JCP Online First, published on May 13, 2015 as 10.1136/jclinpath-2014-202818 Original article
Use of MALDI Biotyper plus ClinProTools mass spectra analysis for correct identification of Streptococcus pneumoniae and Streptococcus mitis/oralis Jonathan H K Chen,1 Kevin K K She,1 Oi-Ying Wong,1 Jade L L Teng,1 Wing-Cheong Yam,1,2 Susanna K P Lau,1,2 Patrick C Y Woo,1,2 Vincent C C Cheng,1,3 Kwok-Yung Yuen1,2 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2014-202818). 1
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong Special Administrative Region, Hong Kong, China 2 Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, Hong Kong, China 3 Infection Control Team, Queen Mary Hospital, Hong Kong Special Administrative Region, Hong Kong, China Correspondence to Professor K-Y Yuen, Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China; [email protected] Received 10 December 2014 Revised 13 April 2015 Accepted 22 April 2015
ABSTRACT Background Differentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system. Methods This study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone. Results The percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification. Conclusions The additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities.
INTRODUCTION To cite: Chen JHK, She KKK, Wong O-Y, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/jclinpath-2014202818
Streptococcus pneumoniae is one of the common human respiratory pathogens causing systemic infections, such as meningitis and endocarditis, in adults and children, with a high mortality rate.1 2 For appropriate antimicrobial therapy, it is clinically important to accurately differentiate S. pneumoniae from other viridans group streptococci (VGS). However, due to the extraordinarily high similarity
in phenotypic and genotypic characteristics among VGS members, especially Streptococcus mitis group species, differentiation of these species is complicated.3 4 In general, laboratory tests (colony morphology examination, optochin susceptibility and bile solubility) are recommended for S. pneumoniae identification.5 However, interpretation of these tests may occasionally lead to incorrect identification.6 7 Genotypic methods are useful for VGS species identification. However, there is no generalised method for genotyping most Streptococcus species. Streptococcus housekeeping genes, such as rpoB, which exhibit >15% sequence divergence among species, were suggested for species differentiation.8 9 Unfortunately, PCR sequencing is relatively expensive and time consuming, which may not be suitable for clinical laboratory use. Recently, matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) has been implemented in clinical laboratories for bacterial identification.10 S. pneumoniae identification by MALDI-TOF MS has been reported to be problematic due to the extraordinarily high homology of the protein mass spectra among S. pneumoniae, S. mitis, S. oralis and S. pseudopneumoniae.11 S. mitis and S. oralis were occasionally reported erroneously as S. pneumoniae by the Bruker MALDI Biotyper system with spectra library V.3.3.1.2 (4613 spectra).12 To improve the accuracy of Streptococcus species identification, the manufacturer incorporated 100 additional VGS mass spectra into the updated spectra library V.4.0.0.1 (5627 spectra) in early 2014. In addition to the MALDI Biotyper for bacterial identification, mass spectra analysis through the use of Bruker ClinProTools software was introduced to discover biomarker mass peaks and to generate classification models for identification of certain phylogenetically related bacterial species, including VGS species.11 13–15 ClinProTools can generate classification models from large numbers of spectra in a rapid and user friendly manner. By using the generated models, a common signature among the spectra of each of the model generation classes can be identified, and spectra from unknown isolates can be classified simply by the model.16 In this study, we tested the performance of the most updated MALDI Biotyper spectra library,
Chen JHK, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2014-202818
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Original article V.4.0.0.1 (5627 spectra), for S. pneumoniae and S. mitis/oralis identification. Then we added the ClinProTools mass spectra analysis after MALDI Biotyper identification. Identification results from the mass spectra analysis were compared with those generated by the MALDI Biotyper alone.
