Metagenomic Deep Sequencing for the Diagnosis of Corneal and External Disease Infections

Seitzman, Gerami D.; Hinterwirth, Armin; Zhong, Lina; Cummings, Susie; Chen, Cindi; Driver, Todd H.; Lee, Michele; Doan, Thuy

doi:10.1016/j.ophtha.2019.06.013

Cited by 27 publications

(28 citation statements)

References 7 publications

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“…Recently, metagenomic deep sequencing (MDS), which involves both DNA and RNA sequencing, has been suggested to have potential for improved diagnostic sensitivity and accuracy [41]. This allows for rapid diagnosis and helps obtain an accurate diagnosis without the need to wait for the fungus to grow.…”

Section: Molecular Diagnostic Methodsmentioning

confidence: 99%

Recent Perspectives in the Management of Fungal Keratitis

Raj

Vanathi

Ahmed

et al. 2021

JoF

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Mycotic keratitis is common in warm, humid regions with a varying profile of pathogenic fungi according to geographical origin, socioeconomic status, and climatic condition. Clinical diagnosis can be challenging in difficult cases and those refractory to treatment. Fungal hyphae on microscopic examination and culture isolation have been the gold standard in the laboratory diagnosis of fungal keratitis. A culture isolate of the aetiological fungus is essential to perform antifungal susceptibility testing. As the culture isolation of fungi is time-consuming, causing delays in the initiation of treatment, newer investigative modalities such as in vivo confocal microscopy and molecular diagnostic methods have recently gained popularity. Molecular diagnostic techniques now help to obtain a rapid diagnosis of fungal keratitis. Genomic approaches are based on detecting amplicons of ribosomal RNA genes, with internal transcribed spacers being increasingly adopted. Metagenomic deep sequencing allows for rapid and accurate diagnosis without the need to wait for the fungus to grow. This is also helpful in identifying new emerging strains of fungi causing mycotic keratitis. A custom-tear proteomic approach will probably play an important diagnostic role in future in the management of mycotic keratitis. Positive repeat cultures are being suggested as an important gauge indicative of a poor prognosis. Positive repeat fungal cultures help to modify a treatment regimen by increasing its frequency, providing the addition of another topical and oral antifungal agent along with close follow-up for perforation and identifying need for early therapeutic keratoplasty. The role of collagen crosslinking in the treatment of fungal keratitis is not convincingly established. Rapid detection by multiplex PCR and antifungal susceptibility testing of the pathogenic fungi, adopted into a routine management protocol of fungal keratitis, will help to improve treatment outcome. Early therapy is essential in minimizing damage to the corneal tissue, thereby providing a better outcome. The role of conventional therapy with polyenes, systemic and targeted therapy of antifungal agents, newer azoles and echinocandins in fungal keratitis has been widely studied in recent times. Combination therapy can be more efficacious in comparison to monotherapy. Given the diversity of fungal aetiology, the emergence of new corneal pathogenic fungi with varying drug susceptibilities, increasing the drug resistance to antifungal agents in some genera and species, it is perhaps time to adopt recent molecular methods for precise identification and incorporate antifungal susceptibility testing as a routine.

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Section: Molecular Diagnostic Methodsmentioning

confidence: 99%

Recent Perspectives in the Management of Fungal Keratitis

Raj

Vanathi

Ahmed

et al. 2021

JoF

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“…Immediately after the swab is obtained, the sample is placed into DNA/RNA stabilization solution, which preserves the genetic material in the sample as well as inactivates the infectivity of all pathogens. The sample is then placed promptly in a −20 °C freezer and transferred to −80 °C when possible [ 166 ]. Freeze thaw cycles are to be minimized as they can degrade the genetic material.…”

