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 Table of Contents  
Year : 2022  |  Volume : 24  |  Issue : 3  |  Page : 36-45

Role of gene sequencing for the diagnosis, tracking and prevention of ocular infections

1 Research and Development Centre, Velammal Medical College Hospital and Research Institute, Madurai, Tamil Nadu, India
2 Department of Ocular Microbiology, Aravind Eye Hospital, Madurai, Tamil Nadu, India

Date of Submission19-Sep-2022
Date of Acceptance29-Sep-2022
Date of Web Publication11-Nov-2022

Correspondence Address:
Prajna Lalitha
Department of Ocular Microbiology, Aravind Eye Hospital, No-1, Anna Nagar, Madurai - 626 020, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jacm.jacm_17_22

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Next-generation sequencing (NGS) is a robust platform which can be employed for pathogen detection. It has the potential to identify pathogens in an unbiased nature including bacteria, fungus, parasites, DNA/RNA viruses and even newer novel pathogens which have been previously reported. The high sensitivity of this technology makes it suitable for ophthalmology applications that rely on very small clinical specimen volumes. Currently, the diagnosis of ocular pathogens relies mainly on conventional procedures such as culturing and microscopy. Traditional methods lack sensitivity, which can be compensated by the application of molecular tools, such as polymerase chain reaction and NGS. In this review, we will look at how NGS can be used to treat ocular infectious diseases such as keratitis, conjunctivitis, trachoma, dacryocystitis, lacrimal sac infection, endophthalmitis and uveitis, with added information on the limitations, advantages and disadvantages on the ocular implication of this NGS technology. NGS-based approaches increase the sensitivity of pathogen detection as well as have the potential to improve healthcare services. NGS still requires advancement in the data analysis tools, establishing standards and reducing turnaround time. We are now closer to realising the full potential of this high-throughput technology. Importantly, NGS also delivers additional data which gives a unique opportunity to explore potential biomarkers of disease pathogenicity or underlying genetic predisposition.

Keywords: Eye infections, next-generation sequencing, polymerase chain reaction

How to cite this article:
Karthikeyan RS, Rameshkumar G, Lalitha P. Role of gene sequencing for the diagnosis, tracking and prevention of ocular infections. J Acad Clin Microbiol 2022;24, Suppl S1:36-45

How to cite this URL:
Karthikeyan RS, Rameshkumar G, Lalitha P. Role of gene sequencing for the diagnosis, tracking and prevention of ocular infections. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Nov 30];24, Suppl S1:36-45. Available from: https://www.jacmjournal.org/text.asp?2022/24/3/36/360979

  Introduction Top

Ocular infection remains one of the major health burdens on our society, especially corneal infection (keratitis) and intraocular infection (endophthalmitis). A variety of pathogens, including viruses, bacteria, fungi and parasites, can cause eye infection.[1] Globally, infectious corneal ulcers constitute up to 3.5% of all cases of blindness.[2] Eye infection without proper clinical intervention will lead to vision loss or even surgical removal of the eye. Further, vision loss leads to an enormous financial burden on the affected family and society due to productivity losses.[3] The source of these infections can occur through the normal flora or external pathogens which gain entrance to the eye through trauma or any invasive procedures such as trabeculectomy, intravitreal injections, cataract surgery, vitreous or retinal surgery or even through an endogenous route via bloodstream infection. The diagnosis of ocular pathogens is extremely difficult due to the limited quantity of specimen retrieved for diagnosis. This limitation will restrict the application of multiple diagnostic tests to be performed. In general, the ophthalmologist relies on the clinical diagnosis based on the unique signs and symptoms for the initial diagnosis and treatment, which is not reliable most of the time.[4],[5] For providing the proper and accurate treatment, rapid and specific identification of the pathogens is very essential.

