Pathogen Surveillance for Acute Infectious Conjunctivitis.

Importance: Acute infectious conjunctivitis is a common ocular condition with major public health consequences. Objective: To assess regional variations and microbial etiologies of acute infectious conjunctivitis to guide treatment. Design, Setting, and Participants: In this cross-sectional study, patients with presumed acute infectious conjunctivitis were enrolled in the study at 5 sites (Honolulu, Hawaii; Los Angeles, San Francisco, and San Diego, California; and Petah-Tikva, Israel) from March 2021 to March 2023. Patients with allergic or toxic conjunctivitis were excluded. Main Outcomes and Measures: Pathogens were identified by unbiased RNA deep sequencing. Results: In all, 52 patients (mean [range] age, 48 [7-80] years; 31 females [60%]) were enrolled at 5 sites (6 patients from Honolulu, 9 from San Diego, 11 from Los Angeles, 13 from San Francisco, and 13 from Petah-Tikva). RNA deep sequencing detected human adenovirus species D in one-quarter of patients (13 of 52). A wide range of pathogens, including human coronavirus 229E, SARS-CoV-2, and herpes simplex virus type 1, was also identified, as well as several bacteria and fungi. Moreover, 62% (32 of 52) of patients presented with purulent discharge, while only 8% (4 of 52) of patients had confirmed bacterial pathogens. Conclusion and Relevance: In this cross-sectional study, pathogens associated with acute infectious conjunctivitis varied between all 5 sites in the US and Israel. Purulent discharge was a common presenting sign in this study, with a low specificity for bacteria-associated conjunctivitis, suggesting that further diagnostic workup may be necessary to inform antibiotic stewardship. Additional research on cost-effectiveness of using RNA deep sequencing is needed to ascertain whether it is better to monitor patients clinically until resolution of disease.

Comprehensive Profile of Pathogens and Antimicrobial Resistance in Conjunctivitis Cases from Niger.

Infectious conjunctivitis outbreaks remain a public health burden. This study focuses on the pathogen and antimicrobial resistance (AMR) profiles identified in Niger. Sixty-two patients with acute infectious conjunctivitis who presented to health posts were enrolled from December 2021 to May 2022. Nasal and conjunctival swabs were obtained from each patient. Unbiased RNA deep sequencing (RNA-seq) was used to identify associated pathogens. A pathogen was identified in 39 patients (63%; 95% CI, 50-74). Of those, an RNA virus was detected in 23 patients (59%; 95% CI, 43-73). RNA viruses were diverse and included human coronaviruses (HCoVs): SARS-CoV-2, HCoV-229E, HCoV-HKU1, and HCoV-OC43. A DNA virus was identified in 11 patients (28%; 95% CI, 17-44). Of those, four patients had a coinfection with an RNA virus and two patients had a coinfection with both an RNA virus and a bacterium. DNA viruses were predominantly human herpesvirus (cytomegalovirus, Epstein-Barr virus, human herpesvirus 8) and human adenovirus species B, C, and F. Eighteen patients (46%; 95% CI, 32-61) had a bacteria-associated infection that included Haemophilus influenza, Haemophilus aegyptius, Staphylococcus aureus, Streptococcus pneumoniae, and Moraxella spp. Antimicrobial resistance determinants were detected in either the conjunctiva or nasal samples of 20 patients (32%; 95% CI, 22-45) and were found to be more diverse in the nose (Shannon alpha diversity, 1.12 [95% CI, 1.05-1.26] versus 1.02 [95% CI, 1.00-1.05], P = 0.01). These results suggest the potential utility of leveraging RNA-seq to surveil pathogens and AMR for ocular infections.

Apollo Rising: Acute Conjunctivitis Outbreak in India, 2022.

Purpose: To identify pathogens associated with the 2022 conjunctivitis outbreak in Tamil Nadu, India. Methods: This prospective study was conducted in November of 2022. Patients with presumed acute infectious conjunctivitis presenting to the Aravind Eye Clinic in Madurai, India were eligible. Anterior nares and conjunctival samples from participants were obtained and processed for metagenomic RNA deep sequencing (RNA-seq). Results: Samples from 29 patients were sequenced. A pathogen was identified in 28/29 (97%) patients. Coxsackievirus A24v, a highly infectious RNA virus, was the predominant pathogen and detected in 23/29 patients. Human adenovirus D (HAdV-D), a DNA virus commonly associated with conjunctivitis outbreaks, was detected in the remaining patients (5/29). Hemorrhagic conjunctiva was documented in both HAdV-D and coxsackievirus A24v affected patients but was not the predominant clinical presentation. Phylogenetic analysis of coxsackievirus A24v revealed a recent divergence from the 2015 outbreak. Conclusions: Coxsackievirus A24v and HAdV-D were co-circulating during the 2022 conjunctivitis outbreak in Tamil Nadu, India. Clinical findings were similar between patients with HAD-V and coxsackievirus A24v associated conjunctivitis. As high-throughput technologies become more readily accessible and cost-effective, unbiased pathogen surveillance may prove useful for outbreak surveillance and control.

