Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Commensal Streptococcus agalactiae isolated from patients seen at University Hospital of Londrina, Paraná, Brazil: capsular types, genotyping, antimicrobial susceptibility and virulence determinants.

BACKGROUND: Streptococcus agalactiae or Group B Streptococci (GBS) have the ability to access various host sites, which reflects its adaptability to different environments during the course of infection. This adaptation is due to the expression of virulence factors that are involved with survival, invasion and bacterial persistence in the host. This study aimed to characterize GBS isolates from women of reproductive age seen at University Hospital of Londrina, according to capsular typing, genetic relatedness, antimicrobial susceptibility profile and occurrence of virulence determinants. RESULTS: A total of 83 GBS isolates were enrolled in this study. Capsular types Ia (42.2%), II (10.8%), III (14.5%) and V (30.1%) were identified in most GBS. One isolate each was classified as type IX and non-typeable.A total of 15 multiple locus variable number of tandem repeat analysis (MLVA) types were identified among the isolates, seven were singletons and eight were represented by more than four isolates. All isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin. Resistance to erythromycin and clindamycin was observed in 19.3 and 13.3% of isolates, respectively. All isolates resistant to clindamycin were simultaneously resistant to erythromycin and were distributed in the capsular types III and V. One isolate showed the constitutive macrolide-lincosamide-streptogramin B (cMLS(B)) phenotype and ten showed the inducible MLS(B) (iMLS(B)) phenotype. The mechanism of resistance to erythromycin and clindamycin more prevalent among these isolates was mediated by the gene ermA, alone or in combination with the gene ermB. The isolates displaying resistance only to erythromycin belonged to capsular type Ia, and showed the M phenotype, which was mediated by the mefA/E gene. All isolates harbored the gene hylB and at least one pilus variant, PI-1, PI-2a or PI-2b. Although cylE was observed in all GBS, four isolates were classified as gamma-hemolytic and carotenoid pigment non-producers. CONCLUSIONS: Our results indicate the potential virulence of commensal GBS isolates, reinforcing the need for continued screening for this bacterium to prevent infections. The distribution of capsular and pili antigens, and MLVA profiles was also identified, which may contribute to the development of new strategies for the prevention and treatment of GBS infection.

Prevalence and antimicrobial susceptibility profile of Streptococcus agalactiae in pregnant women seen at the University Hospital of Londrina, Paraná, Brazil / Prevalência e sensibilidade aos antimicrobianos de Streptococcus agalactiae em gestantes atendidas no Hospital Universitário de Londrina, Paraná, Brazil

A retrospective study of pregnant women seen at the University Hospital of Londrina, Paraná, Brazil was performed to determine the prevalence of Group B Streptococcus (GBS) vaginal-rectal colonization, and the GBS susceptibility for antimicrobials used in intrapartum antibiotic prophylaxis. A vaginal-rectal swab was collected from 2,901 women between 35 and 37 weeks of gestation. Of these, 527 (18.2%) had a positive culture for GBS, and 0.4%, 10.2% and 10% of the isolates were resistant to penicillin, erythromycin and clindamycin, respectively. These results highlight the importance of continuous surveillance of GBS colonization in pregnant women for preventing GBS infections in neonates.

Um estudo retrospectivo foi realizado com gestantes atendidas no Hospital Universitário de Londrina, Paraná, Brasil para determinar a prevalência de colonização vaginal-retal por estreptococos do Grupo B (EGB) e o perfil de sensibilidade de EGB aos antimicrobianos utilizados para a antibioticoterapia profilática intraparto. Swabs vaginais-retais foram coletados de 2.901 mulheres entre a 35ª e 37ª semana de gestação. Destes, 527 (18,2%) apresentaram cultura positiva para EGB, e 0,4%, 10,2% e 10% dos isolados foram resistentes à penicilina, eritromicina e clindamicina, respectivamente. Estes resultados destacam a importância de vigilância contínua da colonização por EGB em gestantes para a prevenção de infecções em neonatos por EGB.