Optimization of triplex real time PCR for detecting Staphylococcus aureus mecA, pvl and nuc genes.

Multiplex polymerase chain reaction (PCR) allows simultaneous detection of two or more genes, using the same reaction conditions, and so it is possible the rapid detection of methicillin resistant Staphylococcus aureus strains (MRSA) in clinical specimens. This study aimed to implement, for the first time in our laboratory, a triplex real time PCR (RT-PCR) technique for detection of genes encoding resistance to oxacillin and synthesis of Panton Valentine leukocidin (pvl), a pathogenicity factor characteristic for community acquired strains (CA-MRSA). The application of this method will permit the epidemiological surveillance of circulating strains and early application of prevention measures.

Triplex real-time pcr for detection of methicillin-resistant staphylococcus aureus and Panton-Valentine leukocidin in North-East Romania.

UNLABELLED: The aim of the present study was to investigate S. aureus isolates for the presence of methicillin-resistance and Panton-Valentine leukocidin (PVL) genes and to further characterize positive strains by means of antibiotic resistance patterns. MATERIAL AND METHODS: We used a triplex Real-Time PCR method for simultaneous detection of nuc, mecA and pvl genes in clinical isolates from 188 patients admitted to "Sf. Parascheva" Infectious Diseases Hospital lasi, during a 3 year period (2008-2010). RESULTS: The study revealed a relatively high rate of PVL-producing strains (23.93%), mainly community-associated (CA-MRSA) (51.11%). Most pvl-positive CA-MRSA isolates were resistant to erythromycin (91.3%), but none was resistant to clindamycin, fluoroquinolones, rifampicin, chloramphenicol or fusidic acid. CONCLUSIONS: Antibiotic susceptibility testing showed a high rate of multidug-resistance among strains classified as CA-MRSA (54.83%), but not among PVL-producers (4.44%). Although resistance to fusidic acid was previously proposed as a marker for PVL-producing CA-MRSA, our data suggest that we cannot rely on resistance to fusidic'acid to screen for PVL-producing CA-MRSA in our setting.

[Use of real time PCR for testing Staphylococcus aureus isolates from the Iasi Infectious Diseases Hospital]. / Utilizarea real time PCR pentru testarea izolatelor clinice de Staphylococcus aureus din Spitalul Clinic de Boli Infectioase Iasi.

UNLABELLED: S. aureus is capable of producing a wide spectrum of diseases and can quickly develop resistance to antibiotics. These features require a careful monitoring of these organisms, by detection of resistance genes and virulence factors, such as Panton-Valentine leukocidin (PVL). AIM: To determine the presence of mecA and pvl genes in S. aureus isolates by a Real Time-PCR technique (RT-PCR) in order to shorten the detection time. MATERIALS AND METHODS: We tested 119 strains isolated from pus, using phenotypic methods for methicillin resistance characterization, according to CLSI 2008-2010 guidelines. Detection of mecA and pvl genes was done with hydrolysis probes. RESULTS: The prevalence rate of methicilin resistant S. aureus (MRSA) was 40,33%, and pvl was detected in 52,08% of those strains. The results of the conventional methods for methicillin resistance detection were validated by those obtained by RT-PCR CONCLUSIONS: RT-PCR is useful in epidemiological surveillance of MRSA and PVL-producing strains and validation of test results for phenotypic resistance to oxacillin.

[Analysis of 62 cases of nosocomial staphylococcal meningitis admitted to the Iasi Hospital of Infectious Diseases over a period of 21 years]. / Analiza asupra a 62 de cazuri de meningita stafilococica nosocomiala internate în Spitalul de Boli Infectioase Iasi într-o perioada de 21 de ani.