Heterogenous pathogen profile associated with acute conjunctivitis in Nepal.

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.

Case Series: Unbiased Deep Sequencing Analysis of Acute Infectious Conjunctivitis in an Ambulatory Eye Center in Berkeley, California.

SIGNIFICANCE: Acute infectious conjunctivitis poses significant challenges to eye care providers. It can be highly transmissible, and because etiology is often presumed, correct treatment and management can be difficult. This study uses unbiased deep sequencing to identify causative pathogens of infectious conjunctivitis, potentially allowing for improved approaches to diagnosis and management. PURPOSES: This study aimed to identify associated pathogens of acute infectious conjunctivitis in a single ambulatory eye care center. CASE REPORTS: This study included patients who presented to the University of California Berkeley eye center with signs and symptoms suggestive of infectious conjunctivitis. From December 2021 to July 2021, samples were collected from seven subjects (ages ranging from 18 to 38). Deep sequencing identified associated pathogens in five of seven samples, including human adenovirus D, Haemophilus influenzae , Chlamydia trachomatis , and human coronavirus 229E. CONCLUSIONS: Unbiased deep sequencing identified some unexpected pathogens in subjects with acute infectious conjunctivitis. Human adenovirus D was recovered from only one patient in this series. Although all samples were obtained during the COVID-19 pandemic, only one case of human coronavirus 229E and no SARS-CoV-2 were identified.

Viruses Associated with Acute Conjunctivitis in Vanuatu.

The first manifestation of a viral infection may be conjunctivitis. There are limited data on the etiology of viral conjunctivitis in Vanuatu, a country in the South Pacific Ocean. Patients presenting to one of two Vanuatu health centers with presumed infectious conjunctivitis were eligible if symptom onset was within 14 days of screening. Conjunctival and anterior nasal swabs were obtained and subjected to unbiased RNA deep sequencing (RNA-seq) to identify DNA and RNA viruses. For samples collected from May to November 2021, RNA-seq identified a viral etiology in 12/48 patients. Human adenovirus species were the most common viruses (58%) detected, followed by human herpes viruses (cytomegalovirus, varicella zoster virus, and human herpes 7 virus). Rhinovirus C, Epstein-Barr virus, and bocavirus were also detected. In summary, the etiology for viral conjunctivitis in Vanuatu appears broad. Unbiased testing may be useful for disease surveillance.

Gut Microbiome among Children with Uncomplicated Severe Acute Malnutrition in a Randomized Controlled Trial of Azithromycin versus Amoxicillin.

Antibiotics are routinely used as part of the management of severe acute malnutrition and are known to reduce gut microbial diversity in non-malnourished children. We evaluated gut microbiomes in children participating in a randomized controlled trial (RCT) of azithromycin versus amoxicillin for severe acute malnutrition. Three hundred one children aged 6 to 59 months with uncomplicated severe acute malnutrition (mid-upper arm circumference < 11.5 cm and/or weight-for-height Z-score < -3 without clinical complications) were enrolled in a 1:1 RCT of single-dose azithromycin versus a 7-day course of amoxicillin (standard of care). Of these, 109 children were randomly selected for microbiome evaluation at baseline and 8 weeks. Rectal swabs were processed with metagenomic DNA sequencing. We compared alpha diversity (inverse Simpson's index) at 8 weeks and evaluated relative abundance of microbial taxa using DESeq2. Of 109 children enrolled in the microbiome study, 95 were followed at 8 weeks. We found no evidence of a difference in alpha diversity between the azithromycin and amoxicillin groups at 8 weeks controlling for baseline diversity (mean difference -0.6, 95% CI -1.8 to 0.6, P = 0.30). Gut microbiomes did not diversify during the study. Differentially abundant genera at the P < 0.01 level included Salmonella spp. and Shigella spp., both of which were overabundant in the azithromycin compared with amoxicillin groups. We found no evidence to support an overall difference in gut microbiome diversity between azithromycin and amoxicillin among children with uncomplicated severe acute malnutrition, but potentially pathogenic bacteria that can cause invasive diarrhea were more common in the azithromycin group. Trial Registration: ClinicalTrials.gov NCT03568643.

Deep sequencing analysis of acute conjunctivitis in Burkina Faso, Africa.

BACKGROUND: Seasonal and epidemic conjunctivitis (pink eye) infections are highly contagious and impose a significant economic burden worldwide. Long-term visual impairment can occur. METHODS: This study used metagenomic deep sequencing to evaluate pathogens causing acute infectious conjunctivitis in Burkina Faso. RESULTS: We found that pathogens causing conjunctivitis in Burkina Faso are diverse, with human adenoviruses responsible for a small fraction of the samples tested. CONCLUSIONS: These results are unexpected and suggest the importance of regional surveillance.

Outpatient human coronavirus associated conjunctivitis in India.

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.

Deep sequencing analysis of clinical samples from patients with acute infectious conjunctivitis during the COVID-19 delta surge in Madurai, India.

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.

Gut Resistome after Antibiotics among Children with Uncomplicated Severe Acute Malnutrition: A Randomized Controlled Trial.

A broad-spectrum antibiotic, typically amoxicillin, is included in many country guidelines as part of the management of uncomplicated severe acute malnutrition (SAM) in children without overt clinical symptoms of infection. Alternative antibiotics may be beneficial for children with SAM without increasing selection for beta-lactam resistance. We conducted a 1:1 randomized controlled trial of single dose azithromycin versus a 7-day course of amoxicillin for SAM. Children 6-59 months of age with uncomplicated SAM (mid-upper arm circumference < 11.5 cm and/or weight-for-height Z-score < -3) were enrolled in Boromo District, Burkina Faso, from June through October 2020. Rectal swabs were collected at baseline and 8 weeks after treatment and processed using DNA-Seq. We compared the resistome at the class level in children randomized to azithromycin compared with amoxicillin. We found no evidence of a difference in the distribution of genetic antibiotic resistance determinants to any antibiotic class 8 weeks after treatment. There was no difference in genetic macrolide resistance determinants (65% azithromycin, 65% placebo, odds ratio, OR, 1.00, 95% confidence interval, CI, 0.43-2.34) or beta-lactam resistance determinants (82% azithromycin, 83% amoxicillin, OR 0.94, 95% CI, 0.33-2.68) at 8 weeks. Although presence of genetic antibiotic resistance determinants to macrolides and beta-lactams was common, we found no evidence of a difference in the gut resistome 8 weeks after treatment. If there are earlier effects of antibiotics on selection for genetic antibiotic resistance determinants, the resistome may normalize by 8 weeks.

Effect of Azithromycin on the Ocular Surface Microbiome of Children in a High Prevalence Trachoma Area.

PURPOSE: The aim of this study was to evaluate the effect of the 4 times per year mass azithromycin distributions on the ocular surface microbiome of children in a trachoma endemic area. METHODS: In this cluster-randomized controlled trial, children aged 1 to 10 years in rural communities in the Goncha Seso Enesie district of Ethiopia were randomized to either no treatment or treatment with a single dose of oral azithromycin (height-based dosing to approximate 20 mg/kg) every 3 months for 1 year. Post hoc analysis of ocular surface Chlamydia trachomatis load, microbial community diversity, and macrolide resistance determinants was performed to evaluate differences between treatment arms. RESULTS: One thousand two hundred fifty-five children from 24 communities were included in the study. The mean azithromycin coverage in the treated communities was 80% (95% CI: 73%-86%). The average age was 5 years (95% CI: 4-5). Ocular surface C. trachomatis load was reduced in children treated with the 4 times per year azithromycin ( P = 0.0003). Neisseria gonorrhoeae , Neisseria lactamica , and Neisseria meningitidis were more abundant in the no-treatment arm compared with the treated arm. The macrolide resistance gene ermB was not different between arms ( P = 0.63), but mefA / E was increased ( P = 0.04) in the azithromycin-treated arm. CONCLUSIONS: We found a reduction in the load of C. trachomatis and 3 Neisseria species in communities treated with azithromycin. These benefits came at the cost of selection for macrolide resistance.

Effect of Oral Azithromycin vs Placebo on COVID-19 Symptoms in Outpatients With SARS-CoV-2 Infection: A Randomized Clinical Trial.

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.

Gut Resistome of Preschool Children After Prolonged Mass Azithromycin Distribution: A Cluster-randomized Trial.

We evaluated the gut resistome of children from communities treated with 10 twice-yearly azithromycin distributions. Although the macrolide resistance remained higher in the azithromycin arm, the selection of non-macrolide resistance observed at earlier time points did not persist. Longitudinal resistance monitoring should be a critical component of mass distribution programs. CLINICAL TRIALS REGISTRATION: NCT02047981.

Comprehensive pathogen detection for ocular infections.

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.

Indirect effect of oral azithromycin on the gut resistome of untreated children: a randomized controlled trial.

BACKGROUND: Antibiotic use by one individual may affect selection for antimicrobial resistance in close contacts. Here we evaluated whether oral antibiotic treatment of one child within a household affected the gut resistome of an untreated cohabiting child. METHODS: Households with at least two children <5 y of age were randomized in a 1:1 fashion to a 5d course of azithromycin or placebo. To evaluate indirect effects of azithromycin treatment on the gut resistome, we randomly assigned one child in the house to azithromycin and one to placebo. In placebo households, each child received placebo. We performed DNA sequencing of rectal swabs collected 5 d after the last antibiotic dose. We estimated risk ratios for the presence of genetic resistance determinants at the class level using modified Poisson models for children in azithromycin households compared with placebo households and assessed the composition of the resistome using permutational analysis of variance (PERMANOVA). RESULTS: Of 58 children (n = 30 azithromycin households, n = 28 placebo households) with post-treatment rectal swabs, genetic resistance determinants were common but there was no significant difference at the class (p = 0.54 for macrolides) or gene (p = 0.94 for structure by PERMANOVA, p = 0.94 for diversity) level between untreated children in azithromycin households compared with placebo households. CONCLUSIONS: The results are encouraging that one child's antibiotic use may not influence the resistome of another child. Trial registration: ClinicalTrials.gov NCT03187834.