Background Metagenomic next-generation sequencing (mNGS) was suggested to potentially replace traditional microbiological methodology because of its comprehensiveness. However, clinical experience with application of the test is relatively limited. Methods From April 2017 to December 2017, 511 specimens were collected, and their retrospective diagnoses were classified into infectious disease (347 [67.9%]), noninfectious disease (119 [23.3%]), and unknown cases (45 [8.8%]). The diagnostic performance of pathogens was compared between mNGS and culture. The effect of antibiotic exposure on detection rate was also assessed. Results The sensitivity and specificity of mNGS for diagnosing infectious disease were 50.7% and 85.7%, respectively, and these values outperformed those of culture, especially for Mycobacterium tuberculosis (odds ratio [OR], 4 [95% confidence interval {CI}, 1.7-10.8]; P < .01), viruses (mNGS only; P < .01), anaerobes (OR, ∞ [95% CI, 1.71-∞]; P < .01) and fungi (OR, 4.0 [95% CI, 1.6-10.3]; P < .01). Importantly, for mNGS-positive cases where the conventional method was inconclusive, 43 (61%) cases led to diagnosis modification, and 41 (58%) cases were not covered by empirical antibiotics. For cases where viruses were identified, broad-spectrum antibiotics were commonly administered (14/27), and 10 of 27 of these cases were suspected to be inappropriate. Interestingly, the sensitivity of mNGS was superior to that of culture (52.5% vs 34.2%; P < .01) in cases with, but not without, antibiotic exposure. Conclusions mNGS could yield a higher sensitivity for pathogen identification and is less affected by prior antibiotic exposure, thereby emerging as a promising technology for detecting infectious diseases.

Microbiological Diagnostic Performance of Metagenomic Next-generation Sequencing When Applied to Clinical Practice.

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(18)30458-6.pdf

Access to pathology and laboratory medicine services: a crucial gap.

Authors/Task Force Members: Alexander Wahba a,b,*,† (Chairperson) (Norway), Milan Milojevic c,d,*,† (Serbia, Netherlands), Christa Boer e (Netherlands), Filip M.J.J. De Somer f (Belgium), Tomas Gudbjartsson g (Iceland), Jenny van den Goor h (Netherlands), Timothy J. Jones i (UK), Vladimir Lomivorotov j (Russia), Frank Merkle k (Germany), Marco Ranucci l (Italy), Gudrun Kunst m,*,† (Chairperson) (UK) and Luc Puis n,*,† (Chairperson) (Belgium)

/pdf/2019-eacts-eacta-ebcp-guidelines-on-cardiopulmonary-bypass-1cd2vlqmtd.pdf

2019 EACTS/EACTA/EBCP guidelines on cardiopulmonary bypass in adult cardiac surgery

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory

2019 EACTS/EACTA/EBCP guidelines on cardiopulmonary bypass in adult cardiac surgery.

Summary Background Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater–cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. Methods We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. Findings Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. Interpretation HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. Funding Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

Merel Langelaar

Papers

Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study