@article {901, title = {Impact of Sample Collection Order on the Diagnostic Performance of Metagenomic Deep Sequencing for Infectious Keratitis.}, journal = {Cornea}, volume = {41}, year = {2022}, month = {2022 Jan 01}, pages = {39-44}, abstract = {

PURPOSE: The purpose of this article was to evaluate the impact of sample collection order on the diagnostic yield of metagenomic deep sequencing (MDS) for determining the causative pathogen in infectious keratitis.

METHODS: We performed a cross-sectional diagnostic test evaluation among subjects with infectious keratitis at Aravind Eye Hospital in Madurai, India. All subjects underwent corneal scrapings of the affected eye to obtain potassium hydroxide smear, Gram stain, bacterial culture, and fungal culture, in this order. The order of MDS specimen collection relative to smear and culture samples was randomized and served as the primary predictor. Outcomes included the normalized copy number of pathogenic RNA detected by MDS, the proportion of MDS samples that were diagnostic, and the agreement of MDS results with cultures.

RESULTS: MDS samples from 46 subjects with corneal ulcers were evaluated. MDS was positive in 33 subjects (76\%) and had 74\% overall agreement with culture results. There was no association between order of MDS sample collection and normalized copy number of genetic material detected (P = 0.62) or the likelihood of MDS positivity (P = 0.46). However, the likelihood of agreement between MDS and cultures decreased when MDS corneal swabs were collected after other diagnostic corneal scrapings (P = 0.05).

CONCLUSIONS: The overall yield of MDS for detecting the cause of infectious keratitis was not affected by sample collection order. However, diagnostic agreement between MDS and cultures decreased when MDS samples were collected after other specimens. Additional investigation is warranted to determine whether this represents increased sensitivity of MDS compared with cultures or higher susceptibility to contaminants.

}, keywords = {Adolescent, Adult, Aged, Aged, 80 and over, Bacteria, Cornea, Cross-Sectional Studies, DNA, Bacterial, DNA, Fungal, Eye Infections, Bacterial, Eye Infections, Fungal, Female, Fungi, High-Throughput Nucleotide Sequencing, Humans, Keratitis, Male, Metagenome, Middle Aged, Specimen Handling, Young Adult}, issn = {1536-4798}, doi = {10.1097/ICO.0000000000002766}, author = {Redd, Travis K and Lalitha, Prajna and Prajna, N Venkatesh and Sikha, Misra and Gunasekaran, Rameshkumar and Hinterwirth, Armin and Chen, Cindi and Zhong, Lina and Liu, Zijun and Lietman, Thomas M and Keenan, Jeremy D and Doan, Thuy and Seitzman, Gerami D} } @article {191, title = {Capnocytophaga Keratitis: Clinical Presentation and Use of Metagenomic Deep Sequencing for Diagnosis.}, journal = {Cornea}, volume = {38}, year = {2019}, month = {2019 Feb}, pages = {246-248}, abstract = {

PURPOSE: To report our experience with 2 cases of Capnocytophaga keratitis.

METHODS: This is a retrospective study of case reports. We present the clinical presentation, diagnosis, and treatment strategies of 2 patients who presented with Capnocytophaga keratitis.

RESULTS: Both patients had risk factors including systemic immune compromise and ocular trauma. Both patients had robust inflammatory keratitis with necrosis. Case 1 demonstrates identification of Capnocytophaga with traditional microbiologic techniques. Case 2 demonstrates the use of unbiased metagenomic deep sequencing for identification of this unusual corneal pathogen.

CONCLUSIONS: Capnocytophaga is a rare and aggressive infection. Even when traditional culture identifies the pathogen rapidly, keratitis can progress to perforation. In cases of severe keratitis in which traditional culture methods are unrevealing, metagenomic deep sequencing has potential to provide actionable diagnoses.

}, keywords = {Capnocytophaga, Eye Infections, Bacterial, Female, Gram-Negative Bacterial Infections, High-Throughput Nucleotide Sequencing, Humans, Keratitis, Male, Metagenome, Microbiological Techniques, Middle Aged, Retrospective Studies}, issn = {1536-4798}, doi = {10.1097/ICO.0000000000001790}, author = {Seitzman, Gerami D and Thulasi, Praneetha and Hinterwirth, Armin and Chen, Cindi and Shantha, Jessica and Doan, Thuy} } @article {151, title = {High-throughput sequencing of pooled samples to determine community-level microbiome diversity.}, journal = {Ann Epidemiol}, volume = {39}, year = {2019}, month = {2019 11}, pages = {63-68}, abstract = {

PURPOSE: Community-level interventions in cluster randomized controlled trials may alter the gut microbiome of individuals. The current method of estimating community diversities uses microbiome data obtained from multiple individual{\textquoteright}s specimens. Here we propose randomly pooling a number of microbiome samples from the same community into one sample before sequencing to estimate community-level microbiome diversity.

