Novel genus-specific PCR-based assays for rapid identification of Neisseria species and Neisseria meningitidis.

This study presents the development of polymerase chain reaction (PCR)-based tests for the identification and detection of Neisseria species and Neisseria meningitidis. Currently, isolating and identifying these pathogens using conventional biochemical methods require 48-72 h. To improve speed and accuracy in diagnosing Neisseria infections, simple PCR-based tests that are specific for the genus Neisseria and the species Neisseria meningitidis have been developed. The genus-specific and species-specific DNA sequences were chosen by selecting and analyzing available database sequences. Neisseria-specific and Neisseria meningitidis-specific primer pairs were derived from the genes asd (coding for the aspartate beta-semialdehyde dehydrogenase) and ctrA (coding for a conserved outer membrane protein), respectively. Both the Neisseria-specific and Neisseria meningitidis-specific PCR assays were specific (they amplified only DNA from the target genus or species, out of 84 bacterial species tested). In addition, the Neisseria-specific assay amplified DNA from 321 of 322 strains tested representing 13 species of Neisseria, while the Neisseria meningitidis-specific assay amplified DNA from all 256 strains tested representing nine serogroups of Neisseria meningitidis. These PCR assays, which can be combined in multiplex, have been adapted to ensure that they are simple and can be performed within approximately 90 min. The tests provide new diagnostic tools for identifying Neisseria infections.

Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis.

Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin (mecA), gentamicin ¿aac(6')-aph(2"), and erythromycin (ermA, ermB, ermC, and msrA). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of beta-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.

Ribavirin potentiates the efficacy and toxicity of 2',3'- dideoxyinosine in the murine acquired immunodeficiency syndrome model.

The efficacy and toxicity of ribavirin (25 or 125 mg/kg/day), 2',3'-dideoxyinosine (ddI) (200 mg/kg/day) and a combination of both drugs at these doses given for 6 weeks were investigated in the murine acquired immunodeficiency syndrome model. Our results showed a significant protection against splenomegaly, lymphadenopathy and hypergammaglobulinemia in mice treated with ribavirin at 25 mg/kg/day alone or in combination with ddI at 200 mg/kg/day. A good synergistic effect was observed with the drug combination, whereas ddI alone (200 mg/kg/day) did not give any protection. Ribavirin/ddI combination protected against the loss of CD8 T cells in spleen and restored the capacity of splenocytes to proliferate after activation with a mitogenic agent. Moreover, the drug combination resulted in a protection of the spleen and cervical lymph node architectures and a regression of germinal centers. Hematotoxicity appeared at a dose of 125 mg/kg of ribavirin alone and increased when used concomitantly with ddI. In conclusion, ribavirin and ddI at low doses are synergistic and effective in the murine acquired immunodeficiency disease model, but at high doses they are toxic.