FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Veterinary ophthalmology2023; 26(6); 500-513; doi: 10.1111/vop.13069

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

Abstract: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). Methods: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. Results: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. Conclusions: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.
© 2023 American College of Veterinary Ophthalmologists.
Get Full Text
Publication Date: 2023-03-21 PubMed ID: 36943705DOI: 10.1111/vop.13069Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study evaluated a commercial next-generation sequencing service’s ability to detect bacterial and fungal pathogens in cases of infectious ulcerative keratitis in animals compared to standard corneal cytology and culture techniques. The sequencing service exhibited comparable sensitivity and identified organisms in samples where traditional methods failed, suggesting its value in diagnosing this disease.

Study Methodology

  • The study was conducted on both canine and equine species – both normal controls and those with infectious ulcerative keratitis.
  • Swabs for corneal aerobic and fungal cultures, as well as DNA swabs for next-generation sequencing, were obtained from each subject.
  • Next-generation sequencing results were compared with the outcomes of bacterial and fungal cultures, to gauge the sensitivity of the sequencing technique.

Study Results

  • In normal controls, the sequencing technique identified bacterial and fungal organisms in five and one cases, respectively, while cultures identified these pathogens in seven and two instances.
  • In cases of infectious ulcerative keratitis, the sequencing technique discovered bacterial and fungal organisms in 18 and one cases, respectively, compared to 20 and five cases identified by culture methods.
  • The sequencing method had a sensitivity of 82.14%, and specificity was 76.47%.
  • There was a high level of concordance (79% for bacterial and 100% for fungal organisms) between the organisms identified by the sequencing method and cultures.
  • Interestingly, the sequencing technique detected pathogens in three culture-negative infectious ulcerative keratitis samples.

Study Conclusions

  • The research determined that a commercial next-generation sequencing service could effectively identify causative agents in infectious ulcerative keratitis cases.
  • The sensitivity of the sequencing method was higher than that reported for aerobic culture in prior studies.
  • The study highlights the need for further testing to clarify the clinical implications of additional organisms isolated by the sequencing method from infected samples, as well as organisms identified in normal corneas.

Cite This Article

APA
Bendlin A, Gemensky-Metzler AJ, Diaz-Campos D, Newbold GM, Miller EJ, Chandler HL. (2023). Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis. Vet Ophthalmol, 26(6), 500-513. https://doi.org/10.1111/vop.13069

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English
Volume: 26
Issue: 6
Pages: 500-513

Researcher Affiliations

Bendlin, Ashley
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
Gemensky-Metzler, Anne J
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
Diaz-Campos, Dubraska
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
Newbold, Georgina M
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
Miller, Eric J
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
Chandler, Heather L
  • Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA.
  • College of Optometry, The Ohio State University, Columbus, Ohio, USA.

MeSH Terms

  • Animals
  • Horses
  • Dogs
  • Corneal Ulcer / diagnosis
  • Corneal Ulcer / veterinary
  • Corneal Ulcer / microbiology
  • Bacteria / genetics
  • Cornea / microbiology
  • High-Throughput Nucleotide Sequencing / veterinary
  • High-Throughput Nucleotide Sequencing / methods
  • Dog Diseases / diagnosis
  • Dog Diseases / microbiology
  • Horse Diseases / microbiology

