Next-Generation Sequencing in Clinical Microbiology: Are We There Yet?

To compare different criteria of Metagenomic Next-Generation Sequencing (mNGS) in bronchoalveolar lavage fluid (BALF) for diagnosing invasive pulmonary aspergillosis (IPA).

We compared the diagnostic agreement and performances of six BALF mNGS-derived criteria (SDSMRN>1, SDSMRN≥3, SMRN≥10, SMRN≥50, RPM ratio≥10, and relative abundance of genus>30 %) in pneumonia patients.

A total of 115 patients were analyzed, with 28 identified with IPA. Diagnostic agreement among the six mNGS-derived criteria was moderate, with a Cohen's kappa of 0.577(P < 0.001). mNGS-derived criteria had low sensitivity ranging from 21.4 % to 57.1 % and high specificity from 88 % to 92 %. The optimal threshold of SDSMRN, SMRN, RPM ratio, and relative abundance of genus for diagnosing IPA were 5, 0.25, 8, and 20 %, respectively. Although using the optimal threshold, the sensitivity of mNGS is lower than 50 %.

All mNGS-derived criteria had low sensitivity for diagnosing IPA. A combination of mNGS and conventional mycological tests may be the best diagnostic strategy.

Whole-genome sequencing (WGS) has been used widely to elucidate transmission of SARS-CoV-2 in acute healthcare settings, and to guide infection, prevention, and control (IPC) responses.

To systematically appraise available literature, published between January 1^st, 2020 and June 30^th, 2022, describing the implementation of WGS in acute healthcare settings to characterize nosocomial SARS-CoV-2 transmission.

Searches of the PubMed, Embase, Ovid MEDLINE, EBSCO MEDLINE, and Cochrane Library databases identified studies in English reporting the use of WGS to investigate SARS-CoV-2 transmission in acute healthcare environments. Publications involved data collected up to December 31^st, 2021, and findings were reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement.

In all, 3088 non-duplicate records were retrieved; 97 met inclusion criteria, involving 62 outbreak analyses and 35 genomic surveillance studies. No publications from low-income countries were identified. In 87/97 (90%), WGS supported hypotheses for nosocomial transmission, while in 46 out of 97 (47%) suspected transmission events were excluded. An IPC intervention was attributed to the use of WGS in 18 out of 97 (18%); however, only three (3%) studies reported turnaround times ≤7 days facilitating near real-time IPC action, and none reported an impact on the incidence of nosocomial COVID-19 attributable to WGS.

WGS can elucidate transmission of SARS-CoV-2 in acute healthcare settings to enhance epidemiological investigations. However, evidence was not identified to support sequencing as an intervention to reduce the incidence of SARS-CoV-2 in hospital or to alter the trajectory of active outbreaks.

The precise diagnosis and treatment of spinal infections (SI) remains challenging for spine surgeons. Identifying the pathogens of SI through metagenomic next-generation sequencing (mNGS) may be a key approach to addressing this challenge.

To evaluate the accuracy and applicability of mNGS in determining the etiology of SI.

Diagnostic test study.

Twenty-five patients who had a clinical suspicion of SI and underwent mNGS testing.

The specificity, sensitivity, and time cost of mNGS and bacterial culture were compared. Clinical outcomes were assessed using the numeric rating scale (NRS) score, Oswestry Disability Index (ODI), and the Japanese Orthopedic Association (JOA) score. Demographic data and laboratory results (blood cell count (WBC), erythrocyte sedimentation rate (ESR), neutrophil percentage (NEUT%), and C-reactive protein level (CRP) were also evaluated.

In this retrospective study, samples were obtained from 25 eligible patients via surgery or CT-guided puncture and subjected to histopathological examination, bacterial culture, and mNGS. The sensitivity and specificity of the bacterial cultures and mNGS were calculated with respect to the histopathological results as a reference. Postoperative antibiotics or antituberculosis drugs were administered on the basis of mNGS results, combined with clinical manifestations, imaging examination, and histopathology. The changes of clinical outcomes and laboratory results after treatment were observed.

Of the 25 patients, 21 had a positive pathology, of which 10 showed a tuberculous pathology, and the remaining 11 showed a nontuberculous inflammatory pathology. The sensitivity of mNGS was higher than that of the bacterial culture. However, the difference in specificity between bacterial culture and mNGS was not significant. Moreover, the time needed to perform mNGS was significantly lower than that of bacterial culture and pathology. All patients were followed up for more than three months, and CRP and NEUT% significantly decreased by three months after treatment. There was no significant difference in WBC and ESR. The ODI, NRS and JOA scores were significantly improved after treatment.

Metagenomic next-generation sequencing technology can play an important role in the detection of pathogens in SI and should be further investigated and applied in future studies.

The genomic analysis of microbial infections entails the use of molecular methods to detect pathogen-specific genetic sequences to diagnose, monitor, or prognose infections (such as qualitative or quantitative nucleic acid amplification methods). Various molecular methods are utilized for the analysis of genetic sequences or arrangements in order to characterize pathogens or to differentiate often epidemiologically unrelated pathogens at the strain level. Molecular diagnostic methods have become the gold standard for diagnosing many infections because of their accuracy and rapid turnaround time compared to conventional methods, such as culture. Molecular strain typing and sequencing methods have become routine because of their accuracy, strain discrimination capability, and wide availability of reagents compared to serologic or phenotypic methods. This chapter provides an overview of molecular approaches to genomic analysis of pathogens in order to diagnose or monitor infections and to characterize the pathogens involved.