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Journal of Clinical Microbiology, August 2008, p. 2751-2758, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02462-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

Kathryn A. Harris,^1* Paul Turner,^2,3 Elaine A. Green,¹ and John C. Hartley¹

Department of Microbiology, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London WC1N 3JH, United Kingdom,¹ Shoklo Malaria Research Unit, P.O. Box 46, 68/30 Ban Toong Road, Mae Sot 63110, Thailand,² Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Old Road, Headington, Oxford OX3 7LJ, United Kingdom³

Received 21 December 2007/ Returned for modification 29 February 2008/ Accepted 9 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of 0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

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Culture of clinical specimens for Streptococcus pneumoniae is slow and is often rendered negative by antibiotic treatment prior to sampling. PCR has been shown to be a useful tool for the rapid identification of S. pneumoniae from both clinical samples and bacterial isolates. Various PCR methods have been used to identify S. pneumoniae, including amplification of the genes coding for two pneumococcal virulence factors, autolysin (lytA) (4, 10) and pneumolysin (ply) (7, 18). However, there are reports that lytA and ply PCR may not be specific for the detection of S. pneumoniae, as some streptococcal species in the S. mitis group (SMG) may also harbor these genes. Such SMG strains appear to be associated with severe disease. Recent studies have found that lytA PCR was more specific than ply PCR for the identification of S. pneumoniae (11, 20, 21).

Penicillin has been the cornerstone of treatment for pneumococcal infections; however, the emergence and spread of reduced susceptibility to penicillin has been noted throughout the world (16). Figures for the year 2000 revealed that 7% of isolates from cases of invasive pneumococcal disease in the United Kingdom had reduced sensitivity to penicillin (3). The definition of MIC breakpoints for pneumococcal isolates to penicillin has been the subject of debate and frequent modification. Currently the Clinical and Laboratory Standards Institute (CLSI) (http://www.clsi.org) and the British Society for Antimicrobial Chemotherapy (BSAC) (http://www.bsac.org.uk) both define strains with penicillin MICs of 0.06 mg/liter as susceptible and those with penicillin MICs of >1.0 mg/liter as resistant. Interpretation of strains with penicillin MICs of 0.12 to 1.0 mg/liter varies according to the site of infection, the route of drug administration, and which advisory body's guidelines are used. Reduced susceptibility to penicillin and other β-lactam antibiotics may be mediated by the production of altered penicillin binding proteins (PBP-1A, -1B, -2A, -2B, -2X, and -3) which have reduced affinities for penicillin. Production of altered PBP-2B as a result of sequence polymorphisms in the pbp2b gene appears to be particularly important (19) and may result in treatment failure if penicillin is used. Agents useful for treatment of penicillin-resistant pneumococcal infections include the expanded-spectrum cephalosporins, rifampin, linezolid, the glycopeptides, and the newer fluoroquinolones (9). All such agents are of a broader spectrum and potentially more toxic or more expensive than penicillin and ideally should be reserved for the treatment of infection with penicillin-resistant organisms.

The increased use of molecular tests such as PCR for the diagnosis of bacterial infection has led in turn to an increased demand for antibiotic susceptibility testing using molecular methods. However, unlike phenotypic testing for antibiotic susceptibility, which examines all resistance mechanisms for a particular antibiotic simultaneously, molecular testing can detect only known resistance mechanisms. To date, the only penicillin resistance mechanism described for S. pneumoniae that relates to treatment failure is an altered pbp2b gene. Several molecular methods for detecting mutations in this gene that predict MIC have been published, including restriction fragment length polymorphism (RFLP) analysis of pbp2b PCR products. Penicillin-susceptible strains were shown to have a characteristic RFLP band pattern when the amplified pbp2b gene was digested with HinfI. Strains with reduced susceptibility gave rise to a number of different patterns (2, 15).

More recently, a real-time PCR assay for the detection of penicillin susceptibility of S. pneumoniae strains has been described (8). The assay amplifies a conserved region of the pbp2b gene in penicillin-susceptible "wild-type" S. pneumoniae; failure to detect a product infers reduced penicillin susceptibility (MICs of >0.06 mg/liter) (5, 8). A real-time pbp2b PCR could provide information on penicillin susceptibility more rapidly than our current PCR-RFLP assay and could feasibly provide prospective clinically applicable data on penicillin susceptibility from culture-negative samples positive for S. pneumoniae DNA by PCR.

