Agnostic metagenomic next-generation sequencing (mNGS) has emerged as a promising single, universal pathogen detection method for infectious disease diagnostics. This methodology allows for identification and genomic characterization of bacteria, fungi, parasites, and viruses without the need for a priori knowledge of a specific pathogen directly from clinical specimens. Although there are increasing reports of mNGS successes, several hurdles need to be addressed, such as differentiation of colonization from infection, extraneous sources of nucleic acid, method standardization, and data storage, protection, analysis, and interpretation. As more commercial and clinical microbiology laboratories develop mNGS assays, it is important for treating practitioners to understand both the power and limitations of this method as a diagnostic tool for infectious diseases.

Extended-spectrum β-lactamase (ESBL)-producing Gram-negative pathogens are a major cause of resistance to expanded-spectrum β-lactam antibiotics. Since their discovery in the early 1980s, they have spread worldwide and an are now endemic in Enterobacterales isolated from both hospital-associated and community-acquired infections. As a result, they are a global public health concern. In the past, TEM- and SHV-type ESBLs were the predominant families of ESBLs. Today CTX-M-type enzymes are the most commonly found ESBL type with the CTX-M-15 variant dominating worldwide, followed in prevalence by CTX-M-14, and CTX-M-27 is emerging in certain parts of the world. The genes encoding ESBLs are often found on plasmids and harboured within transposons or insertion sequences, which has enabled their spread. In addition, the population of ESBL-producing Escherichia coli is dominated globally by a highly virulent and successful clone belonging to ST131. Today, there are many diagnostic tools available to the clinical microbiology laboratory and include both phenotypic and genotypic tests to detect β-lactamases. Unfortunately, when ESBLs are not identified in a timely manner, appropriate antimicrobial therapy is frequently delayed, resulting in poor clinical outcomes. Several analyses of clinical trials have shown mixed results with regards to whether a carbapenem must be used to treat serious infections caused by ESBLs or whether some of the older β-lactam-β-lactamase combinations such as piperacillin/tazobactam are appropriate. Some of the newer combinations such as ceftazidime/avibactam have demonstrated efficacy in patients. ESBL-producing Gram-negative pathogens will continue to be major contributor to antimicrobial resistance worldwide. It is essential that we remain vigilant about identifying them both in patient isolates and through surveillance studies.

The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.