Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae
Pneumonia volume 6, pages 57–66 (2015)
Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.
Streptococcus pneumoniae is an important bacterial pathogen in humans that is recognised as a major cause of pneumonia, meningitis, sinusitis, otitis media, and as an uncommon cause of a variety of other infectious diseases . Pneumococcal disease is under-reported, as only a small portion of presumptive cases can be confirmed by conventional techniques. Isolation of S. pneumoniae from blood occurs in only 20–30% of adult cases of pneumococcal pneumonia and less than 10% of cases among children [2,3]. Even when present in blood, S. pneumoniae may be missed due to a low density of the pathogen, the fastidious nature of the organism, and previous administration of antibiotics . Serologic assays for both antibody and antigen detection lack specificity and sensitivity [5,6].
Accurate and rapid assays are indispensable for prompt diagnosis and effective therapy. Molecular assays with increased sensitivity and specificity are inherently beneficial for detection of infectious agents and are not abated by non-viable organisms . Various molecular assays, including loop-mediated isothermal amplification method (LAMP) , DNA probe test , and TaqMan® quantitative real-time polymerase chain reaction (qPCR) assay  have been developed and employed to assist investigations.
Studies have revealed that genes encoding pneumolysin (ply) , autolysin (lytA) , pneumococcal surface adhesion A (psaA) , penicillin binding protein , and manganese-dependent superoxide dismutase  have been targeted for identification of S. pneumoniae by PCR-based assays. The ply and lytA genes are among the more common gene targets used for screening S. pneumoniae. Although lytA is present in S. pneumoniae as well as related species such as oral streptococci, the lytA gene has sufficient variability that properly designed primers afford specificity for S. pneumoniae [14,15].
Amongst pneumococcus-like viridans group streptococci, a newly recognised species classified as S. pseudopneumoniae which is positive by AccuProbe assay has been described and characterised . Studies have shown that lytA and psaA gene sequences can reliably distinguish S. pneumoniae from S. pseudopneumoniae . Further, Suzuki et al.  developed a highly specific Spn9802 primer set for S. pneumoniae in order to discriminate S. pneumoniae from pneumococcus-like oral streptococci harbouring the ply and lytA genes; however, these Spn9802 primers may not discriminate S. pseudopneumoniae . For the identification of pneumococci, PCR tests have been applied by various researchers, however, there are reports of ambiguous results . Such ambiguities are perhaps not surprising given the diversity that exists within the organisms that are closely related to pneumococci. It is evident that these organisms can harbour pneumococcal virulence determinants such as ply and lytA genes that are notionally used for diagnosis for pneumococci . So, straightforward amplification of ply by PCR or in combination with lytA is unable to resolve the identification of strains with equivocal reactions for serotype, optochin, or bile solubility [5,18]. The development of a diagnostic test based upon single-target identification is an ambitious hope in a clinical laboratory setting. Given the genetic plasticity of the pneumococcus and its naturally transformable relatives, the combination of 3 or more unique loci is needed to reduce the possibility of false positives .
Accurate detection of pneumococcal infection is of importance to estimate disease burden, tracking changes in the epidemiology of the disease and to assess the effectiveness of currently used vaccines. In the present work, we have standardised and evaluated the quantitative multiplex real-time PCR (qmPCR) system for the accurate and rapid identification of S. pneumoniae.
2.1 Bacterial isolates
S. pneumoniae ATCC 49619 (American Type Culture Collection, USA) was used as a reference strain for optimisation of the assay. Twenty-three characterised and typed S. pneumoniae strains were procured from Statens Serum Institute, Denmark (serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F). Twenty invasive S. pneumoniae strains isolated from whole blood samples of children with pneumonia and 29 negative control organisms were obtained from Central Research Laboratory, Kempegowda Institute of Medical Sciences, Hospital and Research Centre, Bangalore. These organisms were cultured under conditions appropriate for each species .