Previous studies at our center found that S. pneumoniae was the most frequent organism detected by broad-range 16S rRNA gene PCR in culture-negative specimens (6) and in particular in culture-negative empyema fluid (17). However, even after broad-range 16S rRNA gene PCR, one-third of culture-negative empyema fluids remain negative. A specific S. pneumoniae real-time PCR that is faster and more sensitive than the broad-range PCR could result in improved detection of microbial DNA in culture-negative samples and, when combined with reliable detection of penicillin susceptibility, could drive the rational prescription of narrow-spectrum antibiotics. The purpose of this study was to develop and validate a duplex real-time PCR for the rapid detection of S. pneumoniae in culture-negative clinical specimens with simultaneous demonstration of penicillin susceptibility (MICs of 0.06 mg/liter).

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Samples. The specimen types tested included blood, cerebrospinal fluid, empyema fluid, pleural fluid, other respiratory samples, pericardial fluid, pus, and tissue. Extracted DNA samples from 246 clinical specimens submitted for broad-range 16S rRNA gene PCR were tested. Forty-six of these were archived DNA extracts (stored at –70°C) used for the validation of the assay. The remaining 200 samples were sent for 16S rRNA gene PCR between January 2006 and August 2007 and were tested prospectively in the assay. Details of the extraction procedure have been described previously (6).

Isolates. A total of 35 streptococcal isolates were examined. Twenty-three of these were isolated in our laboratory, 15 were identified as S. pneumoniae, and 8 were identified as SMG by phenotypic methods, by optochin sensitivity, and by use of API Strep (Biomerieux UK Ltd., Basingstoke, United Kingdom). All eight SMG isolates were considered to be clinically significant; five were isolated from blood cultures, one was isolated from a cerebrospinal fluid sample, and one was isolated from a nasal pharyngeal aspirate. All isolates were eventually identified as SMG, and some S. pneumoniae isolates required 16S rRNA gene sequencing to confirm their identity (6). Twenty-two isolates were from clinical material and 1 (a penicillin-resistant S. pneumoniae isolate) was from a United Kingdom National External Quality Assessment Service (UK NEQAS) sample. DNA was extracted using a previously published method (6). MICs to penicillin for these 23 isolates were determined in our laboratory by the E-test method (Biostat Ltd., Stockport, United Kingdom). The remaining 12 isolates (all S. pneumoniae) were provided as DNA extracts by Stephen Gillespie at The Royal Free Hospital, Hampstead, London, United Kingdom. MICs to penicillin had been determined for the original isolates by use of the agar dilution susceptibility test method (14).

Duplex real-time PCR. An 89-bp fragment of the S. pneumoniae pbp2b gene and a 101-bp fragment of the lytA gene were amplified in the following reaction mixture: 1x Quantitect multiplex master mix (Qiagen); 0.1 µM each of the primers LytA-F, LytA-R, Pbp-2b-F, and either Pbp-2b-R or Pbp-2b-RMOD (Table 1); 0.1 µM each of the probes LytA-probe and Pbp-2b-probe (Table 1); 5 µl of extracted DNA; and PCR-grade water up to a total volume of 40 µl. Reaction mixtures were cycled on the ABI Prism 7000 sequence detection system (Applied Biosystems, Warrington, United Kingdom) as follows: 50°C for 2 min, 95°C for 10 min, and then 45 cycles of 95°C for 15 s and 60°C for 1 min. The primer Pbp-2b-RMOD is a modified version of the published primer pbp-2b-R (8). Positive and negative controls were included in each run as described for the pbp2b PCR-RFLP method. Samples negative for both parts of the assay were subjected to broad-range 16S rRNA gene PCR, which includes a control for PCR inhibition (6).