AIM: To analyze the clinical features, evolution and treatment of nosocomial staphylococcal meningitis admitted to Hospital of Infectious Diseases Iasi between 1988 and 2007. MATERIAL AND METHOD: The data were gathered from the archive of Hospital of Infectious Diseases Iasi. We studied the clinical and laboratory features of 62 cases of nosocomial staphylococcal meningitis admitted in the study period. RESULTS: The development of nosocomial meningitis was subsequent to interventions on the central nervous system for haematoma, ventriculo-peritoneal shunts, tumors, spinal anesthesia. The symptoms were fever, headache, stiff neck and vomiting. The etiological agents were: Staphylococcus aureus 37 in cases and coagulase negative staphylococci in 25 cases. The treatment was according to their susceptibility rates, using systemic quinolones and ceftazidime or colymicin. The evolution was favorable in 52 patients and unfavorable in another 10. CONCLUSION: Nosocomial staphylococcal meningitis remains a serious condition due to the fact that the etiological agents are resistant to the classical antistaphylococcal antibiotics and increasingly resistant to methicillin.

[MecA and pvl genes detection in Staphylococcus aureus strains isolated from lower respiratory tract infections]. / Detectarea genelor mecA si pvl la tulpini de Staphylococcus aureus izolate din infectii ale tractului respirator inferior.

UNLABELLED: Although community-acquired Staphylococcus aureus pneumonia with highly virulent Panton-Valentine leukocidin (PVL)-positive strains, a severe disease with significant lethality, is rare, especially in adult and adolescent patients, recent reports highlight that these infections are on the rise. On the other hand, methicillin-resistant S. aureus (MRSA) is one of the high-risk and potential multi-drug resistant microorganisms. OBJECTIVES: The aim of this study was to investigate the prevalence of mecA and pvl genes in S. aureus strains isolated from lower respiratory tract infections and to assess the antibiotic resistance profile of these strains. MATERIAL AND METHOD: The antibiotic susceptibility testing was performed by the disk diffusion method according to CLSI recommendations in 32 consecutive non-repeated S. aureus strains isolated from sputum specimens and endotracheal aspirates of hospitalized patients over the period from January 2005 to December 2009. Only 20 strains (2008-2009) were tested for the presence of mecA and pvl genes by real-time PCR and detection with specific fluorescence probes. RESULTS: Of the 32 S. aureus isolates, 68.7% were MRSA. MRSA strains showed higher resistance rates to gentamicin, tetracycline, rifampicin, fluoroquinolones comparing to the methicillin susceptible ones. Only one strain produced pvl; it was isolated from a 7 year old child with lethal sepsis with pulmonary and meningeal secondary localisations. CONCLUSIONS: Glicopeptides and linezolid are therapeutic options indicated in the treatment of staphylococcal pneumonia produced either by MRSA strains or pvl positive S. aureus strains.

[Study of nosocomial bacillary meningitis admitted in the Clinic of Infectious Diseases Iasi on a 20 years period]. / Studiu asupra meningitei nosocomiale cu bacili gram- negativi internate in Clinica de Boli Infectioase Iasi pe o perioada de 20 de ani.

AIM: To analyze the clinical features, etiology and evolution of nosocomial bacillary meningitis admitted to the Clinic of Infectious Diseases Iasi between 1988 and 2007. MATERIAL AND METHOD: The data were gathered from the archive of the Clinic of Infectious Diseases Iasi. We studied the clinical and laboratory features of 28 cases of nosocomial bacillary meningitis admitted in the study period. RESULTS: The development of nosocomial meningitis was subsequent to interventions on the central nervous system for hematoma, ventriculo-peritoneal shunts, tumors, rah anesthesia. The symptoms were fever, headache, stiff neck and vomiting. The etiological agents were: Acinetobacter spp--35.71%, Pseudomonas spp.--25%, Klebsiella pneumoniae--17.85%, Escherichia coli--14.28%, and mixed etiology, in 3.57% of the cases each. The treatment was according to their susceptibility, using systemic quinolones and ceftazidime or colymicin. The evolution was favorable in 13 patients and unfavorable in another 15. CONCLUSION: Nosocomial meningitis caused by Gram negative bacilli is a severe infection, with diverse etiology, which raises therapeutical problems due to antibiotic resistance and a high mortality rate.