Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture / 대한임상미생물학회지

BACKGROUND: We evaluated multiplex PCR for species identification and toxin typing to improve the sensitivity and turnaround time of toxigenic Clostridium difficile culture (TCDC). METHODS: We performed multiplex PCR using primers targeting the species-specific gene, tpi, and the toxin genes, tcdA and tcdB. From January to March 2008, 528 stool specimens were tested with direct toxin assay (DT) using C. difficile Tox A/B II (Techlab, Blacksburg, USA) and TCDC. For 288 specimens from early study period, toxin production by C. difficile isolates of TCDC was measured by enzyme immunoassay with culture supernatants using VIDAS C. difficile Toxin A&B (CDAB;bioMerieux, Marcy-l'Etoile, France) and multiplex PCR with isolated colonies. For 240 specimens from late period, only multiplex PCR was used to test toxin production by the isolates. RESULTS: During the early period, 29 C. difficile were isolated and their toxin-positive rates were 65.5% by PCR and 44.8% by CDAB (P<0.05). Among 528 stool specimens, the results of DT+/TCDC+, DT+/ TCDC-, and DT-/TCDC+ were 32 (6.1%), 33 (6.3%), and 10 (1.9%), respectively, when tested with PCR. 13.3% of total 75 positive specimens was detected only by TCDC. Of the 42 toxigenic C. difficile isolates, all were positive for tpi, 30 (71.4%) were tcdA+/tcdB+, and 12 (28.6%) were tcdA-/tcdB+. CONCLUSION: TCDC using multiplex PCR for species identification and toxin typing is sensitive and rapid to be used as a routine diagnostic test.

Evaluation of MRSASelect for Detecting Methicillin-Resistant Staphylococcus aureus from Surveillance Specimens / 병원감염관리

BACKGROUND: A rapid and sensitive surveillance culture has a pivotal role in infection control of methicillinresistant Staphylococcus aureus (MRSA). This study was aimed to compare the performance of MRSASelect (Bio-Rad, France) to that of mannitol salt agar containing 6 microgram/mL of oxacillin (MSA-OX) for detecting MRSA in surveillance cultures. METHOD: From May to June 2006, 86 nasal swabs and 21 sputum specimens were enrolled. All specimens were inoculated onto MRSASelect and MSA-OX, which were incubated for 2 days and 3 days, respectively, and colonies were read daily by a technologist. Pink colonies on MRSASelect and yellow colonies on MSA-OX were examined with Gram stain, Pastorex(R) Staph-plus (Bio-Rad) and mecA-PCR. After the final reading, both media were re-examined by a superviser. RESULTS: Of the 107 specimens cultured, 32 (29.9%) were positive for MRSA. Of these, 27 were detected by both media, one by MSA-OX only, and 4 by re-examination. The day-1 and day-2 sensitivities/specificities of MRSASelect were 78.1%/97.3% and 84.4%/97.3%, respectively, while those of MSA-OX were 53.1%/100% and 78.1%/92.1%, respectively. With MRSASelect, two more positives were detected at day 2, but their incubation was less than 18 hour at day 1. There were six false positive organisms detected: three Enterobacter spp., one Acinectobacter spp., and two coagulase-negative staphylococci (CNS). But, the two CNS grew on MSA-OX only. CONCLUSION: MRSASelect with 1-day incubation showed a sensitivity equivalent to and a specificity better than MSA-OX with 2-day incubation. MRSASelect should be a useful medium for MRSA surveillance when it is read after an incubation of 18-28 hours with the confirmatory Gram stain of screen-positives.