A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum ß-lactamase and carbapenemase genes.

OBJECTIVES: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum ß-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. METHODS: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. RESULTS: The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5-96.5%) and 99.2% (98.8-99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0-99.3%) and 98.5% (98.0-98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. CONCLUSION: We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

Evidence of HPV subtypes linked with cervical cancer in Nepal

Objectives: Cervical cancer is the commonest malignancy among women in Nepal but dataare limited on which subtypes of human papillomavirus (HPV) are associated with cancer in thispopulation. Now that vaccines against HPV types 16 and 18 are available, this evidence is of vitalimportance in obtaining further support for a vaccination programme.Methods: Cervical swabs from 44 histologically confirmed invasive cervical cancer cases wereobtained from two tertiary referral hospitals in Nepal. Evidence of HPV subtypes was identifiedusing an HPV multiplex polymerase chain reaction (PCR), and confirmed at the Scottish HPV VirusReference Laboratory.Results: HPV types 16 and 18 were present in 70% of samples, along with other high-risksubtypes. HPV 6 and 11 were not observed. Epidemiological data assessment appeared to indicatethat patient age, age of marriage and age of first pregnancy were associated with increased HPVinfection in patients.Conclusions: This study provides further evidence of the importance of HPV types 16 and 18 incervical cancer in Nepal and adds support to a nationwide vaccination programme and the use ofHPV detection in screening programmes.