The need for more sensitive and rapid diagnostic tests for ocular infection has been felt most intensely by ophthalmologists, as the infectious aetiologies frequently elude detection with traditional culture and microscopy.[6] Recent advancements in molecular technology will soon confront the standard microbiology diagnostic test. In this concern, next-generation sequencing (NGS) has received the greatest attention from clinicians and researchers. The holy grail of diagnosis is the development of single tests that are rapid and reliable with added information about understanding the disease pathogenesis. NGS provides an all-in-one package of unbiased pathogen identification with the added potential to study antimicrobial susceptibilities, pathogen virulence, molecular epidemiology and strain typing. NGS has opened up new avenues and raised awareness of vast microbial diversity in addition to pathogenic species found in human specimens, including ocular tissue.[7],[8],[9] In addition to pathogen detection in infectious samples, NGS offers additional genetic information, including strain identification and evolution prediction, which are essential for understanding epidemiology, identifying new novel species and also antimicrobial resistance.[7]

  Conventional Mode of Diagnosis Top

The gold standard diagnosis is mainly reliant on microscopic observation and the culture of the infected specimen. The sensitivity and specificity are highly variable and mainly depend on the type of specimen as well as the pathogens to be detected. The conventional identification methods include cultures, which mainly rely on viable organisms present in the specimen, combined with microscopic morphological identification through staining and serological identification of pathogen antigens.[10] The positivity of the culture for the bacterial and fungal growth in the suspected ocular infection ranges from 40% to 70%.[11],[12],[13],[14] These conventional methods take more time, especially for identifying fungal pathogens, which requires at least 48 h and takes up to two to four weeks for sporulation[15],[16] and up to 10 days for anaerobic organisms.[17] The advancements in microscopes, especially the confocal technology, have led to the application of the in vivo confocal imaging of the cornea. Confocal imaging is a non-invasive rapid process that can easily identify certain pathogens, especially parasites (Acanthamoeba) and filamentous fungi.[15],[18],[19],[20] In cases of Acanthamoeba keratitis, in vivo confocal imaging had 100% sensitivity in identifying pathogens compared with cultures, which had only 33% sensitive.[21]

The major difficulty with this technology is that, even though they can detect the presence of pathogens, species-level identification is not possible. The more time it takes for diagnosis, the more destruction of the ocular tissues occurs. Unless the pathogens are identified earlier, optimal therapy cannot be provided promptly to save the eye and prevent any irreversible visual complications. Gene sequencing methods such as targeted Sanger sequencing or high-throughput NGS are more promising than conventional methods in providing an accurate and rapid diagnosis in considerably less time. Although such with advancements, traditional microbiology's constraints remain mostly connected to the necessity to cultivate the organism. A significant number of pathogens were missed due to the constraints of difficulties in culturing slow growing, fastidious nature or prior use of the antibiotics.[22],[23] Even after the growth of the organisms in culture, the identification and characterisation of antibiotic nature complicates the further diagnosis required.

  Molecular Diagnostic Techniques Top

The most significant barrier to ocular infection diagnosis is the scarcity of diagnostic specimens. Adding to the complications, there is no reliable way to confirm the presence of viable organisms in the culture-negative cases. The molecular methods, due to their higher sensitivity, require only very small-volume size tissues. Because of this significant advantage, molecular tools are increasingly being used to diagnose ocular infections. The main methods used for diagnosis include polymerase chain reaction (PCR), Sanger sequencing and, more recently, more advanced NGS.[24],[25]

  Polymerase Chain Reaction Top

In past decades, PCR has proven to be a powerful tool, especially for the diagnosis of ocular infections. PCR relies on the pathogens' specific genetic markers, which can be amplified and used for pathogen detection. PCR is highly sensitive and rapid detecting in pathogens compared to conventional culturing.[14] PCR relies on primer sequences that are highly specific for a particular genetic target in a pathogen. This method is widely employed for detecting ocular pathogens including Cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Toxoplasma gondii,[26],[27] Mycobacterium tuberculosis[28],[29] and even adenovirus.[30] PCR had the added advantage of detecting pathogens even in culture-negative cases. As reported earlier, a study showed that the PCR had a 95% positive rate in detecting bacterial and fungal pathogens using directed 16S or 18S PCR, respectively, in compared with only a 50% culture-positive rate.[31] The low positive rate of culture may be due to the poor sensitivity or limited group of pathogenic organisms that can grow efficiently in the provided nutrient media.

For the identification of bacterial and fungal species, PCR targeting the 16S rRNA and 28S rRNA or internal transcribed spacer region is used, followed by Sanger sequencing.[8],[32],[33] Further including more specific target genes for sequencing for multilocus sequence typing enhances the specificity for identifying the more closely related species, subspecies or clade identification.[33],[34] The major limitation of this technology is that it relies on primers designed to detect only specific pathogens which limits the identification of unknown pathogens. This limitation can be overcome by the use of NGS technology, which can identify any genetic markers present in the samples non-specifically.