METHODS AND MATERIALS Strains A total of 75 clinical isolates, including 28 isolates of S. pneumoniae and 47 isolates of S. mitis/oralis, collected in 2013, were selected for the study. These isolates originated from different types of clinical specimens, including 30 sputum, 21 blood culture, 10 urine, 5 fine needle aspirate tissue, 7 peritoneal fluid, 1 pleural fluid and 1 biopsy. The isolates were cultured on Columbia blood agar, with incubation at 37°C with 5% CO2 for 18 h. Isolated colonies were then used for identification. Another 10 reference strains of S. pneumoniae and 10 reference strains of S. mitis groups were used to build up the mass spectra analysis model in the ClinProTools software.
Routine clinical diagnostic methods The identities of the 75 clinical isolates were initially classified by colony morphologies, optochin susceptibility and bile solubility. Biochemical or enzymatic characteristics were further determined by the VITEK 2 system (BioMerieux, Marcy l’Etoile, France) if necessary. For isolates with an uncertain identification, S. pneumoniae specific lytA real time PCR, 16S rRNA and VGS rpoB gene sequencing were performed for species confirmation.17–19 Sequences were submitted to the NCBI Genbank and the HKU 16SpathDB V.2.0 database for species confirmation.20
Identification by Bruker MALDI Biotyper alone All 75 isolates were prepared for MALDI-TOF MS using the manufacturer’s recommended direct transfer and ethanol– formic acid (EtOH-FA) extraction protocols. A single isolated colony was inoculated directly onto the MSP96 target plate spot by the direct transfer method. The EtOH-FA extraction method was performed according to the Bruker protocol.21 Each bacterial extract was spotted onto the MSP96 target plate after centrifugation. Each spot was overlaid with α-cyano-4-hydroxycinnamic acid matrix (Sigma Aldrich, St Louis, Missouri, USA). The target plate was analysed by the Bruker microflex LT system (Bruker Daltonics, Bremen, Germany). The protein profile of each spot with m/z values of 3000–15 000 generated was analysed by the MALDI Biotyper V.3.1 with the most updated spectra library, V.4.0.0.1 (5627 spectra). The top 10 identification matches were generated along with confidence scores, ranging from 0.0 to 3.0; a score >2.0 indicated promising species level identification.
Mass spectra analysis identification by ClinProTools A mass spectra peak analysis model consisting of 370 S. pneumoniae/mitis/oralis mass spectra was created from 10 identity confirmed S. pneumoniae and 10 identity confirmed S. mitis group reference strains (8 strains of S. mitis and 2 strains of S. oralis) (table 1). For each strain in the model, 18– 24 high quality spectra were prepared by the extraction method and were captured using flexControl V.3.4 software (Bruker Daltonics). The spectra were then imported into the ClinProTools V.3.0 software (Bruker Daltonics) for recognition of mass spectra patterns between S. pneumoniae and other S. mitis species.22 Spectra pretreatment, peak picking and peak calculation operations were performed using the preset configuration. Classification models were generated using all four 2
available algorithms (genetic algorithm, support vector machine (SVM), supervised neural network and QuickClassifier) and compared. For the SVM algorithm, the number of peaks selected in the model was set to 10, while the default settings were left unaltered for the other modelling algorithms. For each model, the recognition capability (RC) and cross validation (CV) percentage was generated to demonstrate the reliability and accuracy of the model. RC and CV percentages were indicators of the model’s performance and useful predictors of the model’s ability to classify test isolates. The model with the highest RC and CV values were used in the analysis. For the 75 clinical isolates, their mass spectra generated from direct transfer and the EtOH-FA extraction method were imported into the ClinProTools after MALDI Biotyper identification for further analysis. Significant differences in identification accuracy among the different methods (MALDI Biotyper with direct transfer method, MALDI Biotyper with EtOH-FA extraction method, ClinProTools with direct transfer method and ClinProTools with EtOH-FA extraction method) were compared using Cochran’s Q test in MedCalc software V.14.10.2 (MedCalc Software, USA).
