Search

The ResPlex I assay (Qiagen) was designed to amplify and detect DNA of six bacterial respiratory pathogens. This assay was compared with real-time PCR assays based upon the same target sequences for the ability detect the target bacteria by use of both stock strains and specimens from respiratory disease patients. The ResPlex I assay is somewhat less sensitive than real-time PCR assays but offers the advantage of multiple assays in a single reaction.

Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia in children and young adults in the United States. Rapid and reliable identification of N. meningitidis serogroups is crucial for judicious and expedient response to cases of meningococcal disease, including decisions about vaccination campaigns. From 1997 to 2002, 1,298 N. meningitidis isolates, collected in the United States through the Active Bacterial Core surveillance (ABCs), were tested by slide agglutination serogrouping (SASG) at both the ABCs sites and the Centers for Disease Control and Prevention (CDC). For over 95% of isolates, SASG results were concordant, while discrepant results were reported for 58 isolates. To resolve these discrepancies, we repeated the SASG in a blinded fashion and employed ctrA and six serogroup-specific PCR assays (SGS-PCR) to determine the genetic capsule type. Seventy-eight percent of discrepancies were resolved, since results of the SGS-PCR and SASG blinded study agreed with each other and confirmed the SASG result at either state health laboratories or CDC. This study demonstrated the ability of SGS-PCR to efficiently resolve SASG discrepancies and identified the main cause of the discrepancies as overreporting of these isolates as nongroupable. It also reemphasized the importance of adherence to quality assurance procedures when performing SASG and prompted prospective monitoring for SASG discrepancies involving isolates collected through ABCs in the United States.

We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.