Published in Cornea

The Potential for Genetic Sequencing for Targeting Infectious Keratitis

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7 min read

Learn how ophthalmologists can employ genetic sequencing to diagnose patients with infectious keratitis. Download the cheat sheet with tips on how to treat the condition.

The Potential for Genetic Sequencing for Targeting Infectious Keratitis
As the fifth leading cause of blindness globally, corneal opacity due to keratitis—or corneal swelling and inflammation—is imperative to clinical management both in developed and developing countries.
Corneal insults (i.e., trauma, infection, degeneration, and nutritional deficiency) can lead to keratitis and visual impairment, with infectious keratitis (IK) being the predominant cause of corneal blindness.1,2

Overview of infectious keratitis

Infectious keratitis can be further classified into two subtypes:
  1. Microbial keratitis, which includes bacterial, fungal, and parasitic.
  2. Viral keratitis, in which herpes simplex is a key player.
IK transmission is often similar among all four categories. Transmission across bacterial, fungal, parasitic, and viral IK includes but is not limited to contact lens wear, ocular surface disease and/or trauma, previous IK infection, and direct contact with infectious secretions and lesions.
When clinically managing patients with IK, key symptoms common to most etiologies to be wary of include photophobia, profuse tearing, corneal clouding and blurred vision, and intense to extreme pain.1,2

Common Infectious Keratitis Pathogens, Diagnostics, and Treatment Modalities Cheat Sheet

Use this cheat sheet to navigate how to identify, diagnose, and treat different forms of infectious keratitis.

Diagnostic approaches for infectious keratitis and limitations

Despite intensive treatments, detrimental complications such as endophthalmitis, corneal perforation, and complete blindness may occur; thus, it is imperative for a prompt and definite diagnosis for a successful treatment and visual outcome.3 The root of diagnosing medical conditions lies in the critical information that is revealed through an extensive and comprehensive history and physical exam.

Beyond these initial measures, diagnostic testing plays a vital role in verifying the insult.

Corneal scrapings may be taken to provide culture/sensitivity results in order to classify the infectious pathogen in question. Polymerase chain reaction (PCR) tests and in vivo confocal microscopy can further verify or strengthen the diagnosis, especially when the culprit is a virus, which is often diagnosed clinically.1
Though corneal culturing is the current “gold standard” that guides us in IK diagnosis and treatment,3 limitations can propose challenges. Relative to systemic infections, IK presents with a low bioburden; thus, the culture yield has low sensitivity and can take up to several weeks for certain pathogens to yield a positive result.3 Prior antimicrobial treatment and poor culturing techniques further lead to negative or false negative culture results.
Additional challenges posed by diagnostic testing for IK include undifferentiated clinical phenotypes, late disease presentations, polymicrobial infection, and antibiotic or antifungal resistance.3

To differentiate among all four etiologies and accurately diagnose IK pathogens, DNA sequencing, molecular genetics, and high-throughput sequencing techniques are superior.

Molecular genetics and IK: Investigating the code

Molecular genetics is an evolving field that has gained much popularity in the past 5 decades,4 and it helps us understand how differences in the structure and expression of DNA come about among all organisms. Thus, molecular genetics is often referred to as an “investigative approach” in terms of clinical diagnosis. As DNA analysis technologies rapidly improve, our understanding of molecular causes of illnesses and diagnostics becomes superior day by day.

DNA sequencing is a general laboratory technique that helps us decipher and understand the function of genes and the genome.

It involves a broad spectrum of methods to determine the exact nucleotide sequence that makes up an organism’s DNA.4 Over many years, scientists all over the world have worked to improve sequencing techniques, and astounding changes have been made throughout the generations of sequencing.5
Among the “first generation” of DNA sequencing techniques includes Sanger’s chain termination sequencing, which was a pioneering method during its time, as well as Maxam and Gilbert's ‘chemical sequencing’ method.5 First-generation sequencing was limited, however, as it was expensive and limited in yield.6 Soon after, the second generation of sequencing took over, which allowed for the large-scale parallelized sequencing efforts to produce thousands of sequences concurrently, thus called high-throughput sequencing (HTS).

What is high-throughput sequencing?