The isolates were identified as S. pneumoniae by colony morphology, alpha haemolysis, Gram stain, optochin sensitivity, and bile solubility tests by standard methods . The identification was confirmed by automated MicroScan WalkAway-40 (Siemens Healthcare Diagnostics Ltd, UK) system with rapid identification panels .
2.2 Serum specimens
Twenty serum samples from patients who were positive for pneumococcal blood culture, 26 from patients who were negative for pneumococcal blood culture, and 30 from healthy subjects were included in the study. The DNA extracted from the serum samples of healthy subjects was subsequently spiked with 4.29 × 102 copies/µl or 1 pg/µl of S. pneumoniae ATCC 49619 genomic DNA. Serum samples were sourced from the Pneumococcal Research Division, Central Research Laboratory, Kempegowda Institute of Medical Sciences, Hospital and Research Centre, Bangalore.
2.3 DNA extraction/or qmPCR analysis
DNA extraction from isolates and serum specimens was performed using QIAamp DNA Mini Kit with automated DNA extracter, QIAcube (Qiagen, Germany), as per manufacturer’s protocol. Briefly, a loopful of the overnight growth from a blood agar plate was suspended in 180 µl of suspension buffer followed by 20 µl proteinase K and incubated at 56°C for 30 min. For serum, 200 µl of lysis buffer was added to 200 µl of clinical material followed by 20 µl of proteinase K and incubated at 70°C for 10 min. After washing steps, DNA was eluted in 100 µl of elution buffer and stored at −20°C.
2.3.1 Quantification and quality determination of extracted DNA
Quantification and quality of the extracted DNA was determined spectrophotometrically at absorbance 260 nm using Nanodrop 2000 (Thermo Fisher Scientific, USA). The optical density of extracted DNA was measured at the wavelengths of 260 and 280 nm. The DNA purity was estimated by measuring the ratio between the absorbance values.
2.4 Primer and hydrolysis probe oligonucleotide design
The oligonucleotide sequences for ply, lytA, psaA, Spn9802, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer-probe sets were obtained from previously published data [2,22,23] and the sequences available in the GenBank database (Table 1). For the Spn9802 probe sequence, extra ATC and TAC bases were added at the 5′ and 3′ ends, respectively. All sequences were analysed for specificity and PCR suitability using the National Centre for Biotechnology Information (NCBI) Primer-BLAST software (https://doi.org/blast.ncbi.nlm.nih.gov/Blast.cgi). Hydrolysis-probes were labeled with the appropriate 5′ reporter dyes (FAM, CAL Fluor Red 610, CAL Fluor Orange 560, Quasar 705, and Quasar 670) and 3′ Black Hole Quencher dyes (BHQ-1, BHQ-2, BHQ-1-plus). BHQ-1-plus quencher was utilised on the Spn9802 probe to improve duplex stability and enhance target specificity. Primers and hydrolysis probes were synthesised from Biosearch Technologies, USA.
The Rotor-Gene Q (Qiagen) thermocycler has 5 preset channels: Green, Yellow, Orange, Red and Crimson, with the detection range from 450 to 712 nm. Each channel detects reporter dyes that emit light at a particular wavelength. Based on the quenching range of BHQ-1 (480–580 nm), BHQ-2 (559–670 nm), emission wavelength of the reporter dye and detection optics of the instrument, we confirmed that each selected reporter dye is compatible and detected by respective detection channels.
