Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility.

نویسندگان

  • Kathryn A Harris
  • Paul Turner
  • Elaine A Green
  • John C Hartley
چکیده

We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of > 1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of < or = 0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Rapid real-time PCR for determination of penicillin susceptibility in pneumococcal meningitis, including culture-negative cases.

A novel real-time PCR-hybridization assay was developed for the rapid (<1 h) detection of penicillin susceptibility in Streptococcus pneumoniae. When applied to 24 pneumococcal DNA-positive cerebrospinal fluid extracts, penicillin-sensitive S. pneumoniae was detected in all instances. Real-time PCR proved more sensitive than culture, microscopy, or antigen detection and provided susceptibility ...

متن کامل

Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae.

BACKGROUND Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macro...

متن کامل

A novel real-time duplex PCR assay for detecting penA and ponA genotypes in Neisseria gonorrhoeae: Comparison with phenotypes determined by the E-test.

BACKGROUND For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved...

متن کامل

545 - 556 Farah Al-Marzooq.pmd

Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multi...

متن کامل

Development of TaqMan Duplex Real-time PCR for Simultaneous Detection of Chlamydia Trachomatis and Mycoplasma Genitalium

Background and Objective: Sexually infections transmitted by bacteria are one of thetherapeutic and social problemsworldwide. The Real-time PCR assay is one of the most sensitive diagnostic and screening methods for these infections. The purpose of this study wassimultaneous detection of Chlamydia trachomatis and Mycoplasma genitaliumusing the TaqMan duplex real-time polymerase chain reaction. ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Journal of clinical microbiology

دوره 46 8  شماره 

صفحات  -

تاریخ انتشار 2008