References

This article includes 58 references
  1. Ledbetter EC, Gilger BC. Disease and surgery of the canine cornea and sclera.. In: Gelatt KN, Gilger BC, Kern TJ, eds. Veterinary Ophthalmology. 5th ed. Wiley-Blackwell Publishing; 2013:976-1049.
  2. Brooks DE, Matthews A, Clode AB. Diseases of the cornea.. In: Gilger BC, ed. Equine Ophthalmology. 3rd ed. Wiley-Blackwell Publishing; 2017:252-368.
  3. Hamor R, Whelan N. Equine infectious keratitis.. Vet Clin North Am Equine Pract 1999;15:623-646.
  4. Andrew S, Brooks D, Smith P. Equine ulcerative keratomycosis: visual outcome and ocular survival in 39 cases (1987-1996).. Equine Vet J 1998;30:109-116.
  5. Moore CP, Fales WH, Whittington P, Bauer L. Bacterial and fungal isolates from equidae with ulcerative keratitis.. J Am Vet Med Assoc 1983;182:600-603.
  6. Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in South Florida and emerging resistance to fluoroquinolones.. Ophthalmology 2000;107:1497-1502.
  7. Sauer P, Andrew SE, Lassaline M, Gelatt KN, Denis HM. Changes in antibiotic resistance in equine bacterial ulcerative keratitis (1991-2000):65 horses.. Vet Ophthalmol 2003;6(4):309-313.
  8. Proietto L, Beatty SS, Plummer CE. Comparison of 3 corneal cytology collection methods for evaluating equine ulcerative keratitis: cytobrush, kimura platinum spatula, and handle edge of scalpel blade.. Vet Ophthalmol 2018;22:153-160.
    doi: 10.1111/vop.12574google scholar: lookup
  9. Tarabichi M, Shohat N, Goswami K. Diagnosis of Periprosthetic joint infection: the potential of next-generation sequencing.. J Bone Joint Surg 2018;100:147-154.
  10. Li Z, Breitwieser FP, Lu J. Identifying corneal infections in formalin-fixed specimens using next generation sequencing.. Investig Ophthalmol Vis Sci 2018;59(1):280-288.
  11. Featherstone HJ, Heinich CL. Part 1: ophthalmic examination and diagnostics.. In: Gelatt KN, Gilger BC, Kern TJ, eds. Veterinary Ophthalmology. 5th ed. Wiley Blackwell; 2013:533-613.
  12. Kim E, Chidambaram JD, Srinivasan M. Prospective comparison of microbial culture and polymerase chain reaction in the diagnosis of corneal ulcer.. Am J Ophthalmol 2008;146:714-723.
  13. Vajpayee RB, Angra S, Sandramouli S. Laboratory diagnosis of keratomycosis: comparative evaluation of direct microscopy and culture results.. Ann Ophthalmol 1993;25:68-71.
  14. McLeod SD, Kolahdouz-Isfahani A, Rostamian K. The role of smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious keratitis.. Ophthalmology 1996;103:23-28.
  15. Massa K, Murphy C, Hartmann F. Usefulness of aerobic microbial cultures and cytologic evaluation of corneal specimens in the diagnosis of infectious ulcerative keratitis in animals.. J Am Vet Med Assoc 1999;215:1671-1674.
  16. Jeffery U, Gervais K, Mowat F, Hostetter S, Whitley D. Ability of corneal cytology to predict bacterial culture results.. American Society for Veterinary Clinical Pathology (ASVCP). 47th Annual Meeting, Seattle, WA, 2012; 2012.
  17. McDonald PJ, Watson DJ. Microbial flora of normal canine conjunctivae.. J Small Anim Pract 1976;17(12):809-812.
  18. Gerding PA Jr, McLaughlin SA, Troop MW. Pathogenic bacteria and fungi associated with external ocular diseases in dogs: 131 cases (1981-1986).. J Am Vet Med Assoc 1988;193(2):242-244.
  19. Murphy JM, Lavach JD, Severin GA. Survey of conjunctival flora in dogs with clinical signs of external eye disease.. J Am Vet Med Assoc 1978;172(1):66-68.
  20. Prado MR, Rocha MF, Brito EH. Survey of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Fortaleza, Ceara, Brazil.. Vet Ophthalmol 2005;8(1):33-37.
  21. Wang L, Pan Q, Zhang L, Xue Q, Cui J, Qi C. Investigation of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China.. Vet Ophthalmol 2008;11(3):145-149.
  22. Banks KC, Ericsson AC, Reinero CR, Giuliano EA. Veterinary ocular microbiome: lessons learned beyond the culture.. Vet Ophthalmol 2019;22:716-725.
