Rapid Detection of Carbapenemase-producing Genes by Multiplex Real-Time PCR with Melting Curve Analysis using a BD MAX™ Open System.

Recently, the global spread of carbapenemase-producing Enterobacterales has become a concern, and rapid detection methods are required. We have developed a rapid and inexpensive multiplex real-time PCR with melting curve analysis using the BD MAX™ system and evaluated it. We used 31 carbapenemase-producing Gram-negative bacteria (blaIMP group, 12; blaGES group, 6; blaNDM group, 5; blaVIM group, 3; blaKPC group, 3; blaOXA-48-like group, 1 strain; and blaIMP group + blaGES, 1 strain), 10 AmpC-producing Gram-negative bacteria, and 10 ESBLproducing Gram-negative bacteria. A BD MAX™ open platform system was used. Carbapenemase-positive and carbapenemase-negative strains were correctly identified 30 of 31 (excluding a blaIMP group and blaGES group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: blaIMP group, 81.2±0.5°C; blaVIM group, 91.8±0.4°C; blaNDM group, 85.4±0.3°C; blaGES group, 90.5±0.3°C; blaKPC group, 94.1±0.5°C; and blaOXA-48-like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the blaGES group could be detected in the strains producing both blaIMP group and blaGES group simultaneously, suggesting that only the genotype with the highest expression level could be captured in cases of simultaneous production. In the carbapenemase-negative strains, no obvious peaks were observed in the 20 AmpC and 20 ESBL-producing Gram-negative bacteria, and even when Tm values were detected, the dF/dT values were low and easily differentiated. This method appears to be very useful as a rapid and inexpensive test that can provide detection in about 2 hours.

Analysis of Clinical Isolates of Extended-Spectrum ß-Lactamase-Producing Bacteria with Primer and Probe Sets Developed to Detect blaCTX-M, blaTEM, and blaSHV Using a Fully Automated Gene Detection System.

In this study, we evaluated extended-spectrum ß-lactamase (ESBL)-producing bacteria with the newly developed primer and probe sets to detect blaCTX-M, blaTEM, and blaSHV using BD MAXTM, a fully automated multiplex polymerase chain reaction assay system. In 36 isolates confirmed by whole-genome sequencing to have blaCTX-M, blaTEM, or blaSHV, the developed primer and probe sets accurately detected each gene without being influenced by the presence of other ß-lactamase genes. In nine control strains that do not harbor either blaCTX-M, blaTEM, or blaSHV no cross-reaction was observed. In 191 strains phenotypically determined to be ESBL-producers by conventional antimicrobial susceptibility tests, 189 strains were blaCTX-M-, blaTEM-, or blaSHV-positive as assessed by BD MAXTM using the developed primer and probe sets, and two strains were negative for these genes. Whole-genome sequencing revealed that these two strains were phenotypically false-positive ESBL-producers. The accuracy of the primer and probe sets seems to be satisfactory, and they may be applicable to detect CTX-M-type ESBL-producing bacteria.

Real-time PCR investigation of the prevalence of Fusobacterium necrophorum in patients with pharyngitis in Japan.

PURPOSE: Recent data suggest an association between Fusobacterium necrophorum infection and pharyngotonsillitis among adolescents and adults. However, existing reports are only from North America and Europe. We aimed to identify and compare the prevalence of F. necrophorum among patients with pharyngitis and asymptomatic controls in Japan and clarify the epidemiological characteristics of pharyngitis. METHODS: Patients aged ≥16 years with pharyngitis and asymptomatic controls were prospectively included. F. necrophorum was detected by using both conventional culture methods and real-time F. necrophorum-specific PCR targeting the rpoB gene. The prevalence of ß-hemolytic streptococci was also identified and compared between groups. RESULTS: Forty-four pharyngitis patients and 31 asymptomatic controls were included. F. necrophorum was identified using PCR in 6 (13.6%) pharyngitis cases and 2 (6.5%) controls, with no significant difference (p = 0.457). The median bacterial load of F. necrophorum identified with real-time PCR was significantly higher in pharyngitis cases than in controls (p = 0.046). Patients with a high Centor Score tended to have a higher bacterial load than those with a low Centor Score and controls. In cases of pharyngitis, the prevalence of F. necrophorum was similar to that of Streptococcus pyogenes (F. necrophorum-positive: 6 [13.6%] vs. S. pyogenes-positive: 5 [11.4%], p = 0.99). CONCLUSION: F. necrophorum was similarly prevalent among pharyngitis cases as S. pyogenes in Japan. The association of higher F. necrophorum bacterial load with symptomatic pharyngitis in accordance with the previous findings from a different geographical region suggests that F. necrophorum is an important causative agent of bacterial pharyngitis.

Fusobacterium necrophorum Subsp. funduliforme in Tonsils from Various Patient Populations in Japan.

Fusobacterium necrophorum has recently been suggested to be associated with tonsillopharyngitis, peritonsillar abscess, and recurrent tonsillitis. Between the 2 subspecies of F. necrophorum, subsp. funduliforme is known to be a major human pathogen. To better understand the epidemiology of F. necrophorum subsp. funduliforme (FNSF), we studied the prevalence of FNSF in the tonsils of patients undergoing elective tonsillectomy (TE) for different indications. Adult patients who underwent elective TE from October 2014 to November 2015 were included. The tonsils were sent for aerobic and anaerobic tissue culture within 30 min of excision; the presence of FNSF was detected using PCR with gyrB primers and 16S rRNA. A total of 32 patients were enrolled. The prevalence of FNSF identified by either culture or gyrB PCR did not significantly differ between infectious and noninfectious TE indications. The constant presence of FNSF might not be associated with recurrent pharyngotonsillitis.