RESULTS Routine biochemical and molecular identification Among the 75 clinical isolates, 28 were confirmed as S. pneumoniae and another 47 were identified as S. mitis or S. oralis using the biochemical and molecular methods. All data are shown in supplementary table 1. All S. pneumoniae isolates were optochin sensitive, bile soluble and lytA gene PCR positive. The other 47 S. mitis/oralis isolates were all optochin resistant and bile resistant. Their 16S rRNA and rpoB gene sequencing results further confirmed them to be either S. mitis or S. oralis. However, due to the high similarity in biochemical responses and genotypic sequences between S. mitis and S. oralis, these two species could not be well differentiated. Therefore, these two species were grouped as S. mitis/oralis in further analyses.
MALDI Biotyper identification alone Using the direct transfer method, 50 of 75 clinical isolates (66.7%) were identified correctly at the species level by the latest version of the MALDI Biotyper system, with scores >2.0 (see supplementary table 1). All 28 clinical isolates of S. pneumoniae (100%) were correctly identified (scores 2.000–2.307). In contrast, 22 of 47 (46.8%) S. mitis/oralis isolates were correctly identified as either S. oralis or S. mitis as the top choice among the top 10 identifications by the MALDI Biotyper (scores 2.019–2.249). The other 13 (27.7%) S. mitis/oralis isolates could only be identified at the genus level by the MALDI Biotyper, with scores of 1.717–1.993. There were another 11 isolates (23.4%) that were misidentified as S. pneumoniae but with scores >2.0 (scores 2.027–2.248). Using the EtOH-FA extraction method, overall 53 of 75 clinical Streptococcus isolates (70.7%) were identified correctly by the MALDI Biotyper system alone (see supplemetary table 1). All 28 isolates of S. pneumoniae (100%) were correctly identified at the species level (scores 2.000–2.405). For the other 47 isolates of S. mitis/oralis, 25 (53.2%) were correctly identified as either S. oralis or S. mitis by the MALDI Biotyper (scores 2.049–2.383), while the other 22 (46.8%) isolates were misidentified as S. pneumoniae (scores 2.004–2.337).
MALDI Biotyper identification plus ClinProTools mass spectra analysis In the ClinProTools, classification models using the genetic algorithm and the SVM algorithm demonstrated values of 100% for Chen JHK, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2014-202818
Original article Table 1 Characteristics of the 20 Streptococcus isolates used to build up the mass spectra analysis model in ClinProTools Reference isolate in mass spectra model Streptococcus pneumoniae C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Streptococcus mitis/oralis C11 C12 C13 C14 C15 C16 C17 C18 C19 C20
Specimen type
VITEK 2 ID
Optochin susceptibility
BS test
lytA PCR
16S rRNA/rpoB sequence
Blood Blood Pleural fluid Blood Blood Blood QC isolate QC isolate Sputum Blood
S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae
S S S S S S S S S S
Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae
QC isolate QC isolate QC isolate Blood Urine Blood Blood Blood Blood Blood
S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S.
R R R R R R R R R R
Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
S. mitis S. mitis S. oralis S. mitis S. mitis S. oralis S. mitis S. mitis S. mitis S. mitis
oralis oralis oralis oralis oralis oralis oralis oralis oralis oralis
BS test, bile solubility test.