  23. Taravati P, Lam D, Van Gelder RN. Role of molecular diagnostics in ocular microbiology.. Curr Ophthalmol Rep 2013;1(4):2-6.
    doi: 10.1007/s40135-013-0025-1google scholar: lookup
  24. Zeiss C, Neanderland M, Yang FC, Terwilliger G, Compton S. Fungal polymerase chain reaction testing in equine ulcerative keratitis.. Vet Ophthalmol 2013;16(9):341-351.
  25. Gaudio P, Gopinathan U, Sangwan V. Polymerase chain reaction-based detection of fungi in infected corneas.. Br J Ophthalmol 2002;86(755-760):11.
  26. Ferrer C. Polymerase chain reaction diagnosis in fungal keratitis caused by Alternaria alternate.. Am J Ophthalmol 2002;133:398-399.
  27. Salzberg SL, Breitwieser FP, Kumar A. Next-generation sequencing in neuropathologic diagnosis of infections of the nervous system.. Neurol Neuroimmunol Neuroinflamm 2016;3(4):e251.
  28. Wilson MR, Naccahe SN, Samayoa E. Actionable diagnosis of neuroleptospirosis by next-generation sequencing.. N Engl J Med 2014;370:2408-2417.
  29. Huang Y, Yang B, Li W. Defining the normal core microbiome of conjunctival microbial communities.. Clin Microbiol Infect 2016;22(7):643.e7-643.e12.
  30. Doan T, Akileswaran L, Andersen D. Paucibacterial micro-biome and resident DNA virome of the healthy conjunctiva.. Invest Ophthalmol Vis Sci 2016;57(13):5116-5126.
  31. Schabereiter-Gurtner C, Maca S, Rolleke S. 16 S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting.. Invest Ophthalmol Vis Sci 2001;42(6):1164-1171.
  32. Austin A, Lietman T, Rose-Nussbaumer J. Update on the management of infectious keratitis.. Ophthalmology 2017;124(11):1678-1689.
  33. Leis ML, Costa MO. Initial description of the core ocular surface microbiome in dogs: bacterial community diversity and composition in a defined canine population.. Vet Ophthalmol 2018;22(3):337-344.
  34. Zimmerman KL, Alam MT, Smith Fleming KM. Use of high-throughput sequencing to define the conjunctival microbiome in bison.. Vet Ophthalmol 2018;22:E30-E31: ABSTRACTS: The 49th Annual Scientific Meeting of the American College of Veterinary Ophthalmologists, Minneapolis, Minnesota, Sept 26-29, 2018.
  35. Alfano N, Courtiol A, Vielgrader H, Timms P, Roca AL, Greenwood AD. Variation in koala micro-biomes within and between individuals: effect of body region and captivity status.. Sci Rep 2015;5:10189.
  36. Thomason CA, Leon A, Kirkpatrick LT, Belden LK, Hawley DM. Eye of the finch: characterization of the ocular microbiome of house finches in relation to mycoplasma conjunctivitis.. Environ Microbiol 2017;19(4):1439-1449.
  37. Weese SJ, Nichols J, Jalali M, Litster A. The oral and conjunctival microbiotas in cats with and without feline immunodeficiency virus infection.. Vet Res 2015;46:21.
  38. Scott EM, Arnold C, Dowell S. Evaluation of the ocular surface microbiome in clinically normal horses before and after topical antibiotic therapy.. Vet Ophthalmol 2018;22:E28. ABSTRACTS: The 49th Annual Scientific Meeting of the American College of Veterinary Ophthalmologists, Minneapolis, Minnesota, Sept 26-29, 2018.
  39. LaFrentz S, Abarca E, Mohammed HH, Cuming R, Arias CR. Characterization of the normal equine conjunctival bacterial community using culture-independent methods.. Vet Ophthalmol 2020;23:480-488.
  40. Rogers CM, Scott EM, Sarawichitr B, Arnold C, Suchodolski JS. Evaluation of the bacterial ocular surface microbiome in ophthalmologically normal dogs prior to and following treatment with topical neomycin-polymyxin-bacitracin.. PLoS One 2020;15(6):e0234313.
  41. Darden JE, Scott EM, Arnold C, Scallan EM, Simon BT, Suchodolski JS. Evaluation of the bacterial ocular surface microbiome in clinically normal cats before and after treatment with topical erythromycin.. PLoS One 2020;14(10):e0223859.
  42. Scott EM, Arnold C, Dowell S, Suchodolski JS. Evaluation of the bacterial ocular surface microbiome in clinically normal horses before and after treatment with topical neomycin-polymyxin-bacitracin.. PLoS One 2019;14(4):e0214877.
