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Utility of MALDI-TOF MS as a new tool for Streptococcus pneumoniae serotyping.

ABSTRACT: Nowadays, more than 95 different Streptococcus pneumoniae serotypes are known, being less than one third responsible for the majority of severe pneumococcal infections. After the introduction of conjugate vaccines, a change in the epidemiology of the serotypes causing invasive pneumococcal disease has been observed making the surveillance of circulating serotypes especially relevant. Some recent studies have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology to identify the most frequent pneumococcal serotypes that cause invasive disease. The objectives of this study were to evaluate the efficacy of previously described discriminatory peaks determined by MALDI-TOF MS for the identification of serotypes 6B, 19F, 19A and 35B using reference and clinical isolates and to try to identify other discriminatory peaks for serotypes 11A, 19F and 19A using transformed pneumococcal strains. Most of the proposed peaks defined in the literature for the identification of serotypes 6B, 19F, 19A, 35B were not found in the spectra of the 10 reference isolates nor in those of the 60 clinical isolates tested corresponding to these four serotypes. The analysis and comparison of the mass spectra of genetically modified pneumococci (transformed strains) did not allow the establishment of new discriminatory peaks for serotypes 11A, 19F, and 19A. MALDI-TOF MS in the usual range of 2,000 to 20,000 m/z did not prove to be a valid technique for direct S. pneumoniae serotyping.

SUBMITTER: Ercibengoa M

PROVIDER: S-EPMC6372175 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Utility of MALDI-TOF MS as a new tool for Streptococcus pneumoniae serotyping.

Ercibengoa María M Alonso Marta M Vicente Diego D Morales Maria M Garcia Ernesto E Marimón Jose María JM

PloS one 20190212 2

Nowadays, more than 95 different Streptococcus pneumoniae serotypes are known, being less than one third responsible for the majority of severe pneumococcal infections. After the introduction of conjugate vaccines, a change in the epidemiology of the serotypes causing invasive pneumococcal disease has been observed making the surveillance of circulating serotypes especially relevant. Some recent studies have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ...[more]

PMID: 30753210

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Project description:BackgroundSerotyping of Streptococcus pneumoniae is important for monitoring of vaccine impact. Unfortunately, conventional and molecular serotyping is expensive and technically demanding. This study aimed to determine the ability of matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry to discriminate between pneumococcal serotypes and genotypes (defined by global pneumococcal sequence cluster, GPSC). In this study, MALDI-TOF mass spectra were generated for a diverse panel of whole genome sequenced pneumococcal isolates using the bioMerieux VITEK MS in clinical diagnostic (IVD) mode. Discriminatory mass peaks were identified and hierarchical clustering was performed to visually assess discriminatory ability. Random forest and classification and regression tree (CART) algorithms were used to formally determine how well serotypes and genotypes were identified by MALDI-TOF mass spectrum.ResultsOne hundred and ninety-nine pneumococci, comprising 16 serotypes and non-typeable isolates from 46 GPSC, were analysed. In the primary experiment, hierarchical clustering revealed poor congruence between MALDI-TOF mass spectrum and serotype. The correct serotype was identified from MALDI-TOF mass spectrum in just 14.6% (random forest) or 35.4% (CART) of 130 isolates. Restricting the dataset to the nine dominant GPSC (61 isolates / 13 serotypes), discriminatory ability improved slightly: the correct serotype was identified in 21.3% (random forest) and 41.0% (CART). Finally, analysis of 69 isolates of three dominant serotype-genotype pairs (6B-GPSC1, 19F-GPSC23, 23F-GPSC624) resulted in the correct serotype identification in 81.1% (random forest) and 94.2% (CART) of isolates.ConclusionsThis work suggests that MALDI-TOF is not a useful technique for determination of pneumococcal serotype. MALDI-TOF mass spectra appear more associated with isolate genotype, which may still have utility for future pneumococcal surveillance activities.

Project description:Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis.In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes.The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database.The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

Project description:BackgroundAnopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used.MethodsTo determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal.ResultsUsing the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS.ConclusionsThe using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.

Dataset Information

Utility of MALDI-TOF MS as a new tool for Streptococcus pneumoniae serotyping.

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Utility of MALDI-TOF MS as a new tool for Streptococcus pneumoniae serotyping.

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