Abstract
BackgroundUnderlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools. Shotgun metagenomics is a broad diagnostic tool that can be useful for identifying multiple microbes simultaneously especially if coupled with lymph node aspirates, a clinical matrix known to house disparate pathogens. The objective of this study was to analyze the utility of this unconventional diagnostic approach (shotgun metagenomics) using clinical samples from human tularemia cases as a test model. Tularemia, caused by the bacterium Francisella tularensis, is an emerging infectious disease in Turkey. This disease commonly manifests as swelling of the lymph nodes nearest to the entry of infection. Because swollen cervical nodes are observed from many different types of human infections we used these clinical sample types to analyze the utility of shotgun metagenomics.MethodsWe conducted an unbiased molecular survey using shotgun metagenomics sequencing of DNA extracts from fine-needle aspirates of neck lymph nodes from eight tularemia patients who displayed protracted symptoms. The resulting metagenomics data were searched for microbial sequences (bacterial and viral).ResultsF. tularensis sequences were detected in all samples. In addition, we detected DNA of other known pathogens in three patients. Both Hepatitis B virus (HBV) and Human Parvovirus B-19 were detected in one individual and Human Parvovirus B-19 alone was detected in two other individuals. Subsequent PCR coupled with Sanger sequencing verified the metagenomics results. The HBV status was independently confirmed via serological diagnostics, despite evading notice during the initial assessment.ConclusionOur data highlight that shotgun metagenomics of fine-needle lymph node aspirates is a promising clinical diagnostic strategy to identify coinfections. Given the feasibility of the diagnostic approach demonstrated here, further steps to promote integration of this type of diagnostic capability into mainstream clinical practice are warranted.
Highlights
Underlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools
Direct whole-genome sequencing of clinical lymph node samples DNA extracts from fine-needle aspirates of lymph nodes from eight de-identified tularemia patients in Turkey [20] were processed in the Pediatric Infectious Disease Unit of the Faculty of Medicine, Hacettepe University hospital in Ankara, Turkey and subjected to direct metagenomics sequence analysis
Molecular confirmation of pathogens detected by bioinformatics analysis To test for the presence of low level hepatitis B (HBV) and human parvovirus B19 (B19) in all eight clinical samples, we employed a nested Polymerase Chain Reaction (PCR) approach using assays developed using information from previous publications [28, 29] (Table 2), and confirmed the pathogen detection by Sanger sequencing of the final PCR amplicons
Summary
Underlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools. Tularemia, caused by the bacterium Francisella tularensis, is an emerging infectious disease in Turkey This disease commonly manifests as swelling of the lymph nodes nearest to the entry of infection. Metagenomics is powerful for its ability to diagnose unsuspected microbial agents [11] It directly analyzes samples in their entirety, which removes the requirement for a priori knowledge to obtain comprehensive information. In this capacity, metagenomics surpasses the limits encountered with traditional diagnostics
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