  Next-Generation Sequencing Top

NGS is the upcoming and newer method which is currently employed widely for the identification of the pathogens in many infections. Sanger sequencing, which is considered the precursor to the NGS technology, relies on DNA polymerase-mediated chain termination due to the insertion of dideoxynucleotide chain terminators with fluorescent nucleotides.[35] The limitations of the Sanger sequencing include the possibility of sequencing only one DNA strand to a maximum of 1000 bp, high cost, labour intensity and prolonged turnaround time. NGS technology in general encompasses a wide range of high-throughput sequencing technologies capable of parallel sequencing of large numbers of nucleic acids within a single specimen.[7],[24] Irrespective of the NGS platform and the samples, the workflow remains almost similar.

NGS is based on high-throughput sequencing technology, which can sequence a large number of small DNA fragments (150–500 bases) in a single reaction.[36],[37] NGS technology is capable of outputting millions of reads or DNA base pairs in a few hours. The technology is also versatile: specimens include body fluids, tissues and even laser-microdissected formalin-fixed and paraffin-embedded (FFPE) tissue can be used.[36],[37] The major limitation with this technology is that the specimen used for the diagnosis will contain the majority of human DNA fragments from the patient and only a small minor fraction will contain (0.1%–8%) the pathogenic organism.[38] The contaminating human DNA can be removed by pre-treatment with saponin or other chemicals[39] or human DNA sequence-specific probes or via Cas9 nuclease digestion.[40] All these methods can reduce human DNA considerably, but there is also a loss of pathogen nucleic acid to an extent.[41] Even without these treatments, this limitation can be overcome by the use of powerful bioinformatic tools to filter and differentiate human DNA from pathogens.[7] Automation in NGS technologies has significantly reduced costs, making it the technique of choice for most applications.[8]

  Next-Generation Sequencing Approach for Diagnosis Top

NGS can provide an unbiased approach to detect any genomic material both DNA and RNA present in a specimen, which is especially useful for pathogen detection in culture-negative or microscopy-negative ocular diseases.[37] There are a number of NGS platforms that are commercially available, with the key difference in the sequencing methodology, reagents, sequence read length and error rate.[42] General approach for the NGS application in the ocular diagnosis is straightforward and follows a similar workflow irrespective of the NGS platform used [Figure 1]. The implications of NGS for the identification of pathogens can be approached by either Targeted Amplicon Sequencing (TAS) or microbial whole-genome sequencing (MWGS).[36]
Figure 1: Common next generation sequencing workflow strategy for the analysis of host or pathogen genomes in the ocular samples

Click here to view

  Targeted Next-Generation Sequencing versus Microbial Whole-Genome Sequencing Methodologies Top

Both of these procedures are commonly used for diagnosis, and each has its own set of benefits and drawbacks. For the targeted amplified sequencing (TAS), PCR is performed targeting conserved genetic regions such as 16S rRNA for bacteria,[43],[44] and 18S rRNA and other targets for eukaryotes,[45],[46] followed by attaching a smaller DNA fragment for barcoding to identify the samples followed by sequencing using the NGS platform. This method is employed to either identify multiple organisms in a single sample, such as bacteria and fungus, or identify specific pathogens in large numbers of samples.[47]

In contrast, MWGS sequences all the available nucleic acids within the given sample. This indiscriminate approach will provide a culture-independent tool for clinical diagnostics,[48],[49] which can be highly instrumental in identifying novel organisms. It also has the potential to detect bacteria, fungi, parasites and both DNA and RNA viruses, from a minimal sample volume using intraocular fluid.[27],[50] MWGS is a valuable tool for detecting pathogens in critically ill patients when other methods of identification have failed.[51],[52],[53],[54],[55],[56] In addition, this will also provide additional genetic information regarding the pathogenesis or even antimicrobial resistance-associated genes.[7] The MWGS approach is generally recommended for clinical specimens with minimal microbial diversity.

Both these approaches have their own advantages, but in terms of diagnosis, TAS is an appropriate tool, which is also a more efficient and cost-effective way to investigate multiple pathogens non-specifically.[37] In the TAS approach, adding unique barcodes to each sample during the library preparation will enable pooling multiple samples and sequencing them simultaneously in a single sequencing run. After sequencing, each sample read can be separated based on the barcode for the data analysis. The sample pooling provides greater advantages, which increase the number of samples to be analysed in a single run without increasing cost or test duration.

