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.

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.

Our findings suggest that CP-CRE may be more virulent than non-CP-CRE and are associated with poorer outcomes. This underscores the added importance of delineating underlying resistance mechanisms of CRE to direct antibiotic treatment decisions.

Metagenomic next-generation sequencing (mNGS) offers an agnostic approach for emerging pathogen detection directly from clinical specimens. In contrast to targeted methods, mNGS also provides valuable information on the composition of the microbiome and might uncover coinfections that may associate with disease progression and impact prognosis. To evaluate the use of mNGS for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or other infecting pathogens, we applied direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing. Nasopharyngeal (NP) swab specimens from 50 patients under investigation for CoV disease 2019 (COVID-19) were sequenced, and the data were analyzed by the CosmosID bioinformatics platform. Further, we characterized coinfections and the microbiome associated with a four-point severity index. SARS-CoV-2 was identified in 77.5% (31/40) of samples positive by RT-PCR, correlating with lower cycle threshold (Ct) values and fewer days from symptom onset. At the time of sampling, possible bacterial or viral coinfections were detected in 12.5% of SARS-CoV-2-positive specimens. A decrease in microbial diversity was observed among COVID-19-confirmed patients (Shannon diversity index, P = 0.0082; Chao richness estimate, P = 0.0097; Simpson diversity index, P = 0.018), and differences in microbial communities were linked to disease severity (P = 0.022). Furthermore, statistically significant shifts in the microbiome were identified among SARS-CoV-2-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae (P = 0.028) and a reduction in the abundance of Corynebacterium accolens (P = 0.025) were observed. Our study corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages rather than the severity of COVID-19. Further, we demonstrate that SARS-CoV-2 causes a significant change in the respiratory microbiome. This work illustrates the utility of mNGS for the detection of SARS-CoV-2, for diagnosing coinfections without viral target enrichment or amplification, and for the analysis of the respiratory microbiome. IMPORTANCE SARS-CoV-2 has presented a rapidly accelerating global public health crisis. The ability to detect and analyze viral RNA from minimally invasive patient specimens is critical to the public health response. Metagenomic next-generation sequencing (mNGS) offers an opportunity to detect SARS-CoV-2 from nasopharyngeal (NP) swabs. This approach also provides information on the composition of the respiratory microbiome and its relationship to coinfections or the presence of other organisms that may impact SARS-CoV-2 disease progression and prognosis. Here, using direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing of NP swab specimens from 50 patients under investigation for COVID-19, we detected SARS-CoV-2 sequences by applying the CosmosID bioinformatics platform. Further, we characterized coinfections and detected a decrease in the diversity of the microbiomes in these patients. Statistically significant shifts in the microbiome were identified among COVID-19-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae and a reduction in the abundance of Corynebacterium accolens were observed. Our study also corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages of disease rather than with severity of disease. This work illustrates the utility of mNGS for the detection and analysis of SARS-CoV-2 from NP swabs without viral target enrichment or amplification and for the analysis of the respiratory microbiome.

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.

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The rapid spread of multidrug-resistant Gram-negative organisms constitutes one of the greatest challenges to global health. While Gram-negative organisms have developed several mechanisms to avert the bactericidal effects of commonly prescribed antibiotic agents, the increasing prevalence of carbapenemase-producing organisms (CPO) is particularly concerning given the rapid spread of mobile genetic elements containing carbapenemase genes, the limited treatment options for infections caused by these organisms, and the high mortality rates associated with CPO infections. Understanding if an organism is carbapenemase producing and, if so, the class of carbapenemase(s) produced has treatment implications, as some agents preferentially have activity against specific carbapenemases. Furthermore, CPO disseminate between patients with greater ease than non-CP-carbapenem-resistant organisms and warrant more intensive infection control measures than would be employed in the absence of carbapenemase production. Phenotypic assays currently used in clinical practice to detect CPO consist of the following: (i) growth-based assays which measure carbapenem resistance based on organism growth in the presence of a carbapenem antibiotic (e.g., modified Hodge test and modified carbapenem inactivation method), (ii) hydrolysis methods which detect carbapenem degradation products (e.g., Carba NP test and matrix-assisted laser desorption-ionization time of flight mass spectrometry), and (iii) lateral flow immunoassays which detect carbapenemase enzymes through the use of specific antibodies. Although there is no single phenotypic test that meets all specifications of the ideal test, as we describe in this review, there are a number of tests that are user-friendly, affordable, accurate, and feasible for implementation in clinical microbiology laboratories of all sizes.

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