Bacterial 16S rDNA sequences were detected in specimens from 8 of the 46 patients. When the same PCR conditions were used, negative control reactions failed to yield a visible product of the expected size. The absence of large amounts of inhibitors of the PCR reaction in specimens from the remaining 38 patients was demonstrated by the ability to amplify human beta-globin sequences from those specimens. The specimen types with positive bacterial 16S rDNA PCR results were CSF (three patients), pleural fluid (two patients), bone marrow aspirate (one patient), and blood culture bottle material (two patients) (Table 1). The sensitivity of this PCR assay with primers fD1mod/16S1RR-B was 5-50 E. coli 16S rDNA gene copies, as assessed by using purified E. coli DNA as template.

CSF was studied from 14 project cases with unexplained meningitis. Specimens from five subjects were analyzed with broad-range bacterial rDNA primer pairs 8F2/806R and 515F/13R. PCR products of approximately the anticipated size (804 and 876 bp, respectively) were generated from specimens from cases XOR34 and XOR06. From case XOR34, a 1342-bp 16S rRNA gene consensus sequence was generated from the overlapping gene fragments. This sequence shared 99.3% similarity (1332/1342 nucleotide positions) with the four identical 16S rRNA gene sequences from each of the recently published complete N. meningitidis serogroup A and B genomes (GenBank accession numbers AL162758 and AE002551); these GenBank sequences were the closest match to the XOR34 case sequence. Direct sequencing of PCR products from case XOR06 generated 654 bp of sequence homologous to the N. meningitidis serogroup A and B 16S rRNA genes. Limited CSF from this case prevented a more complete sequence analysis of the bacterial rDNA in this specimen.

Figure

Figure. Phylogenetic analysis of the bacterial 16S rDNA sequences obtained from cases XEB44 and XMN22. The tree was rooted with Staphylococcus aureus and Escherichia coli as outgroups and constructed with a maximum-likelihood...

CSF from nine other patients was analyzed by using primers fD1mod/16S1RR-B. Two CSF specimens drawn on different days from case XCA73 each generated PCR products whose directly determined sequences were identical (526/526 bp) to the published 16S rDNA sequence from a S. pneumoniae reference strain (NCTC 7465T, AJ001246) and with the 16S rDNA sequences from three S. pneumoniae strains isolated from patients in the same region and from the same time period (SF10014, SF10175 and SF10314); these reference sequences were determined as part of this study (Figure). The sequences of two clones from each of the amplified XCA73 products were identical to the sequences obtained directly from the PCR products. The CSF specimens from cases XOR06, XOR34, and XCA73 had been collected after empiric antibiotic therapy had begun.

From two of the three culture-negative pleural fluid specimens (cases XEB44 and XMN22) DNA products of the expected size (~568 bp) were amplified with bacterial broad range primers fD1mod/16S1RR-B. In addition to analysis of cloned molecules from these products, the PCR product from case XEB44 was sequenced directly; insufficient product was obtained from the pleural fluid of case XMN22 for direct sequencing. From the XEB44 pleural fluid specimen, DNA was also amplified with bacterial broad-range primers 63F/16S1RR-B. Three recombinant plasmid clones from this product were characterized by DNA sequencing (Table 2). One clone sequence from each of the pleural fluid specimens (clones C and G in Table 2) was identical (497/497 and 509/509 bp) to an S. pneumoniae rDNA sequence deposited in GenBank (reference strain NCTC 7465^T, #AJ001246)(Figure). However, four other clones from XEB44 (clones A, B, D, and E) and one from XMN22 (clone F) contained one or two positions with variant nucleotides. This variability was confirmed in case XEB44 by examining the sequence obtained directly from the PCR product (Table 2, original PCR product). In contrast, ambiguities were not seen in directly sequenced PCR products from the three clinical isolates (SF10014, SF10175, SF10314), nor in the sequences obtained from case XCA73 CSF.