RC, CV, sensitivity and specificity through validation (table 2). After validating with other Streptococcus reference strains in the laboratory, the SVM model, analysing 10 peaks of each mass spectrum, was selected for this study (figure 1, table 3). Using the SVM classification model, spectra analysis from the direct transfer method and the EtOH-FA extraction method generated correct identification for 64 (85.3%) and all 75 (100%) clinical isolates, respectively (see supplementary table 1). The additional spectra analysis with the EtOH-FA extraction method significantly improved the identification accuracy of S. pneumoniae and S. oralis/mitis compared with the MALDI Biotyper with the direct transfer method (from 46.8% to 100%), the MALDI Biotyper with the extraction method (from 53.2% to 100%) and the MALDI Biotyper plus spectra analysis with the direct transfer method (from 85.3% to 100%) (Cochran’s Q test, p
Use of MALDI Biotyper plus ClinProTools mass spectra analysis for correct identification of Streptococcus pneumoniae and Streptococcus mitis/oralis Jonathan H K Chen,1 Kevin K K She,1 Oi-Ying Wong,1 Jade L L Teng,1 Wing-Cheong Yam,1,2 Susanna K P Lau,1,2 Patrick C Y Woo,1,2 Vincent C C Cheng,1,3 Kwok-Yung Yuen1,2 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2014-202818). 1
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong Special Administrative Region, Hong Kong, China 2 Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, Hong Kong, China 3 Infection Control Team, Queen Mary Hospital, Hong Kong Special Administrative Region, Hong Kong, China Correspondence to Professor K-Y Yuen, Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special Administrative Region, China; [email protected] Received 10 December 2014 Revised 13 April 2015 Accepted 22 April 2015
ABSTRACT Background Differentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system. Methods This study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone. Results The percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification. Conclusions The additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities.
INTRODUCTION To cite: Chen JHK, She KKK, Wong O-Y, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/jclinpath-2014202818
Streptococcus pneumoniae is one of the common human respiratory pathogens causing systemic infections, such as meningitis and endocarditis, in adults and children, with a high mortality rate.1 2 For appropriate antimicrobial therapy, it is clinically important to accurately differentiate S. pneumoniae from other viridans group streptococci (VGS). However, due to the extraordinarily high similarity
in phenotypic and genotypic characteristics among VGS members, especially Streptococcus mitis group species, differentiation of these species is complicated.3 4 In general, laboratory tests (colony morphology examination, optochin susceptibility and bile solubility) are recommended for S. pneumoniae identification.5 However, interpretation of these tests may occasionally lead to incorrect identification.6 7 Genotypic methods are useful for VGS species identification. However, there is no generalised method for genotyping most Streptococcus species. Streptococcus housekeeping genes, such as rpoB, which exhibit >15% sequence divergence among species, were suggested for species differentiation.8 9 Unfortunately, PCR sequencing is relatively expensive and time consuming, which may not be suitable for clinical laboratory use. Recently, matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) has been implemented in clinical laboratories for bacterial identification.10 S. pneumoniae identification by MALDI-TOF MS has been reported to be problematic due to the extraordinarily high homology of the protein mass spectra among S. pneumoniae, S. mitis, S. oralis and S. pseudopneumoniae.11 S. mitis and S. oralis were occasionally reported erroneously as S. pneumoniae by the Bruker MALDI Biotyper system with spectra library V.3.3.1.2 (4613 spectra).12 To improve the accuracy of Streptococcus species identification, the manufacturer incorporated 100 additional VGS mass spectra into the updated spectra library V.4.0.0.1 (5627 spectra) in early 2014. In addition to the MALDI Biotyper for bacterial identification, mass spectra analysis through the use of Bruker ClinProTools software was introduced to discover biomarker mass peaks and to generate classification models for identification of certain phylogenetically related bacterial species, including VGS species.11 13–15 ClinProTools can generate classification models from large numbers of spectra in a rapid and user friendly manner. By using the generated models, a common signature among the spectra of each of the model generation classes can be identified, and spectra from unknown isolates can be classified simply by the model.16 In this study, we tested the performance of the most updated MALDI Biotyper spectra library,
Chen JHK, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2014-202818
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Original article V.4.0.0.1 (5627 spectra), for S. pneumoniae and S. mitis/oralis identification. Then we added the ClinProTools mass spectra analysis after MALDI Biotyper identification. Identification results from the mass spectra analysis were compared with those generated by the MALDI Biotyper alone.