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TABLE 1. Primer and probe information

Assay sensitivity. A wild-type, penicillin-susceptible (as determined by the BSAC disc sensitivity method [1]) Streptococcus pneumoniae isolate was used to determine the sensitivity of the real-time PCR assay. A suspension (with an optical density at 540 nm of 0.9) of the organism was prepared in 1 ml of sterile saline. The number of CFU in the suspension were estimated using a plate-counting method (12). DNA was extracted from the suspension by a mechanical lysis method as previously described (6). Serial 10-fold dilutions of extracted DNA were amplified in the PCR.

pbp2b PCR-RFLP. The following is a modified version of a published method (2, 15). The PCR mixture was as follows: 1x Accuprime PCR buffer II (Invitrogen, Paisley, United Kingdom), 1 µl of Accuprime Taq polymerase (Invitrogen), 0.4 µM of each of the primers Pbp-2b-JT-F and Pbp-2b-JT-R (Table 1), 5 µl of extracted DNA, and PCR-grade UV-irradiated water to give a final volume of 50 µl. Reaction mixtures were heated to 94°C for 2 min followed by 35 cycles of 94°C for 1 min, 60°C for 2 min, and 68°C for 3 min. A final extension was carried out at 68°C for 5 min. PCRs were electrophoresed through a 2% agarose gel containing 2 µl of 500 nM ethidium bromide at 100 V for 20 min, and bands were visualized by UV transillumination to confirm the presence of a 1,400-bp product. A positive control, DNA extracted from a penicillin-susceptible S. pneumoniae isolate, and a negative control, 5 µl of water, were included in each run.

PCR product (40 µl) was digested with HinfI (1 µl) in 1x buffer II (both from New England Biolabs, Hitchin, United Kingdom) at 37°C for 75 min. The entire digest was mixed with 4 µl of loading dye and run through a 2% agarose gel containing 2 µl of 500 nM ethidium bromide at 100 V for 3 h. Bands were visualized by UV transillumination and RFLP patterns compared to the pattern from the positive control (15).

Sequencing. The entire pbp2b gene was amplified as described above for the PCR-RFLP assay. A 273-bp fragment of the amplicon was sequenced with primers F2 and R4 and 89-bp sections of the resulting sequences were aligned with each other and the corresponding region from S. pneumoniae strain R6 (GenBank AE008520) by use of the CLUSTAL W algorithm in the MEGALIGN program (DNASTAR, Madison, WI). Details of the sequencing procedure have been described previously (6).

GenBank accession numbers. The sequences generated in this study have been deposited in GenBank under accession numbers EU274626 to EU274635 and EU293610 to EU293612.

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Assay validation. (i) Sensitivity. The duplex PCR was applied to a dilution series of DNA prepared from a quantified suspension of S. pneumoniae. The detection limit for both the lytA and pbp2b targets was 0.5 CFU per reaction. This is around 100-fold more sensitive than our broad-range 16S rRNA gene PCR (6).

DNA extracts from 12 penicillin-susceptible S. pneumoniae isolates were amplified in the duplex assay with the published reverse primer Pbp-2b-R (Table 1). All 12 strains were lytA PCR and pbp2b PCR positive; in four cases, however, the pbp2b product was detected around 10 cycles later than that in the lytA PCR, which is equivalent to a reduction in PCR sensitivity of around 1,000-fold. This observation could be explained by mismatches in the primer and probe sequences that have reduced PCR efficiency. To investigate this, the entire pbp2b gene was amplified for these four isolates (S3 to S6), two other susceptible isolates (S1 and S2), two isolates with MICs of >1.0 mg/liter (R1 and R2), and two isolates with MICs of 0.12 to 1.0 mg/liter (I1 and I2). A 273-bp fragment of the resulting amplicons was sequenced in both directions. Figure 1 shows the sequence alignment of the 89-bp sequence corresponding to the real-time PCR product. The four isolates with MICs of >0.6 have several mutations compared to the wild-type R6 sequence. All four of the susceptible isolates that were pbp2b PCR positive, but with a much larger cycle threshold (C_T) value than the lytA PCR run simultaneously (S3 to S6), have a single-nucleotide polymorphism (AT) at position 68 compared to the wild-type sequence from strain R6 and susceptible isolates S1 and S2. This is the 3'-terminal base of the primer Pbp-2b-R, and mismatched primer binding would explain the reduced efficiency of the pbp2b PCR for these four isolates. We modified the primer by shifting it downstream by one nucleotide. This modification does not affect the melting temperature or specificity of the primer and allows efficient amplification of all 12 penicillin-susceptible isolates. The modified primer, Pbp-2b-RMOD (Table 1), was used for the rest of the study.

