Genomics and metagenomics in medical microbiology

Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre‐Edouard

doi:10.1016/j.mimet.2013.10.006

Cited by 56 publications

(29 citation statements)

References 137 publications

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“…The potential application of NGS for the unbiased detection of pathogens through metagenomic sequencing of clinical specimens is an emerging concept. It is expected that the application of this approach will facilitate detection of uncommon, emerging, and unknown pathogens for which no routine testing currently is available (19)(20)(21)(22)(23). However, a number of challenges were identified, that include cost, turnaround time, infrastructure requirements, technical complexities, bioinformatics expertise, standardization, automation, and data quality.…”

Section: Discussionmentioning

confidence: 99%

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

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Section: Discussionmentioning

confidence: 99%

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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“…There is still lack of information about bacterial, viral, fungal, and parasitic sequence composition in CSF of MS patients. A new light on potential infectious etiology of MS may be provided by next-generation sequencing (NGS) based metagenomics which enables a simultaneous analysis of numerous microorganisms (Miller et al 2013;Padmanabhan et al 2013;Sleator et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Metagenomic Analysis of Cerebrospinal Fluid from Patients with Multiple Sclerosis

Perlejewski

Bukowska-Ośko

Nakamura

et al. 2016

Advances in Experimental Medicine and Biology

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Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of central nervous system of unknown etiology. However, some infectious agents have been suggested to play a significant role in its pathogenesis. Next-generation sequencing (NGS) and metagenomics can be employed to characterize microbiome of MS patients and to identify potential causative pathogens. In this study, 12 patients with idiopathic inflammatory demyelinating disorders (IIDD) of the central nervous system were studied: one patient had clinically isolated syndrome, one patient had recurrent optic neuritis, and ten patients had multiple sclerosis (MS). In addition, there was one patient with other non-inflammatory neurological disease. Cerebrospinal fluid (CSF) was sampled from all patients. RNA was extracted from CSF and subjected to a single-primer isothermal amplification followed by NGS and comprehensive data analysis. Altogether 441,608,474 reads were obtained and mapped using blastn. In a CSF sample from the patient with clinically isolated syndrome, 11 varicella-zoster virus reads were found. Other than that similar bacterial, fungal, parasitic, and protozoan reads were identified in all samples, indicating a common presence of contamination in metagenomics. In conclusion, we identified varicella zoster virus sequences in one out of the 12 patients with IIDD, which suggests that this virus could be occasionally related to the MS pathogenesis. A widespread bacterial contamination seems inherent to NGS and complicates the interpretation of results.

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“…In general, studies have demonstrates the prospect of metagenomics as a possible tool for clinical diagnosis. Surveillance and discovery efforts have been focused in studies of normal physiology as well as in the investigation of acute diseases like diarrhea, hemorrhagic fevers, pneumonia, among others classic infectious diseases (26)(27)(28)(29) .…”

Section: Metagenomic: Application In Clinical Contextmentioning

confidence: 99%

Metagenomic approach to study biofilm in medical context

Ribeiro

Castro

Franco

2017

MRAJ

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Microbial organization in biofilms plays an important role in health and disease. This microbial lifestyle can protect its host against pathogens and contribute to the metabolism of nutrients. On the other hand, pathogenic biofilms can increase the morbidity and mortality of patients, raising the economic cost of infections. Here, we describe how biofilm is organized, its role in different scenarios in the human body and discuss how metagenomic studies has contributed to understand the microbial diversity that interact with the host. It is currently known that many biofilms in the human body may have a polymicrobial composition. In the context of infectious diseases, this composition may limit therapeutic approaches to combat biofilms even more. Thus, describing microbial diversity could help to guide therapeutic decisions.

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Genomics and metagenomics in medical microbiology

Cited by 56 publications

References 137 publications

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Metagenomic Analysis of Cerebrospinal Fluid from Patients with Multiple Sclerosis

Metagenomic approach to study biofilm in medical context

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