METHODS AND MATERIALS Strains A total of 75 clinical isolates, including 28 isolates of S. pneumoniae and 47 isolates of S. mitis/oralis, collected in 2013, were selected for the study. These isolates originated from different types of clinical specimens, including 30 sputum, 21 blood culture, 10 urine, 5 fine needle aspirate tissue, 7 peritoneal fluid, 1 pleural fluid and 1 biopsy. The isolates were cultured on Columbia blood agar, with incubation at 37°C with 5% CO2 for 18 h. Isolated colonies were then used for identification. Another 10 reference strains of S. pneumoniae and 10 reference strains of S. mitis groups were used to build up the mass spectra analysis model in the ClinProTools software.
Routine clinical diagnostic methods The identities of the 75 clinical isolates were initially classified by colony morphologies, optochin susceptibility and bile solubility. Biochemical or enzymatic characteristics were further determined by the VITEK 2 system (BioMerieux, Marcy l’Etoile, France) if necessary. For isolates with an uncertain identification, S. pneumoniae specific lytA real time PCR, 16S rRNA and VGS rpoB gene sequencing were performed for species confirmation.17–19 Sequences were submitted to the NCBI Genbank and the HKU 16SpathDB V.2.0 database for species confirmation.20
Identification by Bruker MALDI Biotyper alone All 75 isolates were prepared for MALDI-TOF MS using the manufacturer’s recommended direct transfer and ethanol– formic acid (EtOH-FA) extraction protocols. A single isolated colony was inoculated directly onto the MSP96 target plate spot by the direct transfer method. The EtOH-FA extraction method was performed according to the Bruker protocol.21 Each bacterial extract was spotted onto the MSP96 target plate after centrifugation. Each spot was overlaid with α-cyano-4-hydroxycinnamic acid matrix (Sigma Aldrich, St Louis, Missouri, USA). The target plate was analysed by the Bruker microflex LT system (Bruker Daltonics, Bremen, Germany). The protein profile of each spot with m/z values of 3000–15 000 generated was analysed by the MALDI Biotyper V.3.1 with the most updated spectra library, V.4.0.0.1 (5627 spectra). The top 10 identification matches were generated along with confidence scores, ranging from 0.0 to 3.0; a score >2.0 indicated promising species level identification.
Mass spectra analysis identification by ClinProTools A mass spectra peak analysis model consisting of 370 S. pneumoniae/mitis/oralis mass spectra was created from 10 identity confirmed S. pneumoniae and 10 identity confirmed S. mitis group reference strains (8 strains of S. mitis and 2 strains of S. oralis) (table 1). For each strain in the model, 18– 24 high quality spectra were prepared by the extraction method and were captured using flexControl V.3.4 software (Bruker Daltonics). The spectra were then imported into the ClinProTools V.3.0 software (Bruker Daltonics) for recognition of mass spectra patterns between S. pneumoniae and other S. mitis species.22 Spectra pretreatment, peak picking and peak calculation operations were performed using the preset configuration. Classification models were generated using all four 2
available algorithms (genetic algorithm, support vector machine (SVM), supervised neural network and QuickClassifier) and compared. For the SVM algorithm, the number of peaks selected in the model was set to 10, while the default settings were left unaltered for the other modelling algorithms. For each model, the recognition capability (RC) and cross validation (CV) percentage was generated to demonstrate the reliability and accuracy of the model. RC and CV percentages were indicators of the model’s performance and useful predictors of the model’s ability to classify test isolates. The model with the highest RC and CV values were used in the analysis. For the 75 clinical isolates, their mass spectra generated from direct transfer and the EtOH-FA extraction method were imported into the ClinProTools after MALDI Biotyper identification for further analysis. Significant differences in identification accuracy among the different methods (MALDI Biotyper with direct transfer method, MALDI Biotyper with EtOH-FA extraction method, ClinProTools with direct transfer method and ClinProTools with EtOH-FA extraction method) were compared using Cochran’s Q test in MedCalc software V.14.10.2 (MedCalc Software, USA).