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FIG. 1. Sequence alignment of an 89-bp region of the pbp2b gene from 10 S. pneumoniae isolates and the wild-type R6 strain (GenBank accession number AE008520). S indicates penicillin-susceptible strains (MICs of 0.06 mg/liter), I indicates strains with penicillin MICs of 0.12 of 1.0, and R indicates strains with penicillin MICs of >1.0 mg/liter. CS indicates pbp2b sequences derived from culture-negative clinical samples. The single-base polymorphism (AT) at position 68 is located at the 3' base of the original primer, Pbp-2b-R, is shown in bold type. Primer sequences are shaded gray.

(ii) Specificity. (a) lytA PCR. lytA PCR, as part of the duplex assay, was performed on 35 isolates, i.e., 27 S. pneumoniae and 8 SMG isolates. This assay can be positive (S. pneumoniae detected) or negative (S. pneumoniae not detected). All 27 S. pneumoniae isolates and 3 SMG isolates were lytA PCR positive. Forty-six DNA extracts from clinical samples previously positive for S. pneumoniae by 16S rRNA gene PCR (but not culture) were also tested in the duplex assay, and all of them were positive by lytA PCR.

(b) pbp2b real-time PCR. The 27 S. pneumoniae isolates described above were subjected to a pbp2b real-time PCR (as part of the duplex assay). For the five isolates with penicillin MICs of >1.0 mg/liter, the pbp2b PCR was negative. For the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter, the pbp2b PCR was negative for 9 and positive for 1 (the latter result was later reclassified as equivocal; see below). For the 12 penicillin-susceptible isolates (MICs of 0.06 mg/liter), the pbp2b PCR results were positive. The difference between the C_T values (C_T) for the pbp2b PCR and the lytA PCR that was run simultaneously was calculated for each of the 13 isolates that were pbp2b PCR positive (data not shown). All 12 penicillin-susceptible isolates had a C_T value of 3 cycles or less, and the isolate with a penicillin MIC of 0.12 to 1.0 had a C_T value of 6.6. Based on these results, we devised the following simple algorithm for the interpretation of pbp2b real-time PCR results: a penicillin-susceptible result is pbp2b PCR positive with a C_T value of no more than 3 cycles greater (C_T 3) than that for the lytA PCR run simultaneously, while a reduced penicillin susceptibility result is pbp2b PCR negative. Penicillin susceptibility is not determined for equivocal pbp2b PCR results (positive, but with a C_T value of more than 3 cycles greater [C_T > 3] than that for the lytA PCR run simultaneously) (Fig. 2).

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FIG. 2. Flow chart showing the relationship between drug MICs for 27 S. pneumoniae isolates, pbp2b PCR results, and PCR-RFLP patterns. An equivocal pbp2b PCR result is positive with a CT value of more than 3 cycles greater than the lytA CT value (CT > 3) when the assays were run simultaneously.

Forty-six archived DNA extracts from clinical samples, previously positive for S. pneumoniae by 16S rRNA gene PCR, were tested in the duplex assay. Forty-one samples were pbp2b PCR positive and 5 were negative. The above algorithm was used to interpret the results as follows: 39 samples were penicillin susceptible and 5 had reduced penicillin susceptibility. Penicillin susceptibility was not determined for two samples with equivocal pbp2b PCR results (C_T values of 3.5 and 5.7) (Fig. 3).

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FIG. 3. Flow chart showing how penicillin susceptibility would be assigned by real-time pbp2b PCR (or PCR-RFLP) in 46 cases of culture-negative S. pneumoniae infection. An equivocal pbp2b PCR result is positive with a CT value of more than 3 cycles greater than the lytA CT value (CT > 3) when the assays are run simultaneously.

(c) pbp2b PCR-RFLP. The same 27 S. pneumoniae isolates were subjected to pbp2b PCR-RFLP. This assay can give the penicillin-susceptible RFLP pattern or one of several reduced penicillin susceptibility patterns. The five isolates with penicillin MICs of >1.0 mg/liter all gave a reduced penicillin susceptibility pattern. Of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter, 6 gave a reduced penicillin susceptibility pattern and 4 gave the penicillin-susceptible pattern. All 12 of the penicillin-susceptible isolates (MICs of 0.06 mg/liter) gave the penicillin-susceptible RFLP pattern (Fig. 2).

