Characterisation of Neisseria meningitidis cc11/ET-15 variant by whole genome sequencing

BACKGROUND Neisseria meningitidis strains belonging to clonal complex 11 is the cause of numerous outbreaks and epidemics in the United States, Canada and Europe, accounting for 49.5% of cases of meningococcal disease caused by serogroup C worldwide. In Brazil, it is the second most frequent clonal complex within this serogroup. The genetic characterisation of cc11/ET-15 variants is important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, to support specific actions of Health Surveillance aiming outbreaks control. OBJECTIVES The objective of this study was to identify features in the genome of cc11/ET-15 clones through whole-genome sequencing (WGS), that differ from cc11/non-ET-15 strains that could explain their virulence. METHODS The whole genome of three cc11/ET-15 representative strains were sequenced with a minimum coverage of 100X with the MiSeq System and compared to the genome of cc11/non-ET-15 strains. RESULTS Genome analysis of cc11/ET-15 variants showed the presence of resistance factors, mobile genetic elements and virulence factors not found in cc11/non-ET-15 strains. MAIN CONCLUSIONS Our results show that these strains carry virulence factors not identified in cc11/non-ET-15 strains, which could explain the high lethality rates attributed to this clone worldwide.

Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

Abstract Background Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. Methods A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. Results All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. Conclusions The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Validation of PCR method for mycoplasma detection in the Yellow Fever-vaccine quality control / Validação de método de PCR para detecção de micoplasmas no controle de qualidade da vacina de Febre Amarela

Among the vaccines produced by Bio-Manguinhos, a major centre for manufacturing the immunobiological products in Latin America, stands out the yellow fever (YF) vaccine. To guarantee the excellence and safety of the YF vaccine, the quality control tests has to be performed throughout its production. The World Health Organization (WHO) demands the producers to guarantee the absence of Mycoplasma orale, M. pneumoniae, M. gallisepticum and M. synoviae in the biological products. Mycoplasma is a fastidious microorganism, requiring about 35 days for attaining the conclusive culturing test. In this study PCR methods were selected for amplifying 16S rRNA gene fragments for detecting mycoplasma in the intermediate products of YF vaccine. This standardized methodology was specific and sensitive to detect the low concentrations of mycoplasma in spiked intermediary vaccine products; and the absence of unspecific amplification was also demonstrated. The detection rates ranged from 3.1 to 12.5 colony forming units and showed 100 % of sensitivity and specificity in the tested samples. The PCR protocol for detecting mycoplasmal DNA in YF vaccine was validated by analysing 286 samples. Bio-Manguinhos produces annually 10,000,000 YF vaccine doses, and this method has been successfully employed, complementing the traditional approach in the mycoplasma detection since 2008.

Dentre as vacinas produzidas por Bio-Manguinhos, um importante centro de produção de imunobiológicos da América Latina, destaca-se a vacina de febre amarela (FA) que é produzida em ovos embrionados. Para garantir a excelência e a segurança da vacina, testes de controle de qualidade são realizados durante a produção. A Organização Mundial de Saúde (OMS) exige dos produtores a ausência de Mycoplasma orale, M. pneumoniae, M. gallisepticum e M. synoviae em produtos biológicos. Micoplasmas são micro-organismos fastidiosos, sendo necessários 35 dias para que os testes de cultura sejam conclusivos. Neste estudo foram selecionados métodos de amplificação de fragmentos do gene 16S rRNA para detecção de micoplasmas em produtos intermediários da vacina de FA. Esta metodologia padronizada foi capaz de detectar baixas concentrações de micoplasmas nos produtos intermediários e a ausência de amplificação inespecífica foi demonstrada. O limite de detecção variou entre 3,1 e 12,5 unidades formadoras de colônia; e nas amostras testadas a sensibilidade e a especificidade foram de 100 %. O protocolo de PCR para detecção de micoplasmas na vacina foi validado pela análise de 286 amostras. Bio-Manguinhos produz 10.000.000 doses de vacina de febre amarela por ano e, desde 2008, este método tem sido empregado com sucesso, complementando-se a abordagem tradicional.