  43. Walsh ML, Meason-Smith C, Arnold C, Suchodolski JS, Scott EM. Evaluation of the ocular surface mycobiota in clinically normal horses.. PLoS One 2021;16(2):e0246537.
  44. Leis ML, Madruga GM, Costa MO. The porcine corneal surface bacterial microbiome: a distinctive niche within the ocular surface.. PLoS One 2021;16(2):e0247392.
  45. Banks KC, Giuliano EA, Busi SB, Reinero CR, Ericsson AC. Evaluation of healthy canine conjunctival, periocular haired skin, and nasal microbiotia compared to conjunctival culture.. Front Vet Sci 2020;7:558-568.
  46. Sandmeyer LS, Robinson K, Grahn BH. Diagnostic ophthalmology.. Can Vet J 2014;55(3):281-283.
  47. Fentiman KE, Rankin AJ, Meekins JM, Roush JK. Effects of topical ophthalmic application of 0.5% proparacaine hydrochloride on aerobic bacterial culture results for naturally occurring infected corneal ulcers in dogs.. J Am Vet Med Assoc 2018;253(9):1140-1145.
  48. Roche Molecular Systems. High pure PCR template preparation kit.. Roche Life Science; 2018.https://lifescience.roche.com/en_us/products/high-pure-pcr-template-preparation-kit.html#details.
  49. . The MicroGenDX NGS process.. MicroGen Diagnostics; 2021, microgendx.com/next-generation-sequencing-process/.
  50. Kim D, Hofstaedter CE, Zhao C. Optimizing methods and dodging pitfalls in microbiome research.. Microbiome 2017;5(1):52.
  51. Lowman ME, Tipton CD, Labordère AL, Brown JA. Equine sinusitis aetiology is linked to sinus microbiome by amplicon sequencing.. Equine Vet J 2022. Accepted Author Manuscript.
    doi: 10.1111/evj.13884google scholar: lookup
  52. Kane SP. Sample size calculator.. ClinCalc; 2019. https://clincalc.com/stats/samplesize.aspx.
  53. Ferrer C, Colom F, Frases S, Mulet E, Abad J, Alio J. Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8 S ribosomal DNA typing in ocular infections.. J Clin Microbiol 2020;39(8):2873-2879.
  54. Huzefa RA, Miller AN, Pearce CJ, Oberlies NH. Fungal identification using molecular tools: a primer for the natural products research community.. J Nat Prod 2017;80(3):756-770.
  55. Badotti F, de Oliveira FS, Garcia CF. Effectiveness of ITS and sub-regions as DNA barcode markers for the identification of Basidiomycota (fungi).. BMC Microbiol 2017;17(1):42.
    doi: 10.1186/s12866-017-0958-xgoogle scholar: lookup
  56. Scott EM, Lewin AC, Leis ML. Current ocular microbiome investigations limit reproducibility and reliability: critical review and opportunities.. Vet Ophthalmol 2021;24(1):4-11.
  57. Edwards SG, Maggs DJ, Byrne BA, Kass PH, Lassaline ME. Effect of topical application of 0.5% proparacaine on corneal culture results from 33 dogs, 12 cats, and 19 horses with spontaneously arising ulcerative keratitis.. Vet Ophthalmol 2019;22:415-422.
  58. Ledbetter EC, Scarlett JM. Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals.. Vet Ophthalmol 2008;11:114-122.

Citations

This article has been cited 3 times.
  1. Asao K, Hashida N. Overview of Microorganisms: Bacterial Microbiome, Mycobiome, Virome Identified Using Next-Generation Sequencing, and Their Application to Ophthalmic Diseases. Microorganisms 2025 Jun 3;13(6).
    doi: 10.3390/microorganisms13061300pubmed: 40572188google scholar: lookup
  2. Smart K, Pieper JB, Viall AK, Noxon JO, Berger DJ. Comparison of commercial next-generation sequencing assays to conventional culture methods for bacterial identification and antimicrobial susceptibility of samples obtained from clinical cases of canine superficial bacterial folliculitis. Vet Dermatol 2025 Feb;36(1):14-23.
    doi: 10.1111/vde.13299pubmed: 39323044google scholar: lookup
  3. Toumasis P, Tsantes AG, Tsiogka A, Samonis G, Vrioni G. From Clinical Suspicion to Diagnosis: A Review of Diagnostic Approaches and Challenges in Fungal Keratitis. J Clin Med 2024 Jan 4;13(1).
    doi: 10.3390/jcm13010286pubmed: 38202293google scholar: lookup
Subscribe to our newsletter
Join 99,850 horse owners like you who receive our email updates on horse health and nutrition.