METHODS: We design and analyze an experiment to compare community microbiome diversity (gamma-diversity) estimates derived from 16S rRNA gene sequencing of 1) individually sequenced specimens vs. 2) pooled specimens collected from a community. Pool sizes of 10, 20, and 40 are considered. We then compare the gamma-estimates using Pearson{\textquoteright}s correlation as well as using Bland and Altman agreement analysis for three established diversity indices including richness, Simpson{\textquoteright}s and Shannon{\textquoteright}s.

RESULTS: The gamma-diversity estimates are highly correlated, with most being statistically significant. All correlations between all three diversity estimates are significant in the 10-pooled data. Pools comprising 40 specimens are closest to the line of agreement, but all pooled samples and individual samples fall within the 95\% limits of agreement.

CONCLUSIONS: Pooling microbiome samples before DNA amplification and metagenomics sequencing to estimate community-level diversity is a viable measure to consider in population-level association research studies.

}, keywords = {Administration, Oral, Anti-Bacterial Agents, Azithromycin, Bacteria, Biodiversity, Child, Preschool, Female, Gastrointestinal Microbiome, Gastrointestinal Tract, High-Throughput Nucleotide Sequencing, Humans, Infant, Infant, Newborn, Male, Metagenomics, Microbiota, Niger, Placebos, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1873-2585}, doi = {10.1016/j.annepidem.2019.09.002}, author = {Ray, Kathryn J and Cotter, Sun Y and Arzika, Ahmed M and Kim, Jessica and Boubacar, Nameywa and Zhou, Zhaoxia and Zhong, Lina and Porco, Travis C and Keenan, Jeremy D and Lietman, Thomas M and Doan, Thuy} } @article {186, title = {Metagenomic Deep Sequencing for the Diagnosis of Corneal and External Disease Infections.}, journal = {Ophthalmology}, volume = {126}, year = {2019}, month = {2019 12}, pages = {1724-1726}, keywords = {Adult, Aged, Aged, 80 and over, Animals, Bacteria, Conjunctival Diseases, Corneal Diseases, Eye Infections, Female, Fungi, High-Throughput Nucleotide Sequencing, Humans, Male, Metagenome, Middle Aged, Parasites, Polymerase Chain Reaction, Retrospective Studies, Scleral Diseases}, issn = {1549-4713}, doi = {10.1016/j.ophtha.2019.06.013}, author = {Seitzman, Gerami D and Hinterwirth, Armin and Zhong, Lina and Cummings, Susie and Chen, Cindi and Driver, Todd H and Lee, Michele D and Doan, Thuy} } @article {51, title = {Metagenomic Next-Generation Sequencing for Identification and Quantitation of Transplant-Related DNA Viruses.}, journal = {J Clin Microbiol}, volume = {57}, year = {2019}, month = {2019 12}, abstract = {

Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related double-stranded DNA (dsDNA) viruses (adenovirus [ADV], BK virus [BKV], cytomegalovirus [CMV], Epstein-Barr virus [EBV], human herpesvirus 6A [HHV-6A], HHV-6B, herpes simplex virus 1 [HSV-1], HSV-2, JC virus [JCV], and varicella-zoster virus [VZV]). The mNGS 95\% limit of detection ranged from 14 copies/ml (HHV-6) to 191 copies/ml (BKV), and the lower limit of quantitation ranged from 442 international units (IU)/ml (EBV) to 661 copies/ml (VZV). An evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and quantitative PCR (qPCR) of 89.2\% (306/343), with a κ statistic of 0.725. The positive percent agreement was 84.9\% (73/86), and the negative percent agreement was 90.7\% (233/257). Furthermore, mNGS detected seven subsequently confirmed coinfections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of = 0.953 + 0.075 (95\% confidence interval [CI] of the slope, 0.883 to 1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log concentration; 95\% limits of agreement, -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates analytical and clinical performance comparable to that of qPCR for transplant-related DNA viruses.