Section: Future Directionsmentioning

confidence: 99%

“…To date, only a few studies have evaluated the potential utility of MDS for diagnosing IK [ [166] , [167] , [168] , [169] ]. Seitzman et al [ 166 ] compared the diagnostic efficacy of MDS with conventional culture and viral PCR in patients with IK and demonstrated that MDS was able to unveil all range of causative microorganisms (including bacteria, fungi, Acanthamoeba and herpes simplex virus) in one single assay. The versatility of MDS in diagnosing all types of IK, including culture-negative cases, has been demonstrated in other studies [ [167] , [168] , [169] , [170] ].…”

Section: Future Directionsmentioning

confidence: 99%

“…In these instances, comparative analysis of absolute number of reads, rather than sequence subtraction can be considered. Continued development, refinement, and standardization of bioinformatic tools, techniques, resources, and databases will allow for further increases in the specificity of deep sequencing as a tool for infectious disease diagnosis [ 166 ]. The processing time from sampling to result may take several days and sequencing costs more than current conventional microbiological methods.…”

Section: Future Directionsmentioning

confidence: 99%

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Diagnostic armamentarium of infectious keratitis: A comprehensive review

et al. 2022

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Infectious keratitis (IK) represents the leading cause of corneal blindness worldwide, particularly in developing countries. A good outcome of IK is contingent upon timely and accurate diagnosis followed by appropriate interventions. Currently, IK is primarily diagnosed on clinical grounds supplemented by microbiological investigations such as microscopic examination with stains, and culture and sensitivity testing. Although this is the most widely accepted practice adopted in most regions, such an approach is challenged by several factors, including indistinguishable clinical features shared among different causative organisms, polymicrobial infection, long diagnostic turnaround time, and variably low culture positivity rate. In this review, we aim to provide a comprehensive overview of the current diagnostic armamentarium of IK, encompassing conventional microbiological investigations, molecular diagnostics (including polymerase chain reaction and mass spectrometry), and imaging modalities (including anterior segment optical coherence tomography and in vivo confocal microscopy). We also highlight the potential roles of emerging technologies such as next-generation sequencing, artificial intelligence-assisted platforms. and tele-medicine in shaping the future diagnostic landscape of IK.

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“…In one case, the MDS identified the organism not supposed to be a cause of infectious keratitis. The case was culture positive for Purpureocillium lilacinum was identified as the second most abundant organism and, the most abundant organism in the sample was Auricoccus indicus, which is not known to cause ocular infections and not even listed in the University of California San Francisco's mass spectrometry's database for identifiable organisms [73].…”

Section: Metagenomic Deep Sequencingmentioning

confidence: 99%

Fungal Keratitis: Recent Advances in Diagnosis and Treatment

Suman¹,

Kumar²,

Saxena³

et al. 2021

Infectious Eye Diseases - Recent Advances in Diagnosis and Treatment

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Fungal keratitis or fungal corneal ulcer is potentially blinding infection of cornea, is considered one of the major cause of ocular morbidity, particularly in developing countries. It is a common cause of infectious keratitis, especially in tropical and subtropical countries. Fungal keratitis is notoriously challenging to diagnosis and difficult to treat. Delay in diagnosis may result in irreversible sequelae of corneal fungal infections, which can be preventable. Fungal keratitis often have worse treatment outcomes than bacterial keratitis, Delayed diagnosis and scarcity of effective antifungal agents are the major factors for poor outcome. In the recent years considerable advancement in the diagnosis and treatment has been occurred. In this chapter, we will discuss the recent advances in diagnosis and management of fungal keratitis with a brief discussion on pathogenesis and future therapeutic models.

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Metagenomic Deep Sequencing for the Diagnosis of Corneal and External Disease Infections

Abstract: 2012 (start date of EHR) to March 30, 2018 were explored under Institutional Review Board approval to identify the subset of subjects who interacted with an eye care provider. No animal subjects were used in this study.

Cited by 27 publications

References 7 publications

Recent Perspectives in the Management of Fungal Keratitis

Recent Perspectives in the Management of Fungal Keratitis

Diagnostic armamentarium of infectious keratitis: A comprehensive review

Fungal Keratitis: Recent Advances in Diagnosis and Treatment

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