FREE SHIPPING over $40
Horse Supplements USA English
Horse Supplements Canada EnglishSuppléments Équins Canada French
My Account
View Cart My Horses Track Orders Account Details My Subscriptions Update Address Payment Methods
Register/Login
0
Home/Equine Research Bank/Vet Ophthalmol/Evaluation of a commercial NGS service for detection of bact...
Veterinary ophthalmology2023; 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.
LinkedIn
  • 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. https://doi.org/10.1111/vop.13069

Publication

Veterinary ophthalmology
ISSN: 1463-5224
NlmUniqueID: 100887377
Country: England
Language: English

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.

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.12574
  9. Tarabichi M, Shohat N, Goswami K, et al. 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, et al. 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, et al. 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/s40135-013-0025-1
  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, et al. 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, et al. 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, et al. 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, et al. 16u2009S 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, et al. Evaluation of the ocular surface microbiome in clinically normal horses before and after topical antibiotic therapy. Vet Ophthalmol. 2018;22:E28.u00a0ABSTRACTS:u00a0Theu00a049th Annual Scientificu00a0Meeting of the American College of Veterinary Ophthalmologists,u00a0Minneapolis,u00a0Minnesota,u00a0Septu00a026-29,u00a02018.
  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. Microbiology: The MicrogenDX NGS Process. Technologies: the MicroGenDX NGS process. MicroGen Diagnostics; 2021, microgendx.com/next-generation-sequencing-process/
  50. Kim D, Hofstaedter CE, Zhao C, et al. Optimizing methods and dodging pitfalls in microbiome research. Microbiome. 2017;5(1):52.
  51. Lowman ME, Tipton CD, Labordu00e8re 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.13884
  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 5.8u2009S 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, et al. 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-x
  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 0 times.

Secure Payments

PayPal Visa Mastercard American Express

Satisfaction Guarantee

We offer a 60-day guarantee to ensure you are completely satisfied.

Fast & Free Shipping

We provide fast and free shipping on all orders over $40.

Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists