Heterogenous pathogen profile associated with acute conjunctivitis in Nepal

Abstract Background Infectious conjunctivitis is common in Nepal. Materials and Methods This prospective study recruited 60 patients with presumed acute infectious conjunctivitis from the B.P. Koirala Lions Center for Ophthalmic Studies in Kathmandu, Nepal. Swabs from the conjunctiva and anterior nares were processed for metagenomic RNA deep sequencing (RNA-seq). Results Pathogens were identified in 55% of cases. RNA viruses were the most common pathogen class identified. Severe acute respiratory syndrome coronavirus 2 was the most common RNA virus identified. Conclusions Acute infectious conjunctivitis varies by location. Contrary to expectations, RNA viruses predominated. Repeat surveillance may be useful and RNA-seq allows for detection of the unexpected pathogen including RNA viruses.


Introduction
Conjunctivitis is a common health problem in Nepal.In 2003, Nepal experienced a nationwide outbreak of acute hemorrhagic conjunc tivitis, affec ting nearly one-half of the entire population.Both human adenovirus and coxsackievirus viruses were identified, 1 suggesting that epidemics can be caused by more than one pathogen and that DNA and RNA viruses can comingle.A 2011 prospective Kathmandu-based study identified Streptococcus pneumonia as the most common isolated organism in young patients presenting with acute conjunctivitis.

Materials and Methods
This research was approved by the Institutional Review Boards of both BPKLCOS in Kathmandu, Nepal, and the UCSF, and adhered to the tenets of the Declaration of Helsinki.Samples were obtained from August 2021 through September 2022 at the BP-KLCOS eye clinic.Inclusion criteria required signs and symptoms suggestive of acute infec tious conjunc tivitis for a duration of < 14 d in any age group.Sterile polyester applicators (Puritan) were used to swab the lower fornix of each eye and each anterior nasal passage.Swabs were placed in a DNA/RNA-Shield (Zymo Research) and stored in a -20°C freezer prior to shipping to UCSF for processing.Sample processing, library preparation, sequencing and bioinformatic algorithm for pathogen identification have been previously described. 3The prespecified criteria for pathogen identification were: (1) organism known to be a human pathogen and representing the most abundant matched reads after water background subtraction; or for viral pathogens (2) ≥2 unique reads covering separate regions in DNA virus genomes; or (3) ≥1 unique reads matching RNA virus genomes.

Discussion
In this case series, pathogens associated with acute conjunctivitis in Katmandu Nepal are diverse.Interestingly, SARS-CoV-2 was the most frequent pathogen associated with acute infectious conjunctivitis.Even although three of the five patients in whom SARS-CoV-2 was identified from the conjunctival swabs also had this virus present in their nasal swabs, none of these participants reported any systemic symptoms.This study is limited by a lack of normal controls and no follow-up data.Like many sites in the SCORPIO study, roughly one-third of the patients presented using topical antimicrobial drops, which limit bacterial and some fungal pathogen identification.This study highlights the importance of location in disease surveillance, as different pathogens appear to predominate in different areas of the world.Repeat surveillance, especially with seasonal variation, is important.Lastly, the use of RNA-deep sequencing emphasises the importance of RNA virus detection in disease surveillance.
M. Chaudhary et al.
analyses.GDS and TD wrote the initial draft and all coauthors reviewed the manuscript and agreed to publication.
Funding: Research reported in this manuscript was supported by the National Eye Institute of the National Institutes of Health under Award Number R01EY032041 (TD), a Research to Prevent Blindness Unrestricted Grant and the Peierls Foundation.The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated, or of the National Institutes of Health.
Competing interests: None declared.
Ethical approval: This research was approved by the Institutional Review Boards of both BPKLCOS in Kathmandu, Nepal, and University of California, UCSF, and adhered to the tenets of the Declaration of Helsinki.

Figure 1 .
Figure 1.(A) Demographics and clinical signs and symptoms of study participants.(B) Pathogen class identified by RNA-seq.(C) Specific pathogens identified in conjunctiva and nasal swabs.