To understand better the variation observed in these pneumococcal sequences, a model of the secondary structure of the S. pneumoniae 16S rRNA was constructed, based on the published secondary structure of the Bacillus subtilis homologue (17) (supplemental data available at http://relman.stanford.edu). Two of the five polymorphic positions are predicted to participate in a “stem” structure and can therefore be assessed for compensatory changes at the paired position. Of the two, only the polymorphism at position 442 creates a noncanonical pairing, thereby suggesting a possible Taq polymerase incorporation error. Intrachromosomal allelic variability was another possible explanation for the observed polymorphisms in these pleural fluid S. pneumoniae 16S rDNA sequences. However, the complete genome sequence of each of two S. pneumoniae strains contains four identical copies of the 16S rRNA operon (16) (unpub. data, Michael A. Loretto and Martin Rosenberg). Therefore, our data are consistent with the hypothesis that patient XEB44 was infected in the pleural space by at least two different S. pneumoniae strains. Insufficient amounts of data from the patient XMN22 specimen hamper speculation of a similar nature for this case.

PCR analysis of a bone marrow aspirate from a case of fatal respiratory disease (XOR63) showed a 16S rDNA sequence that was 99.8% (527/528 bp) similar to 16S rDNA sequences from Stenotrophomonas maltophilia (strain ATCC 13637, #AB008509). This sequence was obtained directly from the PCR product as well as from two recombinant clones; all were identical. A bone marrow aspirate from another patient in our study did not reveal bacterial 16S rDNA sequences.

Two blood culture bottle specimens (from cases XOR56 and XCT29) of nine that were studied gave positive results for bacterial rDNA using PCR primer pair fD1mod/16S1RR-B. All these specimens had been collected from inoculated culture bottles that had failed to exhibit signs of bacterial growth during routine incubation. From one of the two positive specimens (case XOR56), sequences obtained directly from the PCR product and from two recombinant clones were identical (at 535 positions) to the 16S rDNA sequence of a Staphylococcus epidermidis type strain (ATCC 14990T, #D83363). From the other PCR-positive blood culture specimen (case XCT29), direct sequencing of the PCR products indicated the presence of multiple, different DNA molecules. Six recombinant clones were sequenced. Two clones were 99.8% (545/546 bp) similar to 16S rDNA sequences from Enterococcus faecium (strain DSM20477, #AJ276355) and E. durans (strain DSM20633, #AJ276354). One clone was 99.8% similar to a sequence from Bacillus stearothermophilus (strain IF012550, #AB021196), one was 98.6% similar (509/516) to a sequence from a Halomonas sp. (strain ML-028, #AF139994), and one clone with a truncated sequence was 96.4% similar (323/335 bp) to several Bacillus species. The clinical relevance of the rDNA findings in the etiology of these two cases remains uncertain.

In this study, we applied a molecular diagnostic method, broad-range bacterial rDNA PCR to 59 specimens from 46 UNEX cases. Positive findings were obtained for eight of the cases. In six of these cases, the organism detected in the specimens is a known cause of the type of syndrome the patient had.

The diagnosis of infectious diseases with molecular data, rather than from cultivation or serology, requires a careful examination of criteria for causation (18) and may require new molecular surveys of host microbial ecology. Streptococcus pneumoniae and N. meningitidis are common causes of bacterial meningitis in the United States (19), and S. pneumoniae is a well-known cause of pneumonia and empyema. Despite some unconfirmed reports of S. pneumoniae DNA in the serum of healthy persons (20–22), our failure to find other bacterial sequences in the two pleural fluid specimens with broad-range primers leads us to conclude that S. pneumoniae was the cause of these two cases of respiratory tract disease. In CSF, DNA from these organisms has been detected only in meningitis cases. The response of project patients to appropriate antibiotics further supports these conclusions. Similarly, S. maltophilia was the only bacterial species detected in a privileged anatomic site (bone marrow) in case XOR63. The known association of this organism with lower respiratory tract disease and septicemia strongly suggests that it caused at least some of this patient’s respiratory disease. However, additional specimens were not available for confirmation; in addition, S. maltophilia is ubiquitous in nature and, despite our negative controls, may in theory contaminate laboratory reagents. We speculate that it may have been acquired after the patient's admission to the hospital and contributed to the late stages of the patient’s illness.