The 46 DNA extracts were also subjected to pbp2b PCR-RFLP, and all 39 samples that were penicillin susceptible by pbp2b PCR and the 2 samples with equivocal pbp2b PCR results gave the penicillin-susceptible RFLP pattern. Of the five samples that were classified as having reduced penicillin susceptibility by pbp2b PCR, only one gave a reduced penicillin susceptibility RFLP pattern, while the remaining four samples gave the penicillin-susceptible pattern (Fig. 3).

The validation of the assay has demonstrated that the PCR-RFLP assay could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter, and therefore it was not applied in prospective analysis of clinical samples.

(d) pbp2b sequence data. A 273-bp fragment of the pbp2b gene was sequenced for one isolate (I3) that had a penicillin MIC of 0.12 to 1.0 mg/liter and an equivocal pbp2b real-time PCR result. This revealed two mutations in the 89-bp region of interest, one of which was within the probe site (Fig. 1).

Sequencing of the pbp2b gene was also performed for two DNA extracts from clinical samples that gave equivocal real-time PCR results. One of these samples (CS1) had the wild-type pbp2b sequence (C_T = 3.5), and the other (CS2) had two mutations within the primer and probe binding sites (C_T = 5.7) (Fig. 1).

Prospective routine clinical application of the assay. Two hundred culture-negative clinical specimens sent to the laboratory for broad-range 16S rRNA gene PCR were also tested as part of the routine diagnostic algorithm in the duplex real-time S. pneumoniae PCR if S. pneumoniae infection was suspected from the clinical details. One hundred six of these samples were lytA PCR negative and S. pneumoniae DNA negative by broad-range 16S rRNA gene PCR (22 samples were positive for another organism). Ninety-four specimens (from 70 patients) were lytA PCR positive, 37 of these were 16S rRNA gene PCR negative (or in two cases positive for another organism), 17 samples did not have a 16S rRNA gene PCR performed (because the lytA PCR was strongly positive), and 40 samples were also 16S rRNA gene PCR positive. Of these 40 samples, 13 were confirmed as S. pneumoniae positive by 16S rRNA gene sequencing, while the remaining 27 samples were presumed to be S. pneumoniae positive by 16S rRNA gene PCR because of the strongly positive lytA PCR result.

Of the 94 lytA PCR-positive samples, 70 samples (from 57 patients) were also pbp2b PCR positive, with C_T values within 3 cycles of the lytA PCR run simultaneously (C_T 3), and therefore were interpreted as penicillin susceptible. Nine samples (from eight patients) gave equivocal pbp2b PCR results, that is, results that were positive but with a C_T value of greater than 3; penicillin susceptibility was not determined. Fifteen lytA PCR-positive samples (from 11 patients) were pbp2b PCR negative, and 14 of these were interpreted as having reduced penicillin susceptibility. The remaining sample was lytA PCR positive, with a C_T value of 40; therefore, susceptibility to penicillin could not be determined due to the unreliable performance of PCR after so many cycles (Fig. 4).

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FIG. 4. Flow chart showing the interpretation of penicillin susceptibility for 94 clinical specimens (from 70 patients), all S. pneumoniae positive (by lytA PCR). A positive real-time PCR result implies susceptibility to penicillin (MICs of 0.06 mg/liter) and a negative result implies reduced penicillin susceptibility (MICs of >0.06 mg/liter). Penicillin susceptibility was not determined for equivocal pbp2b PCR results, that is, results that were positive with a CT value of more than 3 cycles greater than the lytA CT value (CT > 3) when the assays were run simultaneously.

