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Serotyping of Streptococcus pneumoniae based on capsular genes polymorphisms

ABSTRACT: Diagnostic primer extension assay to serotype Streptococcus pneumoniae. Assay validation. Background: Monitoring of Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. To simplify S. pneumoniae serotyping, a novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Results: Autolysin (lytA), pneumolysin (ply) and eight genes located in the capsular operon (cps) were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system that transforms genetic typing data into capsular serotype identification. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. In addition, the assay was tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitidis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. The assay presented with no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. Conclusion: This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction.

ORGANISM(S): Streptococcus pneumoniae

PROVIDER: GSE45865 | GEO | 2013/12/31

SECONDARY ACCESSION(S): PRJNA196513

REPOSITORIES: GEO

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Project description:Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. Background Dengue is a mosquito-borne viral infection causing a major public health problem globally. Dengue virus (DENV) is the causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF) and includes four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). DENV-2 and DENV-3 have been associated with severe dengue disease, consequently, laboratory testing for DENV is needed to confirm the diagnosis of DENV infection, serotype and to differentiate dengue from other febrile tropical illnesses. In addition, surveillance of mosquitoes infected with DENV is needed to monitor the infection rates within vector mosquito populations harboring specific serotype to provide an early warning sign to predict epidemics. Results In this work we have applied microarray analysis to simultaneously determine the serotype of multiple RNA samples from human or mosquitoes. The proposed microarray method can be used for i) rapid and reliable dengue diagnosis; ii) serotyping and iii) surveillance of mosquitoes infected with dengue. These microarrays were useful to confirm the presence of DENV-2 in 94 serum samples, DENV-3 in three samples from Juchitan, Oaxaca and one case from Juchitan, Oaxaca contained DENV-2 and -3. Moreover by using these microarrays we also determined DENV in pools of gravid females mosquitoes collected in several sites of nineteen Mexican states in 2005. Mosquito pools from 31 cities in the states of Yucatan, Campeche, Tabasco, Chiapas, Veracruz, Oaxaca, Guerrero, Tamaulipas and Colima were infected with DENV-2, six cities in Yucatán, Tabasco, Morelos, Tamaulipas, Colima, and Nayarit with DENV-1, three from Tabasco, Veracruz and Oaxaca with DENV 3 and two with two serotypes simultaneously (Ciudad Mante with DENV-1 and DENV-2, and Tavela with DENV-2 and DENV-3). Conclusion Here we show the success of applying microarrays assay to provide a consistently robust qualitative detection of dengue serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) in serum samples from patients or in pools of gravid female mosquitoes collected in the field of nineteen Mexican states. Interestingly, we did not detect any mosquito or serum sample containing DENV-4.

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Serotyping of Streptococcus pneumoniae based on capsular genes polymorphisms

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