broad-range bacterial PCR can be recommended for samples obtained during antimicrobial treatment and/or by a demanding clinical procedure, especially if routine cultures remain negative. The findings presented here may be used as a basis for the development of faster and less labor-intensive molecular methodologies for the direct identification of bacterial pathogens.
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ACKNOWLEDGMENTS |
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Pentti Huovinen is acknowledged for critical reading of the manuscript. We thank Päivi Sandberg and Tuula Närä for computational assistance. Tiina Haarala, Kirsi Sundholm, Merja Mikkola, and Anne Peippo are acknowledged for excellent technical work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland. Phone: 358-2-3337423. Fax: 358-2-2330008. E-mail: kaisu.rantakokko{at}utu.fi.
Present address: Department of Microbiology and Immunology,
Stanford University School of Medicine, VA Palo Alto Health Care System
154T, Palo Alto, CA 94304.
Present address: National Public Health Institute, 20520 Turku, Finland.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, January 2000, p. 32-39, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Wellinghausen, N., Kochem, A.-J., Disque, C., Muhl, H., Gebert, S., Winter, J., Matten, J., Sakka, S. G.
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