  Ocular Applications of Next-Generation Sequencing Top

The use of NGS technology in the treatment of ocular diseases is becoming increasingly common as the technology becomes more accessible and less expensive. There are a number of reports about polymicrobial eye infections[57],[58],[59],[60],[61],[62][57],[58],[59],[60],[61],[62] and some of them associated with normal environmental organisms.[63],[64] Whenever there is a polymicrobial infection or unsuspected pathogens, the conventional methods will fail considerably in the identification of multiple organisms.

  Next-Generation Sequencing in the Diagnosis of Ocular Surface Infections Top

The external infection in cornea, conjunctiva and sclera has a greater advantage for direct observation and sample collection. Irrespective of this advantage, the isolation of the viable pathogen from the infected surface, especially the cornea, is only 60%.[65] Clinically differentiating infectious from non-infectious inflammatory scleritis is highly challenging. The NGS application is ideal for the diagnostic application for ocular surface infection. The greater challenge will be the differentiation of normal microflora from the actual pathogenic infection.

  Keratitis Top

Li et al. evaluated the implication of the NGS in 16 cases of keratitis using the formalin-fixed tissue section.[38] Their methodology used ten-micron thick tissue sections from FFPE specimens. The DNA was extracted and obtained at 20–46 million/sample using an Illumina NextSeq platform. Most of the sequences they obtained were human DNA, and only 1.7% represented microbial sequences. They found four samples infected with bacteria, five fungal-infected samples, three Acanthamoeba infections and three viral infections.[38]

Contact lens-associated keratitis infections are due to the contamination of the contact lens or storage solution with diverse environmental bacteria in addition to the actual pathogens. These microbial communities are difficult to culture, mainly due to inactivation by the antimicrobial and preservatives in the storage solutions.[66] Even though they are not detected, there are greater chances that they may prevail in the clinical samples. Eguchi et al. applied a 16S rDNA library analysis to the CL storage case of a patient with Pseudomonas aeruginosa keratitis.[67] The culture methods only identified three isolates, such as Bacillus sp., Pseudomonas putida and P. aeruginosa, in the CL storage solution. Whereas the 16S rDNA library analysis detected 23 genera, indicating the contact lens (CL) storage case was contaminated with a greater number of bacteria. These uncultivable coexisting organisms might have a role in keratitis pathophysiology. Further, additional investigation by the team identified more bacteria coexisting with the CL storage cases from patients with Acanthamoeba keratitis. Acanthamoeba are known to have symbiotic associations with the bacteria, which might have an influence on the disease.[67],[68]

  Lacrimal Sac Infections Top

Eguchi et al. and their team employed the MiSeq 3000 platform (Illumina, Inc., Tokyo, Japan) for metagenomic microbiome analysis using NGS in lacrimal sac secretion.[67] In their study, they included two acute dacryocystitis patients and one chronic dacryocystitis patient. They identified that both the NGS and the culture had similar identification of bacterial groups in the two acute dacryocystitis cases. In the case of chronic dacryocystitis, NGS identified Veillonella sp. (Gram-negative anaerobes) in addition to Haemophilus parainfluenzae and Staphylococcus sp. identified by both culture and NGS. These findings suggest that NGS has an advantage over traditional culture methods in identifying difficult-to-grow pathogens in ocular infection. Dacryocystitis is an infection of the lacrimal sac known to have a higher microbial load, leading to disease pathology. Mycoplasma sp. is one of the most difficult to culture or diagnose in acute dacryocystitis, which can be identified by metagenomic analysis. Further polymicrobial infections were also reported in 30% of the cases with multiple isolates.[62] The polymicrobial infection should be considered when deciding on an antimicrobial treatment. An additional proof-of-concept study conducted by Seitzman et al., using total RNA extracted from the swabs collected from keratitis, scleritis and conjunctivitis patients.[69] They used HiSeq 4000 or NovaSeq using 150-nucleotide paired-end sequencing and analysed the data using an in-house developed pipeline to identify potential pathogens. Furthermore, they used the contralateral unaffected eye as a control for background subtraction to rule out common microflora. The organism is considered a pathogen only if it has the most abundant reads after the background subtraction. They identified a wide variety of pathogenic organisms in this study, including HSV-1, adenovirus, Acanthamoeba and Aspergillus species, which correlate with microbiologic and molecular testing. A similar study by Lalitha et al. using extracted RNA from conjunctival swabs identified 86% (12 out of 14 eyes tested) positive for human pathogens with the most common abundance of adenovirus and also identified the very rare and unusual fungal pathogen, Vittaforma corneae.[70]