2.5 qmPCR assay
A 20X primer-probe mix comprising 10 µM of each forward primer, 10 µM of each reverse primer, and 4 µM of each hydrolysis probe was prepared in DNase/RNase free water (Qiagen) from 100 µM stock solutions of each primer and hydrolysis probe. PCR was performed in 25 µl volumes using the 2X Rotor-Gene Multiplex PCR Master Mix (Qiagen) containing HotStar TaqPlus DNA polymerase (Qiagen), MgCl2 and deoxynucleotide triphosphates (dNTPs: dATP, dCTP, dGTP, dTTP), 20X primer-probe mix, template DNA and water, as described in Table 2. Final optimal forward/reverse primer and probe concentrations were 0.5 µM and 0.2 µM, respectively. The qmPCR assays were performed on the Rotor-Gene® Q (Qiagen) thermocycler using 200 µl PCR tubes and 36 well rotor. Optimal performance of the assay was achieved by following the thermocycling conditions as recommended by the manufacturer. It consisted of 1 cycle of denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s and combined annealing/extension step at 60°C for 15 s. Data analysis was performed by using Rotor-Gene® Q software (Qiagen). Analysis settings (threshold values, background settings, dynamic tube normalisation, outlier removal) for every reporter dye detection channel were adjusted in every run to obtain accurate quantification data as suggested by the manufacturer and as has been described elsewhere .
2.5.1 Standard curve and lower limit of detection
Standard curves (Figure 1) for each gene target in the qmPCR assay was generated by 10-fold serial dilutions of S. pneumoniae ATCC 49619 DNA equivalent to 4.29 × 105 to 4.29 × 101 genome copies/µl (1 ng/µl to 100 fg/µl). Each standard dilution was run in triplicate.
For assessments of the lower limit of detection (LLD), serial 10-fold dilutions equivalent to from 4.29 × 105 to 0.4 genome copies/µl (1 ng/µl to 1 fg/µl) of purified DNA were prepared and the aliquots were tested by the ply-lytA-, psaA-, and Spn9802-specific primer-probe sets in multiplex reaction [2,7].
2.5.2 Sensitivity and specificity on culture isolates
Sensitivity of the qmPCR assay was determined by testing 10 ng/µl of DNA from 43 confirmed S. pneumoniae isolates. Specificity determination was made by testing 10 ng/µl of DNA extracted from 29 non-pneumococcal isolates in the multiplex reaction. We could not test S. pseudopneumoniae due to non-availability of the strain.
2.5.3 Sensitivity and specificity on serum specimens
DNA extracted from 30 serum samples of healthy subjects spiked with 1 pg/µl of S. pneumoniae ATCC 49619 genomic DNA corresponding to 4.29 × 102 genome copies/µl and 20 S. pneumoniae culture positive serum samples were tested by the qmPCR for sensitivity determination. Twenty-six culture-negative serum samples were examined by the qmPCR for specificity determination. The extracted DNA from each sample (5 µl) was used as a template. GAPDH-specific primer-probe set was used to amplify the endogenous internal control in multiplex reactions.
A no template control (NTC) was included in every run. Positive samples were defined as those which showed amplification for ≥3 target specific sequences and LLD of ≥4 genome copies/µl. Culture and serum specimens were run in duplicates.
2.6 Ethics statement
The study was approved by the Kempegowda Institute of Medical Science independent ethics committee (Approval ID: ECR/216/Inst/Kar/2013). The study was conducted according to the guidelines and recommendations of Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from each participant or legal guardian as applicable.
2.7 Statistical analysis
Sensitivity and specificity of qmPCR on culture and serum specimens were calculated using MedCalc Software bvba version 15.6 (Belgium).
3.1 qmPCR standard curves
A linear standard curve was acquired for each of the ply, lytA, psaA and Spn9802 primer-probe sets within the qmPCR between 4.29 × 105−4.29 × 101 genome copies/µl (1 ng/µl–100 fg/µl) of DNA from the reference strain S. pneumoniae ATCC 49619. The slope of the curves ranged from −3.399 to −3.271 with the R2 value ≥0.97. The efficiency of the qmPCR assay for all the detection channels were very similar and ranged from 97% to 100% (0.97–1.0). The former was contributed by psaA, and the latter by ply, lytA and Spn9802. The amplification profile for each target is provided in Figure 2.
