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Background Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. Methods DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. Results The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. Conclusions The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay’s suitability for direct screening of clinical specimens.

Objectives: To evaluate the performance of the ResistancePlus GC (beta) assay for the simultaneous detection of Neisseria gonorrhoeae and gyrA S91 markers of resistance (S91F) and susceptibility (WT) to ciprofloxacin, from both clinical specimens and isolates. Methods: Performance was assessed on several sample banks, including N. gonorrhoeae isolates (n = 822), non-gonococcal isolates (n = 110), N. gonorrhoeae-positive clinical specimens (n = 402) and N. gonorrhoeae-negative specimens (n = 290). Results were compared with previous testing data, including S91 genotyping and phenotypic resistance profiles. Results: Overall, the assay demonstrated 100% sensitivity for N. gonorrhoeae detection in clinical isolates. For gyrA S91 mutation detection in clinical isolates, the assay showed 100% sensitivity/specificity compared with the genotype, and >99%/>97% sensitivity/specificity when compared with phenotype. For positive clinical specimens, the assay demonstrated >96% sensitivity for N. gonorrhoeae detection and 100% sensitivity/specificity for gyrA S91 mutation detection. The assay demonstrated >99% specificity for N. gonorrhoeae detection against non-gonococcal isolates and 100% specificity for negative clinical specimens. Conclusions: The ResistancePlus GC (beta) assay is suitable for the detection of N. gonorrhoeae and gyrA markers associated with resistance/susceptibility to ciprofloxacin directly in clinical samples. This assay could be implemented for the individualized treatment of gonorrhoea infections as well as to enhance current antimicrobial resistance surveillance methods.

Introduction Syphilis is a well-known STI caused by the bacterium Treponema pallidum. It can result in genital lesions and substantial morbidity and mortality. Recently, there has been an alarming global resurgence of syphilis with infections rising to unprecedented rates. As such, it is increasingly pertinent to test genital lesions for syphilis.Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and Varicella zoster virus (VZV) cause lesions in cutaneous and mucocutaneous sites. While HSV-1 and HSV-2 are commonly known to cause genital lesions, recent publications have also found VZV in genital specimens. This unexpected finding suggests that reactivation of VZV in this atypical presentation is not as uncommon as previously believed. Using our highly specific PlexZyme technology that enables efficient multiplexing in real-time PCR (qPCR), we have developed a genital lesion assay for the detection of syphilis, HSV-1, HSV-2 and VZV to facilitate prompt and correct treatment of these STI pathogens. Methods To develop a genital lesion assay, the syphilis target was added to our existing HSV-1, HSV-2 and VZV assay (also containing a DNA extraction/amplification control) in a single-well multiplex qPCR. The performance of the assay was evaluated on 90 genital specimens for which in-house PCR results for syphilis had been determined. Results The genital lesion assay showed robust performance in multiplex with sensitive detection to 10 copies for all targets. The multiplexed assay detected 54/57 syphilis positives, corresponding to a sensitivity and specificity of 94.7% and 100.0%, respectively. The assay also detected 4 HSV-1 and 2 HSV-2 infections (2 and 1 syphilis co-infections, respectively). Conclusion The lesion assay offers simultaneous detection and differentiation of pathogens that cause genital lesions. In response to the current emerging syphilis outbreak, this assay could provide a rapid and effective method of determining the infectious agent responsible for genital lesions, supporting earlier detection and rapid treatment to reduce morbidity or worse outcomes.