In the cases of multisystem failure (XOR56) and cardiac disease (myocarditis, XCT29), the role played by the organisms detected in the blood culture specimens in causing disease is much less clear. Although S. epidermidis was also cultivated from the blood of case XOR56 by local physicians, it is a well-recognized blood culture contaminant. Determining its clinical relevance requires additional data. Bacillus sp. are also occasional blood culture contaminants, and their DNA may contaminate blood culture media (23). An Enterococcus sp. is the most likely disease-causing agent among those detected in case XCT29. In general, with reliance on molecular methods, conclusions about causation are most reliably drawn after 1) repeated detection of the same organism in separate specimens from the same patient collected only at clinically relevant time points; 2) direct detection of the organism in situ in areas of pathology (24); 3) documentation of response to appropriate therapy; and 4) assessment of specific immune responses. Many of the same concerns and goals are applicable to cultivation-based diagnosis. In practice, especially when case investigation occurs after resolution of illness or death, as in this project, the preferred type and amount of specimens are not always available.

Our findings from these “unexplained” cases raise several important questions. Why weren’t these well-known bacterial pathogens detected during the routine care of these patients? Specimens are not always collected from the appropriate anatomic site, at the appropriate time, in a sufficient amount, nor processed in an optimal fashion. In addition, the sensitivity of cultivation and serology is imperfect, even for organisms amenable to these approaches. Most of the project patients had received broad-range antibiotics before specimens were collected for this study.

[In applying broad-range rDNA PCR to clinical specimens, one confronts the problem of 16S rDNA sequence microheterogeneity. Small discrepancies between directly amplified sequences and reference sequences in public databases pose difficulties for the definition of taxon boundaries, and complicate clinical interpretation of laboratory data (25,26). In our investigation, the finding of Streptococcus pneumoniae 16S rDNA sequence microheterogeneity in the pleural fluid of a patient with culture-negative empyema suggests that some cases of pneumococcal disease, and perhaps other bacterial disease, may be caused by multiple concurrent strains of the same species.

Why have we failed to explain most of these project cases? Are some unexplained cases caused by novel or previously unrecognized pathogens? Our experiments addressed only the possibility of a bacterial cause with bacterial domain-specific broad-range PCR primers; current efforts include broad-range primers for various families of viruses, and for fungi and the Archaea. Multiple broad-range primer pairs may be necessary for each group (as we describe for the bacteria) to detect unrecognized organisms with polymorphisms in conserved primer recognition sites.

In addition to the specimen problems listed above, PCR inhibitors may cause some false-negative results. The timing of specimen acquisition may also be relevant, since the average delay between onset of illness and specimen collection was 9 days for our PCR-positive cases versus 16 days for all cases studied with broad-range PCR. Although bacterial DNA persists longer than cultivatable organisms after therapy is initiated (27), the former still has a limited half-life. Finally, it must be kept in mind that some of these cases may not have a microbial cause.

Applying broad-range bacterial PCR to this set of difficult clinical cases raised several important points about the future use of this diagnostic approach. First, certain specimen types, such as CSF (3/16 specimens positive), pleural fluid (2/3 positive), and bone marrow (1/2 positive), may provide higher diagnostic yield in bacterial disease than other types, such as serum and blood. Second, the microbial sequence “background” in clinical specimens from healthy persons (e.g., blood) must be better characterized before findings from ill persons can be reliably interpreted. Third, sequence microheterogeneity in anatomically isolated sites of microbial disease may be more common than previously assumed. Our findings from the XEB44 pleural fluid illustrate this point. There are several possible explanations for the S. pneumoniae 16S rDNA heterogeneity in this specimen (see above); however, we believe that at least some of the heterogeneity is best explained by the presence of two (or more) S. pneumoniae strains. To our knowledge this is the first case of invasive disease associated with multiple S. pneumoniae strains, although dual infections with other pathogens have been described (28,29). In fact, carriage of multiple strains of Haemophilus influenzae was recently shown to correlate with an increased risk for otitis media in children (30), and mixed-strain infections with Mycobacterium tuberculosis have also been reported (31). Multiple-strain infections with S. pneumoniae may have been previously missed with culture-based methods because of the common laboratory practice of subculturing a single representative when there is only one apparent colony morphotype.

The search for microbial causes of unexplained illnesses and deaths must continue to integrate traditional and molecular methods and will inevitably challenge assumptions about the mechanisms of microbial disease causation. Refined techniques, novel approaches, and study of other populations such as immunocompromised or impaired hosts are likely to provide new insights into the spectrum of infectious diseases.