RESULTS Routine biochemical and molecular identification Among the 75 clinical isolates, 28 were confirmed as S. pneumoniae and another 47 were identified as S. mitis or S. oralis using the biochemical and molecular methods. All data are shown in supplementary table 1. All S. pneumoniae isolates were optochin sensitive, bile soluble and lytA gene PCR positive. The other 47 S. mitis/oralis isolates were all optochin resistant and bile resistant. Their 16S rRNA and rpoB gene sequencing results further confirmed them to be either S. mitis or S. oralis. However, due to the high similarity in biochemical responses and genotypic sequences between S. mitis and S. oralis, these two species could not be well differentiated. Therefore, these two species were grouped as S. mitis/oralis in further analyses.
MALDI Biotyper identification alone Using the direct transfer method, 50 of 75 clinical isolates (66.7%) were identified correctly at the species level by the latest version of the MALDI Biotyper system, with scores >2.0 (see supplementary table 1). All 28 clinical isolates of S. pneumoniae (100%) were correctly identified (scores 2.000–2.307). In contrast, 22 of 47 (46.8%) S. mitis/oralis isolates were correctly identified as either S. oralis or S. mitis as the top choice among the top 10 identifications by the MALDI Biotyper (scores 2.019–2.249). The other 13 (27.7%) S. mitis/oralis isolates could only be identified at the genus level by the MALDI Biotyper, with scores of 1.717–1.993. There were another 11 isolates (23.4%) that were misidentified as S. pneumoniae but with scores >2.0 (scores 2.027–2.248). Using the EtOH-FA extraction method, overall 53 of 75 clinical Streptococcus isolates (70.7%) were identified correctly by the MALDI Biotyper system alone (see supplemetary table 1). All 28 isolates of S. pneumoniae (100%) were correctly identified at the species level (scores 2.000–2.405). For the other 47 isolates of S. mitis/oralis, 25 (53.2%) were correctly identified as either S. oralis or S. mitis by the MALDI Biotyper (scores 2.049–2.383), while the other 22 (46.8%) isolates were misidentified as S. pneumoniae (scores 2.004–2.337).
MALDI Biotyper identification plus ClinProTools mass spectra analysis In the ClinProTools, classification models using the genetic algorithm and the SVM algorithm demonstrated values of 100% for Chen JHK, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2014-202818
Original article Table 1 Characteristics of the 20 Streptococcus isolates used to build up the mass spectra analysis model in ClinProTools Reference isolate in mass spectra model Streptococcus pneumoniae C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Streptococcus mitis/oralis C11 C12 C13 C14 C15 C16 C17 C18 C19 C20
Specimen type
VITEK 2 ID
Optochin susceptibility
BS test
lytA PCR
16S rRNA/rpoB sequence
Blood Blood Pleural fluid Blood Blood Blood QC isolate QC isolate Sputum Blood
S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae
S S S S S S S S S S
Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble Soluble
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae
QC isolate QC isolate QC isolate Blood Urine Blood Blood Blood Blood Blood
S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S. S. mitis/S.
R R R R R R R R R R
Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble Not soluble
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
S. mitis S. mitis S. oralis S. mitis S. mitis S. oralis S. mitis S. mitis S. mitis S. mitis
oralis oralis oralis oralis oralis oralis oralis oralis oralis oralis
BS test, bile solubility test.
RC, CV, sensitivity and specificity through validation (table 2). After validating with other Streptococcus reference strains in the laboratory, the SVM model, analysing 10 peaks of each mass spectrum, was selected for this study (figure 1, table 3). Using the SVM classification model, spectra analysis from the direct transfer method and the EtOH-FA extraction method generated correct identification for 64 (85.3%) and all 75 (100%) clinical isolates, respectively (see supplementary table 1). The additional spectra analysis with the EtOH-FA extraction method significantly improved the identification accuracy of S. pneumoniae and S. oralis/mitis compared with the MALDI Biotyper with the direct transfer method (from 46.8% to 100%), the MALDI Biotyper with the extraction method (from 53.2% to 100%) and the MALDI Biotyper plus spectra analysis with the direct transfer method (from 85.3% to 100%) (Cochran’s Q test, p