The qmPCR assay illustrated LLD equivalent to 4 genome copies/µl or 20 genome copies/reaction for ply-, lytA-, psaA- and Spn9802-specific primer-probe sets. There was an increase in quantification cycle (Cq) values with the decrease in template concentration in subsequent reactions.
3.2 Standardisation of qmPCR on culture isolates
The qmPCR was positive for all 43 S. pneumoniae strains, representing 23 different serotypes, indicating 100% sensitivity for known isolates. The specificity of each of the specific primer-probe sets was evaluated and the specificities were compared by amplifying DNA extracted from 29 strains of non-pneumococcal bacteria. These strains represented several genera of Gram-positive and Gram-negative bacteria, some of which inhabit the oral cavity (Table 3). There was no amplification of any non-pneumococcal bacteria by any of the primer-probe sets (ply, lytA, psaA, and Spn9802) indicating 100% specificity for this cross-reactivity panel (Table 4).
3.3 Standardisation of qmPCR on serum specimens
All 30 spiked DNA extracts were qmPCR positive at the expected concentration (4.29 × 102 genome copies/µl or 1 pg/µl) demonstrating that the DNA extraction eluted from serum samples was free from PCR inhibitors.
All 20 serum samples previously shown as blood culture positive for S. pneumoniae were positive for each target (ply, lytA, psaA, and Spn9802) in the qmPCR, indicating 100% sensitivity. Quantification data of the 20 S. pneumoniae positive serum samples for each target is described in Table 5. All 26 serum samples, previously shown as blood culture negative for S. pneumoniae, were negative for each target (ply, lytA, psaA, and Spn9802) in the qmPCR, indicating 100% specificity (Table 4). Endogenous internal control GAPDH was amplified in all the multiplex reactions along with the S. pneumoniae-specific primer-probe sets.
Real-time PCR assays have immense potential to serve as sensitive diagnostic tests for the detection of invasive S. pneumoniae. Currently, there is no simple and dependable method to assess its performance; hence, the diagnostic capability of qPCR should be critically evaluated to acquire reliable results [15,25]. In this study, we evaluated a 4 target qmPCR for accurate detection of S. pneumoniae in culture and serum specimens.
Our qmPCR targeting ply, lytA, psaA, and Spn9802 sequences had 100% sensitivity for detecting S. pneumoniae in culture isolates. The assay also had 100% specificity against a cross-reaction panel of 29 organisms representing diverse genera, including 6 streptococcal species. The present study shows greater sensitivity and specificity than reported by Falquera et al.  (78% sensitivity, 93% specificity), Toikka et al.  (44% sensitivity, 100% specificity), and Michelow et al.  (92% sensitivity, 95% specificity). However, our results are in concordance with the findings of Carvalho et al. , McAvin et al. , and Messmer et al. , who also reported 100% sensitivity and specificity.
qPCR for pneumococci in serum has been reported to be challenging due to the presence of inhibitors in blood and the low number of genomic copies [4,29]. In the present study, qmPCR testing of 30 spiked DNA extracts and 20 pneumococcal culture-positive serum specimens correlated completely with the culture results, indicating 100% sensitivity. High correlation results were also reported by Bayram et al.  (97.2%) and Stralin et al.  (94%). None of the 26 culture-negative serum samples were positive by qmPCR, suggesting 100% specificity. Similar results were reported by Ismail et al. , Carvalho et al.  and McAvin et al. .
In the present study, all the primer-probe sets showed a lower limit of detection equivalent to 4 genome copies/µl. This is similar to that reported by McAvin et al.  and Rudolph et al. . Scott et al.  and Carvalho et al.  reported 1 genome copy/µl and <10 genome copies/µl, respectively. Even though the PCR could detect as little as one genome copy of target DNA as has been described by Scott et al. , the sensitivity of pneumococcal PCR may be poor in clinical evaluations due to the presence of inhibitors. Taq polymerase is highly sensitive to porphyrin inhibitors that are generated from the breakdown of haemoglobin. Most of the inhibitors can be eliminated by an efficient DNA extraction method. In our study, human GAPDH used as an endogenous internal control amplified in all the reactions, suggesting optimal DNA extraction, qmPCR efficiency, and absence of inhibitors.