}, keywords = {Computational Biology, DNA Virus Infections, DNA Viruses, High-Throughput Nucleotide Sequencing, Humans, Metagenomics, Molecular Diagnostic Techniques, Sensitivity and Specificity, Transplantation}, issn = {1098-660X}, doi = {10.1128/JCM.01113-19}, author = {Carpenter, Meredith L and Tan, Susanna K and Watson, Thomas and Bacher, Rowena and Nagesh, Vaishnavi and Watts, Alain and Bentley, Gordon and Weber, Jenna and Huang, ChunHong and Sahoo, Malaya K and Hinterwirth, Armin and Doan, Thuy and Carter, Theodore and Dong, Queeny and Gourguechon, St{\'e}phane and Harness, Eric and Kermes, Sean and Radhakrishnan, Srihari and Wang, Gongbo and Quiroz-Z{\'a}rate, Alejandro and Ching, Jesus and Pinsky, Benjamin A} } @article {126, title = {Unbiased Pathogen Detection and Host Gene Profiling for Conjunctivitis.}, journal = {Ophthalmology}, volume = {126}, year = {2019}, month = {2019 08}, pages = {1090-1094}, abstract = {

PURPOSE: The etiology of conjunctivitis is often misdiagnosed. An ideal diagnostic test would identify all possible infectious causes. In this study, we apply unbiased metagenomic RNA deep sequencing (MDS) to identify pathogens causing conjunctivitis.

DESIGN: Molecular study of prospectively collected conjunctival swabs from patients with presumed infectious conjunctivitis.

PARTICIPANTS: Patients with presumed acute infectious conjunctivitis.

METHODS: Conjunctival swabs were collected from patients presenting with acute conjunctivitis. Swabs were processed for MDS. Pathogens were identified using a rapid computational pipeline to analyze the nonhost sequences obtained from MDS. Differential gene expression analysis was performed to evaluate for host transcriptome signatures for infectious types. Clinical samples were deidentified, and laboratory personnel handling the samples and interpreting the data were masked.

MAIN OUTCOME MEASURES: Pathogens and differential transcripts identified by MDS.

RESULTS: Metagenomic RNA deep sequencing detected pathogens in 86\% (12/14) of the patients tested. Swabs from 10 of 14 patients were positive for human adenovirus (HAdV) while swabs from 2 of 14 patients were positive for Vittaforma corneae (a parasitic fungal species of the microsporidia group). Samples positive for HAdV by RNA-seq were independently verified in a CLIA-certified laboratory. Pathogen-directed polymerase chain reaction confirmed the presence of V.\ corneae genome in the samples positive by RNA-seq. Local host transcriptome analysis identified 12 differentially expressed genes that provided distinct expression signatures for patients infected with HAdV compared with V.\ corneae.

CONCLUSIONS: Metagenomic RNA deep sequencing can reliably detect and quantify common and rare pathogens causing conjunctivitis, and identify strains. The unbiased nature of metagenomic RNA deep sequencing allowed an expanded scope of pathogen detection, including fungal species not commonly associated with acute conjunctivitis. In addition, the identification of infection type-specific local host transcriptome signatures may allow for pathogen detection even when the pathogen load is too low for direct identification.

}, keywords = {Adult, Aged, Conjunctivitis, DNA, Bacterial, DNA, Fungal, DNA, Viral, Female, High-Throughput Nucleotide Sequencing, Humans, Male, Metagenomics, Middle Aged, Young Adult}, issn = {1549-4713}, doi = {10.1016/j.ophtha.2019.03.039}, author = {Lalitha, Prajna and Seitzman, Gerami D and Kotecha, Ritesh and Hinterwirth, Armin and Chen, Cindi and Zhong, Lina and Cummings, Susie and Lebas, Elodie and Sahoo, Malaya K and Pinsky, Benjamin A and Lietman, Thomas M and Doan, Thuy} } @article {251, title = {Metagenomic deep sequencing of aqueous fluid detects intraocular lymphomas.}, journal = {Br J Ophthalmol}, volume = {102}, year = {2018}, month = {2018 01}, pages = {6-8}, abstract = {

INTRODUCTION: Currently, the detection of pathogens or mutations associated with intraocular lymphomas heavily relies on prespecified, directed PCRs. With metagenomic deep sequencing (MDS), an unbiased high-throughput sequencing approach, all pathogens as well as all mutations present in the host{\textquoteright}s genome can be detected in the same small amount of ocular fluid.

METHODS: In this cross-sectional case series, aqueous fluid samples from two patients were submitted to MDS to identify pathogens as well as common and rare cancer mutations.

RESULTS: MDS of aqueous fluid from the first patient with vitreal lymphoma revealed the presence of both Epstein-Barr virus (HHV-4/EBV) and human herpes virus 8 (HHV-8) RNA. Aqueous fluid from the second patient with intraocular B-cell lymphoma demonstrated a less common mutation in the gene associated with B-cell lymphoma.