The HTS subtypes vary depending on the organism from which the sample was acquired, tissue type, disease state, and experimental conditions.6
HTS is imperative for studying and sequencing exomes, genomes, epigenomes, transcriptome profiling, and DNA-protein interactions, and for the ophthalmology field when dealing with IK, internal transcribed spacer genes. Additionally, a key advantage of HTS that we are interested in is the ability to identify RNA classes, structure, as well as RNA-protein interactions.6

IK diagnosis with HTS of internal transcribed spacer genes

As mentioned previously, routine gram stains and culturing may lead to (false) negative IK identification and antibiotic/antifungal resistance; therefore, internal transcribed spacer (ITS) regions are known as an excellent source for identifying unknown or non-culturable bacteria and fungi.7 ITS regions are the spacer DNA segments between the small and large ribosomal RNA (rRNA) subunits or the corresponding transcribed region in polycistronic rRNA precursor transcript.
Both rRNA gene sequence analysis and PCR can successfully identify common and unusual microbial pathogens of the eye. Per Daroy et al., ITS sequence PCR identified organisms in 19 culture-negative samples, thus proving to be an important factor in pathogen detection.7

Although the ITS region is not translated into proteins, they play a crucial role in rRNA functionality and development.

These regions vary in sequence among species, with minimal variability among strains within species.8 Thus, ITS sequencing can identify bacteria that may be difficult or impossible to culture, such as H. influenzae,7 especially within a shorter duration between sample collection and pathogen diagnosis.9
In a 2005 study by Bagyalakshmi et al., culturing ocular fungi varied from 1 to 15 days, averaging 3 days, whereas the results of PCR were available within 5 hours of specimen collection with greater sensitivity and specificity.9 As described in both Daroy et al. and Bagyalakshmi et al. studies, HTS of ITS regions allows for rapid pathogen detection and treatment.7,9

In conclusion

Worldwide, infectious keratitis can be a devastating disease that leads to blindness. Although culturing is important to distinguish IK etiology, many drawbacks, such as delayed diagnosis and antibiotic/antifungal resistance, have led to the development and use of high-throughput sequencing in the world of ophthalmology.
Not only has HTS been proven to be more sensitive and specific, but it has highlighted genetic sequencing as a method for prompt etiological diagnosis and resultant effective treatment early in the disease course.

For a breakdown of different etiologies of infectious keratitis and the various treatment options, DOWNLOAD THE CHEAT SHEET now.

  1. Cabrera-Aguas M, Khoo P, Watson SL. Infectious keratitis: A review. Clin Exp Ophthalmol. 2022 Jul;50(5):543-562. doi: 10.1111/ceo.14113. Published June 3, 2022.
  2. American Optometric Association. Keratitis. American Optometric Association. Published February 28, 2023.
  3. Ting DSJ, Chodosh J, Mehta JS. Achieving Diagnostic Excellence for Infectious Keratitis: A Future Roadmap. Frontiers. Published September 20, 2022.
  4. Ishida C, Zubair M, Gupta V. Molecular Genetics Testing. [Updated 2022 Oct 26]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. Published January 2022.
  5. Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics. 2016 Jan;107(1):1-8. doi: 10.1016/j.ygeno.2015.11.003. PMID: 26554401; PMCID: PMC4727787. Published November 10, 2015.
  6. Ting DSJ, Ho CS, Deshmukh R, et al. “Infectious keratitis: an update on epidemiology, causative microorganisms, risk factors, and antimicrobial resistance.” Eye. 2021;35:1084–1101.
  7. Daroy MLG, Lopez JS, Torres BCL, et al. “Identification of Unknown Ocular Pathogens in Clinically Suspected Eye Infections Using Ribosomal RNA Gene Sequence Analysis.” Clinical Microbiology and Infection. 2011;17(5):776–779.
  8. Kumar M, Shukla PK. Use of PCR targeting of internal transcribed spacer regions and single-stranded conformation polymorphism analysis of sequence variation in different regions of rrna genes in fungi for rapid diagnosis of mycotic keratitis. J Clin Microbiol. 2005 Feb;43(2):662-8.
  9. Bagyalakshmi RMS, Therese KL, Madhavan HN. Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens. Indian Journal of Ophthalmology. 55(4):p261-266, Jul–Aug 2007.
Puneet Singh, MS
About Puneet Singh, MS

Puneet Singh is a current medical student at the New York Institute of Technology College of Osteopathic Medicine (NYITCOM). She graduated summa cum laude from the University of Massachusetts Amherst, where she studied Biochemistry and Molecular Biology with a minor in psychology; she has since been conducting research in a variety of academic settings. Then she received her MS in Applied Molecular Biotechnology and prior to attending medical school, she worked at New England College of Optometry.

Puneet’s current interest in ophthalmology stems from her pursuit in viewing the human body as a whole and understanding the effects of systemic disease on every organ system.

Outside of her academic and research pursuits, Puneet enjoys traveling, hiking, baking, and dancing.

Puneet Singh, MS
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