Accurate, fast and cost-effective simultaneous detection of bacterial meningitis by qualitative PCR with high-resolution melting.

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are leading causes of meningitis and acute invasive infections. PCR-based methods are widely used for the diagnosis and surveillance of bacterial pathogens because of their high sensitivity, specificity and high-throughput capabilities compared with conventional laboratory methods. This study evaluated a high-resolution melting qualitative PCR analysis method for the simultaneous detection of these three pathogens. The assay has been optimized to detect three species-specific genes of each organism isolated from clinical samples, enabling accurate identification of the etiological agent. The method proved to be highly sensitive and cheaper than the real-time PCR TaqMan® system because it is probe-free; it could be used for the diagnosis of invasive diseases in public health laboratories of developing countries.

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

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.

Fatal case of co-infection with dengue virus and Neisseria meningitidis during a dengue epidemic in the state of Rio de Janeiro, Brazil.

INTRODUCTION: Dengue and meningococcal disease are caused by two different agents: a flavivirus and a Gram-negative bacterium, respectively. The first symptoms of both diseases can be indistinct and a rapid and accurate diagnosis is crucial, considering that both diseases are associated with high morbidity and mortality, representing a major public-health problem in Brazil. CASE PRESENTATION: We report a fatal case of co-infection of dengue virus (DENV) and Neisseria meningitidis in a 54-year-old patient. The serum tested positive for DENV NS1 antigen, and N. meningitidis serogroup C was detected by nspA-PCR. Following the initial positive result for DENV infection, rRT-PCRwas performed and DENV-4 was confirmed. CONCLUSION: Our report highlights the importance of accurate differential diagnosis during periods of high circulation of DENV, in order to provide adequate management and an improved outcome.

PorA variable antigenic regions VR1, VR2, and VR3 of Neisseria meningitidis serogroups B and C isolated in Brazil from 1999 to 2004.

The high genetic diversity found among the PorA regions VR1 and VR2 of 101 Neisseria meningitidis isolates from patients with meningococcal disease and healthy carriers in Brazil contrasts with the stability found in the PorA VR3 of these isolates. The presence of VR3 epitope variant 35 or 36 on the surfaces of 87% of the strains analyzed suggests that these antigens should be considered for inclusion in new formulations of vaccines against serogroup B meningococci in Brazil.