Twenty-four patients had more than one sample that was lytA PCR positive. For 19 of these patients, the interpretation of penicillin susceptibility based on the pbp2b PCR result was the same for all samples. The remaining five patients (with between two and seven samples each) had at least one discrepant result. Two patients each had one sample interpreted as penicillin susceptible and another as equivocal; one patient had two penicillin-susceptible samples and one equivocal sample; one patient had two penicillin-susceptible samples, one equivocal sample, and one sample for which penicillin susceptibility could not be determined; and one patient had five reduced-penicillin-susceptibility samples and two samples that were equivocal.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The aim of this study was to create a sensitive duplex PCR assay that can simultaneously detect lytA and pbp2b gene targets in a single real-time PCR, providing rapid diagnosis of Streptococcus pneumoniae infection, along with penicillin susceptibility data, for culture-negative clinical specimens. Initial validation of a previously published real-time pbp2b PCR (8) in our laboratory revealed that it does not amplify all penicillin-susceptible S. pneumoniae strains with equal efficiencies, due to a single-nucleotide mismatch at the 3' end of the reverse primer. Modification of the primer resulted in a sensitive PCR for the detection of all penicillin-susceptible strains.

The turn-around time for the assay, including extraction of DNA from the sample, is around 3 hours. This is faster than any conventional PCR and significantly faster than broad-range 16S rRNA gene PCR, which requires sequencing of PCR products. With additional PCR-RFLP analysis of the pbp2b gene, the entire procedure takes around 72 h (2, 6, 15).

During assay validation, all 27 S. pneumoniae isolates tested in the assay and 46 clinical samples tested that were S. pneumoniae positive by broad-range 16S rRNA gene PCR were lytA PCR positive. This demonstrates the ability of the assay to detect S. pneumoniae DNA. It has been reported in the literature that strains of closely related oral streptococci, in particular the SMG strains, may also harbor the lytA gene in addition to other S. pneumoniae-specific virulence factors such as pneumolysin (20, 21). These atypical isolates often display unusual phenotypic characteristics, such as optochin sensitivity, that make them appear more like S. pneumoniae isolates. It must be noted, though, that these "atypical" oral streptococci are often highly virulent and isolated from patients with significant pneumococcal infection-like clinical conditions (21). In this study, we tested eight SMG isolates, all of which were causing clinically significant infections, were felt to be "atypical" or "S. pneumoniae-like," and had required 16S rRNA gene sequencing for identification. Three of these isolates were lytA PCR positive. These data support the idea that the lytA gene may not be specific for S. pneumoniae. However, if the lytA PCR gives false-positive results for SMG organisms that are causing serious clinical infections, we would argue that this "nonspecificity" does not render the assay any less useful from a clinical perspective.

During assay validation, five isolates with penicillin MICs of >1.0 mg/liter and 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter were tested. Fourteen of these isolates were pbp2b real-time PCR negative, and one isolate (MIC of 0.12 to 1.0 mg/liter) gave an equivocal result (C_T = 6.6) (Fig. 2). Sequencing of the pbp2b gene for the equivocal isolate (I3) revealed two mutations compared to the wild-type sequence in the region analyzed (Fig. 1). One of these was within the probe sequence and would have affected probe binding. None of these 15 isolates with penicillin MICs of >0.06 mg/liter would have been classified as penicillin susceptible by the duplex assay (Fig. 2). During assay validation, 46 DNA extracts were tested. Thirty-nine samples were pbp2b real-time PCR positive and can be interpreted as penicillin susceptible. Five samples were pbp2b real-time PCR negative and would be interpreted as having reduced penicillin susceptibility, while two samples gave an equivocal pbp2b real-time PCR result, and no interpretation of penicillin susceptibility is possible (Fig. 3). However, one of these equivocal samples (CS1) had a C_T value of close to 3, and it is likely that this sample contains DNA from a fully sensitive organism. The other equivocal sample (CS2) had a C_T value of greater than 5 and is therefore was more likely to derive from an isolate with a penicillin MIC of >0.06 mg/liter. The sequence data for these two samples also support this, with the first isolate (C_T = 3.5) having a sequence identical to that of the wild-type strain in the region of the pbp2b gene analyzed. The other isolate (C_T = 5.7) had two mutations in the region of the pbp2b gene analyzed, one of which would have affected probe binding (Fig. 1).

