Illuminating Uveitis: Metagenomic Deep Sequencing Identifies Common and Rare Pathogens

Abstract

SUMMARYBackgroundOcular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~ 50% of presumed infectious uveitis cases.1,2 We aimed to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis.MethodsThis is a proof-of-concept study, in which intraocular fluid samples were obtained from 5 subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS.FindingsUnbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n=4) and without (n=1) uveitis. Rubella virus (RV) was identified in one case of chronic bilateral idiopathic uveitis. The subject’s strain was most closely related to a German RV strain isolated in 1992, one year before he developed a fever and rash while living in Germany.InterpretationMDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular RV infection highlights the eye’s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.

Metagenomic Sequencing Detects Respiratory Pathogens in Hematopoietic Cellular Transplant Patients

“…Despite this, the etiologic pathogens often remain unidentified because of reduced yields of traditional diagnostics in the setting of prophylactic antimicrobials, reduced antibody titers, and infections from uncommon opportunistic microorganisms. Metagenomic next-generation sequencing (mNGS) may offer enhanced diagnostic capabilities by providing culture-independent, comprehensive measurement of the microbial composition of clinical samples (2,3). By capturing both microbial and human RNA, mNGS also permits simultaneous transcriptional profiling of the host immunologic response and can deliver rapid (,48 h) and actionable microbiologic data for precise infectious disease diagnosis (2).…”

“…RNA and DNA sequencing libraries were constructed from 250 ml BAL from each patient and underwent paired-end Illumina sequencing according to established methods (4). Pathogen detection leveraged a custom bioinformatics pipeline that discriminates pathogens from background microbial contaminants in clinical samples (2,3). A ranking score consisting of the nucleotide reads aligned per million reads sequenced (rpM) multiplied by the sum of the nt and nr Z-scores for each genus relative to no-template controls was used [score = rpM nt 3 (Z nt 1 Z nr )] (4).…”

“…Our understanding of transcriptomic responses in acute respiratory distress syndrome (ARDS) is derived, almost exclusively, from studies that have used samples procured from the peripheral circulation as opposed to the alveolar space (1)(2)(3)(4). However, it is not known to what degree genomic signatures captured from circulating leukocytes accurately reflect the gene expression patterns of leukocytes in the lung.…”

Inhibition of rubella virus replication by the broad-spectrum drug nitazoxanide in cell culture and in a patient with a primary immune deficiency

“…Chronic recurrent rubella-associated arthritis can develop after immunization of adults (Fraser et al, 1983;Tingle et al, 1985). Granulomas in persons with primary immune deficiencies (PID) and Fuchs's uveitis are newly suggested diseases associated with decades-long persistent RV infections (Abernathy et al, 2015;Bodemer et al, 2014;Doan et al, 2016;Perelygina et al, 2016). Currently, there are no antiviral drugs to treat rubella infections and identification of treatments for chronic rubella diseases will be beneficial.…”

Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

“…Unlike assays that target the 16S and/or 28S/ITS ribosomal RNA amplicons, unbiased metagenomic next-generation sequencing (mNGS) can detect bacteria, viruses, and fungi and has shown promising results for diagnosing neurologic and ocular infections [8][9][10]. However, the identification of filamentous molds such as Aspergillus spp.…”