The efficiency, accuracy, sensitivity, and dynamic range of the PCR assay is determined by the standard curve which is independent of variables associated with the sample preparation. The standard curves generated in this study showed efficiency of 97% to 100%. Similar findings were reported by Carvalho et al. . The high efficiency of this protocol signifies that the amount of PCR product is doubling during each cycle and there is an absence of PCR inhibitors.
By targeting the ply gene, S. pneumoniae DNA in blood samples has been detected with sensitivity ranging from 35% to 100% [15,33]. A matter of concern for the ply PCR is that it is unable to distinguish S. pneumoniae from other streptococcal species . In view of the low specificity of the ply PCR, the lytA PCR has been used and found to have higher specificity [2,34], and pneumococcal DNA was not detected in the blood of healthy subjects irrespective of carrier status . Sequencing and high resolution DNA typing of S. pneumoniae illustrated conservation of the lytA gene. It has been shown that lytA differentiates S. pneumoniae from genotypically related species . Monoclonal antibody studies suggest that psaA is expressed in all 90 serotypes of S. pneumoniae, and PCR-restriction fragment length polymorphism analysis of the 23 vaccine serotypes demonstrated the conservation of the gene . Recently a new S. pneumoniae specific target, the gene fragment Spn9802, has been reported to discriminate S. pneumoniae from pneumococcus-like strains .
Application of multiplex real-time PCR in serum specimens has emerged as a valuable clinical diagnostic tool that offers an opportunity to readdress the problem of the diagnosis of S. pneumoniae infections. The problems associated with microorganisms that are low in number, difficult to culture, or antigenically similar has been minimised with the advancement of sensitive and specific multiplex PCR assays [30,36]. It has several positive outcomes over the singleplex assay as it reduces test costs, eliminates well-to-well variability, conserves precious samples, increases test throughput, and improves turnaround times .
Multiplex PCR assays are popularly adopted for simultaneous detection of various pathogens in the clinical specimens. In the present protocol, multiple specific primer-probe sets were used for detection of a single pathogen to eliminate the spurious negative and positive results. Our study establishes that—with high sensitivity, specificity, and rapidity—the qmPCR assay is a valid platform for detection of pneumococci from serum specimens in clinical laboratories. Finally, the present methodology has broader applications beyond the scope of the present study that demands further investigation. Future studies with large sample sizes should seek to replicate our findings across different geographical locations, particularly in regions with high prevalence of pneumococcal infections.
The qmPCR assay targeting ply, lytA, and psaA genes and Spn9802 DNA fragment is a sensitive and specific assay for the rapid identification of S. pneumoniae. This technology should offer an added advantage when it is used in conjunction with other assays for pneumococcal disease diagnosis. The efficacy of the DNA extraction procedure, degree of sensitivity, and specificity supports the use of this procedure for the direct detection of S. pneumoniae in patient specimens.
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The authors would like to thank Dr. Anjana Gopi, Kempegowda Institute of Medical Sciences, Bangalore, for providing the non-pneumococcal isolates.
Funding source: The authors have no funding to report.
Competing interests: All authors have no competing interests to declare
Author contributions: All the authors met ICMJE authorship criteria. FAG designed the research plan, performed experiments and conducted the data analysis. VG collected data. RK supervised the study. FAG wrote the first draft of the manuscript. All authors critically reviewed the manuscript for important intellectual content. All authors agree with the manuscript results and conclusions.
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Ganaie, F.A., Govindan, V. & Ravi Kumar, K.L. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae. Pneumonia 6, 57–66 (2015). https://doi.org/10.15172/pneu.2015.6/559
- multiplex real-time PCR
- Streptococcus pneumoniae
- pneumococcal surface adhesin A