@article {1056, title = {Deep sequencing analysis of clinical samples from patients with acute infectious conjunctivitis during the COVID-19 delta surge in Madurai, India.}, journal = {J Clin Virol}, volume = {157}, year = {2022}, month = {2022 Oct 05}, pages = {105318}, abstract = {

BACKGROUND: Seasonal outbreaks of infectious conjunctivitis remain a public health issue. Determination of outbreak etiologies in the context of a worldwide pandemic may provide useful information to guide public health strategies. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge.

METHODS: This prospective study was conducted from April 2021 to September 2021. All outpatients presenting to the Aravind Eye Center (Madurai, India) with signs and symptoms consistent with acute infectious conjunctivitis were eligible. Three swabs were obtained from each participant: one from each conjunctiva and one from the anterior nares. Samples were processed for metagenomic RNA deep sequencing (RNA-seq).

RESULTS: Samples from 106 study participants were sequenced. The most common presenting symptoms were tearing (86\%) and itching (71\%). Preauricular lymphadenopathy was present in 38\% of participants. 20\% of participants had close contacts with similar symptoms. Systemic symptoms such as coughing, runny nose, vomiting or diarrhea were uncommonly reported. 60\% of all participants used some medicated eye drops upon enrollment. 75\% of study participants demonstrated infection with human adenovirus D (HAdV-D). 11\% of conjunctivitis was associated with SARS-CoV-2. 15\% had no definitive pathogen detected. 8\% of all participants had codetection of more than one pathogen on RNA-seq.

CONCLUSIONS: During the COVID-19 Delta surge in India, HAdV-D was the most common pathogen associated with infectious conjunctivitis. SARS-CoV-2 was the second most common associated pathogen. Seasonal surveillance may be necessary for the determination of emerging and reemerging pathogens responsible for infectious conjunctivitis.

}, issn = {1873-5967}, doi = {10.1016/j.jcv.2022.105318}, author = {Lalitha, Prajna and Prajna, N Venkatesh and Gunasekaran, Rameshkumar and Teja, Gonugunta Vishnu and Sharma, Sankalp S and Hinterwirth, Armin and Ruder, Kevin and Zhong, Lina and Chen, Cindi and Deiner, Michael and Huang, ChunHong and Pinsky, Benjamin A and Lietman, Thomas M and Doan, Thuy and Seitzman, Gerami D} } @article {966, title = {Interepidemic Respiratory Syncytial Virus during the COVID-19 Pandemic.}, journal = {Microbiol Spectr}, year = {2022}, month = {2022 Apr 25}, pages = {e0094722}, issn = {2165-0497}, doi = {10.1128/spectrum.00947-22}, author = {Huang, ChunHong and Sahoo, Malaya K and Verghese, Michelle and Sibai, Mamdouh and Solis, Daniel and Mfuh, Kenji O and Kurzer, Jason and Hogan, Catherine A and Doan, Thuy A and Pinsky, Benjamin A} } @article {1046, title = {Outpatient human coronavirus associated conjunctivitis in India.}, journal = {J Clin Virol}, volume = {157}, year = {2022}, month = {2022 Sep 27}, pages = {105300}, abstract = {

BACKGROUND: Viral conjunctivitis (pink eye) can be highly contagious and is of public health importance. There remains significant debate whether SARS-CoV-2 can present as a primary conjunctivitis. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge.

METHODS: This prospective study was conducted in the spring and summer months of 2021. 106 patients with acute conjunctivitis who presented to the Aravind Eye Center in Madurai, India were included. One anterior nasal swab and one conjunctival swab of each eye were obtained for each enrolled patient. Samples were subsequently processed for unbiased metagenomic RNA deep sequencing (RNA-seq). Outcomes included clinical findings and codetection of other pathogens with SARS-CoV-2 in patients with conjunctivitis.

RESULTS: Among the 13 patients identified with human coronavirus RNA fragments in their swabs, 6 patients had SARS-CoV-2 infection, 5 patients had coinfections of SARS-CoV-2 and human adenovirus (HAdV), 1 patient had a coinfection with human coronavirus OC43 and HAdV, and 1 patient had a coinfection of Vittaforma corneae and SARS-CoV-2. 30\% had bilateral disease and symptoms on presentation. Petechial hemorrhage was noted in 33\% of patients with SARS-CoV-2 infection. No patients with SARS-CoV-2 or SARS-CoV-2 and HAdV infections had subepithelial infiltrates on presentation. All patients denied systemic symptoms.

CONCLUSIONS: Among the patients presented with conjunctivitis associated with human coronavirus infection, over 50\% of the patients had co-infections with other circulating pathogens, suggesting the public-health importance of broad pathogen testing and surveillance in the outpatient conjunctivitis population.

}, issn = {1873-5967}, doi = {10.1016/j.jcv.2022.105300}, author = {Prajna, N Venkatesh and Lalitha, Prajna and Teja, Gonugunta Vishnu and Gunasekaran, Rameshkumar and Sharma, Sankalp S and Hinterwirth, Armin and Ruder, Kevin and Zhong, Lina and Chen, Cindi and Deiner, Michael and Huang, ChunHong and Pinsky, Benjamin A and Lietman, Thomas M and Seitzman, Gerami D and Doan, Thuy} } @article {946, title = {Vaccine-Associated Measles Encephalitis in Immunocompromised Child, California, USA.}, journal = {Emerg Infect Dis}, volume = {28}, year = {2022}, month = {2022 Apr}, pages = {906-908}, abstract = {

We report a fatal case of vaccine-associated measles encephalitis in an immunocompromised child in California, USA. The infection was confirmed by whole-genome RNA sequencing of measles virus from brain tissue. We observed biased matrix-gene hypermutation consistent with persistent measles virus central nervous system infection.

}, issn = {1080-6059}, doi = {10.3201/eid2804.212357}, author = {Costales, Cristina and Sahoo, Malaya K and Huang, ChunHong and Guimaraes, Carolina V and Born, Donald and Kushner, Lauren and Gans, Hayley A and Doan, Thuy A and Pinsky, Benjamin A} } @article {731, title = {Comprehensive pathogen detection for ocular infections.}, journal = {J Clin Virol}, volume = {136}, year = {2021}, month = {2021 Feb 11}, pages = {104759}, abstract = {

BACKGROUND: Molecular diagnostics such as pathogen-directed PCRs have transformed testing for ocular infections since the late 1990s. Although these assays remain important diagnostic tools for samples with low biomass, the lack of diagnostic range motivates alternative molecular approaches for ocular infections. The aim of this study was to determine the performance of a high-throughput RNA sequencing approach, RNA-seq, to detect infectious agents in ocular samples from patients with presumed ocular infections.

METHODS: We compared the performance of RNA-seq to pathogen-directed PCRs using remnant nucleic acids from 41 aqueous or vitreous samples of patients with presumed ocular infections. Pathogen-directed PCRs were performed at the CLIA-certified Stanford Clinical Virology Laboratory. RNA-seq was performed in a masked manner at the Proctor Foundation at the University of California San Francisco. Percent positive and negative agreement between the two testing approaches were calculated. Discordant results were subjected to orthogonal testing.

RESULTS: The positive percent agreement between RNA-seq and pathogen-directed PCRs was 100\% (95\% confidence interval (CI): 78.5\%-100\%). The negative percent agreement was 92.6\% (95\% CI: 76.6\%-97.9\%). RNA-seq identified pathogens not on the differential diagnosis for 9.7\% (4/41) of the samples. Two pathogens solely identified with RNA-seq were confirmed with orthogonal testing.

CONCLUSIONS: RNA-seq can accurately identify common and rare pathogens in aqueous and vitreous samples of patients with presumed ocular infections. Such an unbiased approach to testing has the potential to improve diagnostics although practical clinical utility warrants additional studies.

}, issn = {1873-5967}, doi = {10.1016/j.jcv.2021.104759}, author = {Doan, Thuy and Sahoo, Malaya K and Ruder, Kevin and Huang, ChunHong and Zhong, Lina and Chen, Cindi and Hinterwirth, Armin and Lin, Charles and Gonzales, John A and Pinsky, Benjamin A and Acharya, Nisha R} } @article {851, title = {Effect of Oral Azithromycin vs Placebo on COVID-19 Symptoms in Outpatients With SARS-CoV-2 Infection: A Randomized Clinical Trial.}, journal = {JAMA}, year = {2021}, month = {2021 Jul 16}, abstract = {

Importance: Azithromycin has been hypothesized to have activity against SARS-CoV-2.

Objective: To determine whether oral azithromycin in outpatients with SARS-CoV-2 infection leads to absence of self-reported COVID-19 symptoms at day 14.

Design, Setting, and Participants: Randomized clinical trial of azithromycin vs matching placebo conducted from May 2020 through March 2021. Outpatients from the US were enrolled remotely via internet-based surveys and followed up for 21 days. Eligible participants had a positive SARS-CoV-2 diagnostic test result (nucleic acid amplification or antigen) within 7 days prior to enrollment, were aged 18 years or older, and were not hospitalized at the time of enrollment. Among 604 individuals screened, 297 were ineligible, 44 refused participation, and 263 were enrolled. Participants, investigators, and study staff were masked to treatment randomization.

Interventions: Participants were randomized in a 2:1 fashion to a single oral 1.2-g dose of azithromycin (n = 171) or matching placebo (n = 92).

Main Outcomes and Measures: The primary outcome was absence of self-reported COVID-19 symptoms at day 14. There were 23 secondary clinical end points, including all-cause hospitalization at day 21.

Results: Among 263 participants who were randomized (median age, 43 years; 174 [66\%] women; 57\% non-Hispanic White and 29\% Latinx/Hispanic), 76\% completed the trial. The trial was terminated by the data and safety monitoring committee for futility after the interim analysis. At day 14, there was no significant difference in proportion of participants who were symptom free (azithromycin: 50\%; placebo: 50\%; prevalence difference, 0\%; 95\% CI, -14\% to 15\%; P \> .99). Of 23 prespecified secondary clinical end points, 18 showed no significant difference. By day 21, 5 participants in the azithromycin group had been hospitalized compared with 0 in the placebo group (prevalence difference, 4\%; 95\% CI, -1\% to 9\%; P = .16).

Conclusions and Relevance: Among outpatients with SARS-CoV-2 infection, treatment with a single dose of azithromycin compared with placebo did not result in greater likelihood of being symptom free at day 14. These findings do not support the routine use of azithromycin for outpatient SARS-CoV-2 infection.

Trial Registration: ClinicalTrials.gov Identifier: NCT04332107.

}, issn = {1538-3598}, doi = {10.1001/jama.2021.11517}, author = {Oldenburg, Catherine E and Pinsky, Benjamin A and Brogdon, Jessica and Chen, Cindi and Ruder, Kevin and Zhong, Lina and Nyatigo, Fanice and Cook, Catherine A and Hinterwirth, Armin and Lebas, Elodie and Redd, Travis and Porco, Travis C and Lietman, Thomas M and Arnold, Benjamin F and Doan, Thuy} } @article {166, title = {Dual-target, real-time PCR for the diagnosis of intraocular infections.}, journal = {Br J Ophthalmol}, volume = {103}, year = {2019}, month = {2019 04}, pages = {569-572}, abstract = {

BACKGROUND: is the most common infectious cause of posterior uveitis worldwide. Two multicopy targets (B1 and Rep529) are commonly used in PCR assays, but studies evaluating these targets in ocular fluid samples are limited. Herein, we determine the analytical characteristics of a single-reaction, internally controlled, dual-target, real-time PCR and evaluate the clinical performance of this assay in intraocular fluid samples obtained at a reference ophthalmologic centre in the USA.

METHODS: Lower limits of detection for the B1 and Rep529 components of the dual-target assay were determined using serial dilutions of cultured strain Z185. The dual-target assay was then used to test 148 archived intraocular samples (132 vitreous,16 aqueous humour) collected at the Francis I. Proctor Foundation between January 2010 and December 2015 for testing by a nested, conventional PCR targeting the B1 gene.

RESULTS: The 95\% lower limits of detection for the dual-target assay was determined to be 1.05 tachyzoites/mL for B1 and 0.83 tachyzoites/mL for Rep529. Using archived clinical intraocular specimens, the dual-target assay demonstrated 97.2\% positive per cent agreement (n=35/36; 95\% CI 83.7\% to 99.9\%) and 99.1\% negative per cent agreement (n=111/112; 95\% CI 94.4\% to 100\%) compared with the nested, conventional B1 PCR.

CONCLUSION: This single-reaction, internally controlled, dual-target (B1, Rep529) real-time PCR for the detection of DNA in intraocular specimens demonstrated excellent agreement with nested, conventional, B1 PCR. The dual-target design may ensure detection when variation is present in one of two target regions.

}, keywords = {Animals, Aqueous Humor, DNA, Protozoan, Eye Infections, Parasitic, Humans, Real-Time Polymerase Chain Reaction, Toxoplasma, Toxoplasmosis, Ocular, Uveitis, Posterior}, issn = {1468-2079}, doi = {10.1136/bjophthalmol-2018-313064}, author = {Gomez, Carlos A and Sahoo, Malaya K and Kahn, Ghazala Yasmeen and Zhong, Lina and Montoya, Jos{\'e} G and Pinsky, Benjamin A 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 {241, title = {Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections.}, journal = {Ophthalmology}, volume = {124}, year = {2017}, month = {2017 08}, pages = {1247-1248}, keywords = {Cytomegalovirus, Cytomegalovirus Infections, DNA, Protozoan, DNA, Viral, Eye Infections, Parasitic, Eye Infections, Viral, Herpes Simplex, Herpes Zoster Ophthalmicus, Herpesvirus 3, Human, Humans, Metagenomics, Polymerase Chain Reaction, Sensitivity and Specificity, Sequence Analysis, DNA, Simplexvirus, Toxoplasma, Toxoplasmosis, Ocular, Uveitis, Vitreous Body}, issn = {1549-4713}, doi = {10.1016/j.ophtha.2017.03.045}, author = {Doan, Thuy and Acharya, Nisha R and Pinsky, Benjamin A and Sahoo, Malaya K and Chow, Eric D and Banaei, Niaz and Budvytiene, Indre and Cevallos, Vicky and Zhong, Lina and Zhou, Zhaoxia and Lietman, Thomas M and DeRisi, Joseph L} } @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} }