Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common causes of sexually transmitted infections (STI). Infection by these microorganisms can lead to clinical syndromes ranging from mild urethritis to pelvic inflammatory disease. However, both organisms can also cause asymptomatic infections in women and men; these infections can proceed in women to an ascending infection of the fallopian tubes (3,10,14). The outcome of the ascending infection can be reduced fertility, infertility, or even extrauterine pregnancies. Therefore, early diagnosis is necessary to prevent these complications and control the spread of infection.Several studies have shown that nucleic acid amplification tests are superior to conventional tests for the detection of C. trachomatis and N. gonorrhoeae (4,5,8). Because these organisms are regularly found together, the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae (CA CT/NG) multiplex PCR test (Roche Diagnostics, Mannheim, Germany) provides a useful platform for STI evaluation. An internal control greatly reduces false-negative results. The CA CT component has proven to be a useful diagnostic tool for the detection of C. trachomatis in cervical scraping and urine specimens (13,22,23). The CA NG component, however, lacks specificity, as some non-N. gonorrhoeae Neisseria species (NgNS) (8, 16) and even lactobacilli (23) have been reported to give falsepositive results. Therefore, confirmation by a second, more specific PCR assay is required, especially in low-prevalence communities (8,15,22). This second PCR assay should be convenient and fast to ensure that the delay introduced by required confirmation is kept ...

The increased incidence of infections by vancomycin-resistant Enterococcus (VRE) causes an accumulation of patients who are either colonized with VRE or flagged as potentially colonized with VRE. Since such patients require precautionary isolation upon admission to a hospital, rapid methods to establish VRE colonization status would improve patient care and optimize hospital operation. We evaluated van quantitative PCR (qPCR) on one enrichment broth as a VRE-screening approach. We obtained 255 sets of five rectal specimens from 243 patients. The specimens were cultured using an amoxicillin-containing enrichment broth. Subsequently, a chromogenic agar was incubated and suspect colonies were inoculated on a blood agar plate and characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), followed by a vancomycin Etest in cases in which Enterococcus spp. were detected. The culturing results were compared with the outcome of van qPCR on all enrichment broths of the first rectal swab. The van qPCR was positive for 43% of the sample sets (vanA, n ϭ 5; vanB, n ϭ 101; vanA and vanB, n ϭ 3). Based on culture data, 20 (7.8%) of the sets were VRE positive in at least one of five samples. The negative predictive value of van qPCR on the first enrichment broth was 99.3%. With a cutoff quantification cycle (C q ) value of Ͼ35 to discriminate negative and positive samples, 87% of the negative patients can be identified within a day after obtaining the sample, compared to 7 days in the culturing approach. VRE screening using qPCR on one enrichment broth can quickly identify non-VRE-colonized patients and therefore decrease costs and limit unnecessary isolation restrictions. KEYWORDS VRE, culturing, qPCR In Europe, the rate of vancomycin-resistant Enterococcus (VRE) colonization on intensive care unit (ICU) admission is estimated at 2.7%, compared to 12.3% in the United States (1). However, VRE prevalence increased significantly in Europe between 2011 and 2014, from 6.2% to 7.9% (2). Although VRE prevalence in The Netherlands is low at 0.9%, 16 VRE hospital outbreaks have been reported to the Signaling Consultation of Hospital acquired Infections and AntiMicrobial Resistance (SO-ZI/AMR) in 2015 (3). Infection control practices for VRE differ globally, but most hospitals give patients who are VRE colonized a VRE flag (4), and this flag stays with the patient upon hospital dismissal until the criteria for removing the flag are met. In The Netherlands, it is common practice to preemptively flag patients who have been in contact with known VRE-colonized patients in an outbreak situation (5). Large outbreaks can result in thousands of patients