CONCLUSION: MDS detects pathogens that, in some instances, may drive the development of intraocular lymphomas. Moreover, MDS is able to identify both common and rare mutations associated with lymphomas.

}, keywords = {Adult, Aqueous Humor, Biomarkers, Tumor, Cross-Sectional Studies, DNA Mutational Analysis, DNA, Neoplasm, Female, High-Throughput Nucleotide Sequencing, Humans, In Situ Hybridization, Intraocular Lymphoma, Lymphoma, B-Cell, Male, Middle Aged, Mutation, Myeloid Differentiation Factor 88}, issn = {1468-2079}, doi = {10.1136/bjophthalmol-2017-311151}, author = {Gonzales, John and Doan, Thuy and Shantha, Jessica G and Bloomer, Michele and Wilson, Michael R and DeRisi, Joseph L and Acharya, Nisha} } @article {211, title = {Current and future molecular diagnostics for ocular infectious diseases.}, journal = {Curr Opin Ophthalmol}, volume = {27}, year = {2016}, month = {2016 Nov}, pages = {561-567}, abstract = {

PURPOSE OF REVIEW: Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections.

RECENT FINDINGS: Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection.

SUMMARY: Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

}, keywords = {Acanthamoeba, Amebiasis, Chlamydia Infections, Chlamydia trachomatis, Eye Infections, High-Throughput Nucleotide Sequencing, Humans, Metagenome, Molecular Diagnostic Techniques, Polymerase Chain Reaction, Toxoplasma, Toxoplasmosis}, issn = {1531-7021}, doi = {10.1097/ICU.0000000000000311}, author = {Doan, Thuy and Pinsky, Benjamin A} } @article {266, title = {Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens.}, journal = {Genome Med}, volume = {8}, year = {2016}, month = {2016 08 25}, pages = {90}, abstract = {

BACKGROUND: Ocular 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. The objective of this study is to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis.

METHODS: This is a proof-of-concept study, in which intraocular fluid samples were obtained from five 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 those from conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS.

RESULTS: Unbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n = 4) and without (n = 1) uveitis. Samples positive for Cryptococcus neoformans, Toxoplasma gondii, and herpes simplex virus 1 as tested by a Clinical Laboratory Improvement Amendments-certified laboratory were correctly identified with MDS. Rubella virus was identified in one case of chronic bilateral idiopathic uveitis. The subject{\textquoteright}s strain was most closely related to a German rubella virus strain isolated in 1992, one year before he developed a fever and rash while living in Germany. The pattern and the number of viral identified mutations present in the patient{\textquoteright}s strain were consistent with long-term viral replication in the eye.

CONCLUSIONS: MDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular rubella virus infection highlights the eye{\textquoteright}s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.

}, keywords = {Aqueous Humor, Cryptococcosis, Cryptococcus neoformans, Herpes Simplex, Herpesvirus 1, Human, High-Throughput Nucleotide Sequencing, Humans, Metagenomics, Phylogeny, Rubella, Rubella virus, Toxoplasma, Toxoplasmosis, Uvea, Uveitis, Virus Replication}, issn = {1756-994X}, doi = {10.1186/s13073-016-0344-6}, author = {Doan, Thuy and Wilson, Michael R and Crawford, Emily D and Chow, Eric D and Khan, Lillian M and Knopp, Kristeene A and O{\textquoteright}Donovan, Brian D and Xia, Dongxiang and Hacker, Jill K and Stewart, Jay M and Gonzales, John A and Acharya, Nisha R and DeRisi, Joseph L} } @article {256, title = {Paucibacterial Microbiome and Resident DNA Virome of the Healthy Conjunctiva.}, journal = {Invest Ophthalmol Vis Sci}, volume = {57}, year = {2016}, month = {2016 Oct 01}, pages = {5116-5126}, abstract = {

Purpose: To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques.

Methods: Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses.

Results: Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65\% of all conjunctiva samples tested.

Conclusions: Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.).

}, keywords = {Adult, Bacteria, Conjunctiva, DNA, Bacterial, Eye Infections, Bacterial, Female, Healthy Volunteers, High-Throughput Nucleotide Sequencing, Humans, Male, Microbiota, Middle Aged, Prospective Studies, Real-Time Polymerase Chain Reaction, Sequence Analysis, DNA}, issn = {1552-5783}, doi = {10.1167/iovs.16-19803}, author = {Doan, Thuy and Akileswaran, Lakshmi and Andersen, Dallin and Johnson, Benjamin and Ko, Narae and Shrestha, Angira and Shestopalov, Valery and Lee, Cecilia S and Lee, Aaron Y and Van Gelder, Russell N} }