  Trachoma Top

Trachoma is one of the severe infections of the eye caused by ocular Chlamydia trachomatis. There is an increase in the application of whole-genome sequencing (WGS) of C. trachomatis.[71],[72],[73] Last et al. completed a genome-wide association study on 81 ocular C. trachomatis isolates using the Illumina GAII or HiSeq 2000 platform to identify genomic markers for trachoma disease severity.[74] Their study identified a greater diversity of the circulating C. trachomatis ocular pathogen than expected in trachoma-endemic West African communities. Alkhidir et al. did a similar investigation, using WGS to sequence 20 ocular C. trachomatis isolates from Gadarif State, Sudan.[75] They used the Illumina NextSeq platform for sequencing and successfully sequenced all 20 samples with high-quality reads. They selected 12 isolates that had more than 98% genome coverage for further analysis. The phylogenetic analysis reveals that all these isolates grouped closely with the subclade within the T2-trachoma clade but were phylogenetically distinct from isolates from other geographic regions. In addition, this study also revealed the absence of macrolide resistance alleles, which indicates that the macrolide antibiotic group is still effective for treatment. Azithromycin is the drug of choice for the treatment of C. trachomatis, but even after prolonged treatment, the ocular infections often persist.[76],[77] This might be due to the other co-founding factors that may contribute to antibiotic resistance. Both these studies demonstrated the WGS potential in terms of epidemiological understanding as well as disease outcome.

  Next-Generation Sequencing in the Diagnosis of Intraocular Infections Top

Aqueous and vitreous fluids are primarily used for diagnosis of intraocular infections such as uveitis, retinitis, and invasive or endogenous ocular infection. The small volume of specimens collected for diagnosis is one of the reasons for the low diagnostic positivity for identifying pathogens.[26],[78],[79] A proof-of-concept study by Doan et al.[80] demonstrated that MWGS can accurately detect viral (HSV-1), fungal (Cryptococcus neoformans) and protozoan (T. gondii) infections. In addition, they also identified the presence of rubella virus in one subject who had chronic idiopathic bilateral uveitis for more than 16 years.[80] Their study demonstrated the unbiased nature of the MWGS system in detecting fungi, parasites and DNA and RNA viruses in minimal samples.[50] Using metagenomic DNA sequencing (DNAseq), the same team was able to identify intraocular pathogens in vitreous samples that were negative for CMV, VZV, HSV and T. gondii through PCR.[27] In this study, they employed the Illumina HiSeq 4000 platform with an average read depth of 15 × 106 reads/sample. The author used their in-house developed software for analysing the NGS data using the National Center for Biotechnology Information as a reference database.[27] They identified eight samples that were positive for one or more of the following pathogens such as CMV,[50],[81] human herpesvirus-6, HSV-2,[82] HTLV-1, Klebsiella pneumoniae and Candida dubliniensis.[26] All of these pathogens have been previously reported to be associated with intraocular infection.

Lee et al. employed targeted 16S amplification in ocular fluids of patients with infectious endophthalmitis.[83] In this study, they identified that the sequencing results are in good concordance with the bacterial and fungal cultures in the seven control patients, with additional findings of the presence of Streptococcus and Pseudomonas sp. in culture-negative specimens.[83] In another study by Lee et al., using whole genome sequencing, found no correlation between the S. epidermidis burden and endophthalmitis severity.[84] Further, they also identified the presence of non-pathogenic human anellovirus, torque teno virus (TTV) in all the culture-negative samples, 57.1% positivity in the culture-positive specimens and none in the control samples.[83] TTV is mostly reported in patients with an altered immune system[85] and also has a significantly higher prevalence in endophthalmitis patients who have undergone multiple ocular invasive procedures such as intraocular injection or surgery,[86] as well as in seasonal hyperacute panuveitis patients.[87] The patient with the TTV also had a higher risk of second vitrectomy,[83] suggesting that the presence of the TTV indicates the severity of the ocular disease, which can be considered a biomarker. Similarly, Seitzman et al identified an unculturable gram-negative bacterium Capnocytophaga in aqueous fluid using metagenomic sequencing.[88] Deshmukh et al. amplified the V3-V4 regions of the bacterial 16S rRNA and the ITS2 region of bacterial and fungus, respectively, followed by sequencing on an Illumina HiSeq 2500 Machine on 34 patients with endophthalmitis and thirty non-infectious controls with presumed infectious endophthalmitis.[79] In their research, they reported that conventional culture-based diagnosis was only 44% (15/34) positive while NGS positivity was 88% (30/34) positive. In addition, NGS identified a mixed infection in two patients, with bacteria in 26 and fungal infection in 2 patients. NGS outperformed the culture methods with additional identification of Streptococcus sp., Staphylococcus sp., Pseudomonas sp., Gemella sp., Haemophilus sp. and Acinetobacter sp. in the culture-negative cases. NGS had a specificity of 100% when compared to the 20% with culture, but the sensitivity remained almost similar between both methods (87.5% and 88%, respectively).

Another major advantage of NGS is that, in addition to pathogen detection, by employing the bioinformatic tools, simultaneously analysing mutations in genes is also possible. In their study, Doan et al. were able to detect mutations in the UL97 gene coding for phosphotransferase that confer resistance to Ganciclovir and Valganciclovir,[27] and similar findings were reported by Kirstahler et al., in post-operative endophthalmitis patients.[89] In going further, Gonzales et al. identified the lymphoma associated with infectious aetiology of both Epstein–Barr virus and human herpesvirus-8 in one patient and the S243N mutation in MYD88, which has been associated with B-cell lymphoma in another patient.[90] Both of these diagnoses are only possible with NGS because these markers are not primary targets for conventional diagnosis. All these recent findings suggest that the mNGS application can extend far beyond the infectious diagnosis and can be applied to non-infectious ocular diseases.

  Alternate Approach for Pathogen Detection Top

DNA-based molecular tools, including NGS, cannot distinguish between viable and non-viable organisms. This problem can be overcome by RNA sequencing, with the added advantage of detecting RNA viruses.[14],[50] In the case of infection, the presence of a pathogen can also be detected indirectly by monitoring the RNA profile or specific gene expression. This approach has proven to be complementary to direct diagnosis when isolation of the pathogens is not possible or inaccessible for collecting specimens. This approach has proven to be effective in identifying unique transcriptome profiles in peripheral blood in respiratory patients.[91],[92] The effectiveness of the RNA sequence was also proven recently by Wecker et al., who showed the identification of more than 150 microRNAs in the very small volume of aqueous samples of patients undergoing cataract surgery.[93] This alternative strategy still requires more studies and it has the potential to be employed as the alternate methods whenever the direct detection of pathogens is difficult.

  Conclusion Top

There are a number of studies that confirmed that NGS results are concordant with the standard diagnostic methods, including cultures, microscopy and PCR. Further, NGS has also proven to be effective in small-volume samples such as aqueous and vitreous fluids, various body tissues, other body fluids and even treated specimens such as formalin-embedded or frozen specimens. NGS has shown that it has the ability to greatly enhance the detection of infectious pathogens in an unbiased manner, allowing it to detect bacteria, fungus, protozoan and even DNA/RNA viruses. In addition, NGS can provide a plethora of information such as antibiotic resistance, virulence nature, epidemiological prevalence and even gene mutation associated with other diseases. NGS-based approach will definitely complement the present ophthalmology diagnostic strategy. Even though NGS is the latest and still evolving technology, analysis and interpretation of the results is still a daunting task. It is still unwise to make diagnostic decisions exclusively based on NGS data, and all results, including those from conventional microbiological methods, must be thoroughly interpreted for a better understanding. NGS has proven to be highly sensitive, which further adds to the problem of amplifying or picking up the contamination. The ocular surface has a rich microflora in addition to pathogens that can interfere with the sample collection and have a huge impact on diagnostic results. In order to overcome those issues, lots of quality of checkpoints should be placed in each and every step. Data processing in the NGS still requires major advancement and simplification, and the sheer volume of data can overwhelm ophthalmologists and microbiologists. In most clinical settings, NGS is still prohibitively costly, and to become a standard routine test, the cost of testing must be decreased further.

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