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. 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. 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. 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.

To investigate the occurrence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and their clonal relationships from hospital and municipal wastewater treatment plants (WWTPs). Eighteen K. pneumoniae strains recovered from three WWTPs were identified by MALDI-TOF. The antimicrobial susceptibility were evaluated by disk-diffusion and the carbapenemases production by Carbapenembac®. The carbapenemases genes were investigated by real-time PCR and the clonal relationship through multi-locus sequence typing (MLST). Thirty nine percent (7/18) of isolates were classified as multidrug-resistant (MDR), 61.1% (11/18) extensively drug-resistant (XDR) and 83.3% (15/18) showed carbapenemase activity. Three carbapenemase-encoding genes were found, bla_KPC (55%), bla_NDM (27.8%) and bla_OXA-370 (11.1%) as well five sequencing types ST11, ST37, ST147, ST244 and ST281. ST11 and ST244, sharing four alleles were grouped into clonal complex 11 (CC11). Our results show the importance of monitoring antimicrobial resistance in WWTPs effluents to minimize the risk of spreading bacterial load and ARGs in aquatic ecosystems, using advanced treatment technologies to reduce these emerging pollutants at WWTPs.

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.

Coccidioidal pericarditis is a condition found in approximately 1–5 % of patients infected by Coccidioides species. It is associated with widely diverse clinical symptoms. This paper reports a case of coccidioidal pericarditis diagnosed by an in-house Coccidioides posadasii antigen and confirmed with mycological and molecular methods. From February to September 2005, the patient suffered from fever, weight loss, a non-productive cough, thoracic pain and tachycardia. He received a positive diagnosis of coccidioidal pericarditis only in October 2005. The macromorphological examination of the culture showed a whitish felt-like colony, which became brownish with age. Preparations in lactophenol cotton blue stain showed hyaline septate hyphae with fragmentation and thin arthroconidia-like structures. Pericardial fluid and sera samples were positive for Coccidioides antibodies by immunodiffusion and ELISA with a C. posadasii in-house antigen preparation. The C. posadasii identification was confirmed by nested PCR of the antigen 2/proline-rich antigen (Ag2/PRA) encoding gene.