PCR-RFLP analysis did not detect all of the isolates with penicillin MICs of 0.12 to 1.0 mg/liter; four were classified as penicillin susceptible. Additionally, four of the five DNA extracts that were determined to indicate reduced penicillin susceptibility by real-time pbp2b PCR gave the penicillin-susceptible RFLP pattern. We concluded that PCR-RFLP is not a reliable method for determining penicillin susceptibility (MICs of 0.06 mg/liter). However, this validation has demonstrated that the real-time pbp2b PCR can accurately differentiate between penicillin-susceptible isolates (MICs of 0.06 mg/liter) and isolates with reduced susceptibility to penicillin (MICs of >0.06 mg/liter) and could provide clinically useful data on penicillin susceptibility in S. pneumoniae PCR-positive, culture-negative clinical specimens.

The assay was applied prospectively to 200 culture-negative clinical samples sent to the laboratory for routine analysis by broad-range 16S rRNA gene PCR. Ninety-four samples (from 70 patients) were lytA PCR positive. Thirty-seven of these samples were broad-range PCR negative (or in two cases positive for another organism), demonstrating an improvement in the diagnosis of S. pneumoniae in culture-negative specimens due to the increased sensitivity of the real-time PCR compared to that of the broad-range assay.

Seventy of the lytA PCR-positive samples (from 57 patients) were also pbp2b PCR positive (C_T 3). The validation of the assay allows us to be confident in our interpretation of these 70 samples as positive for penicillin-susceptible S. pneumoniae (MICs of 0.06 mg/liter). Fifteen lytA PCR-positive samples (from 11 patients) were pbp2b PCR negative, and 14 of these were interpreted as having reduced penicillin susceptibility (MICs of >0.06 mg/liter). The remaining sample was very weakly positive in the lytA PCR (C_T = 40). The pbp2b PCR result for this sample was negative but penicillin susceptibility could not be determined because amplification of the pbp2b target may not be reliable in the last few cycles of a PCR assay.

The remaining nine lytA PCR-positive samples (from eight patients) gave equivocal pbp2b PCR results (that is, positive with a C_T value of >3). Three of these samples (from two patients) had very large C_T values (greater than 7) and could be considered to have probable reduced susceptibility to penicillin based on the observation during the validation of the assay that one penicillin isolate with an MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b PCR result, with a C_T value of 6.6.

Interestingly, four of the nine pbp2b PCR-equivocal samples (C_T values of between 3 and 5) were from patients who also had at least one other sample interpreted as penicillin susceptible. A further two pbp2b PCR-equivocal samples (C_T values of greater than 7) were from a patient who had five other samples that were pbp2b PCR negative and therefore were interpreted as indicating reduced penicillin susceptibility.

Two samples that were positive for reduced-penicillin-susceptibility S. pneumoniae were from two patients that subsequently yielded S. pneumoniae, reported as penicillin susceptible, upon culture from specimens taken at their referring hospitals. Review of susceptibility testing in the referring hospital showed that one isolate had a penicillin MIC of 0.12 to 1.0 mg/liter by Vitek (Biomerieux UK Ltd., Basingstoke, United Kingdom) and was penicillin susceptible by the BSAC disc sensitivity method (1). The other isolate was classified as having reduced penicillin susceptibility (MIC of >0.06 mg/liter), but a transcriptional error had occurred during reporting.

This study has shown that a duplex PCR for lytA and pbp2b gene targets can be applied to culture-negative samples to improve the diagnosis of S. pneumoniae infection. The assay can confidently deduce penicillin susceptibility (MICs of 0.06 mg/liter) in the majority (89%) of S. pneumoniae PCR-positive samples; the assay is fast and reliable enough to use to facilitate the rational reduction of antibiotic spectrum for the appropriate treatment of S. pneumoniae infection.

 
We thank Joseph Tsedeke and Sarah Furrows, who carried out the early work optimizing and validating the PCR-RFLP method, and all the staff in the main microbiology laboratory at Great Ormond Street Hospital for the phenotypic characterization of isolates and susceptibility testing. We also thank Stephen Gillespie for proving isolates and MIC data.

 
* Corresponding author. Mailing address: Department of Microbiology, Level 4 Camelia Botnar Laboratories, Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London WC1N 3JH, United Kingdom. Phone: 020 7405 9200. Fax: 0207 813 8268. E-mail: harrik2{at}gosh.nhs.uk

Published ahead of print on 18 June 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Clinical Microbiology, August 2008, p. 2751-2758, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02462-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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