Prospective comparison of conventional microbiological diagnostic methods and direct polymerase chain reaction genotyping in diagnosis of suspected bacterial keratitis in Ilrorin

Abstract

Background: Infectious keratitis is a leading cause of monocular blindness. Diagnostic information from corneal sampling that is thought to help guide therapeutic interventions and the culturing methods are inadequate. The purpose of this study is to compare the diagnostic performance of conventional microbiological methods and direct polymerase chain reaction (PCR) genotyping for bacterial keratitis (IK). Methods: This is an observational prospective comparative cross-sectional study of patients who presented with bacterial keratitis at the selected hospitals Ophthalmology Clinics in Ilorin between July 2015 and July 2018. Gram staining of the smears for presence of Gram-positive or Gram-negative bacteria, Gram’s iodine stained (mordant) for cellular morphology. Brain heart infusion broth (BHIB) for staphylococcus and streptococcus species, Blood agar for isolation of Haemophilus or Niesseria species, MacConkey agar for isolation of Escherichia coli, Enterococcus, Pseudomonas species and direct polymerase chain reaction (PCR) were done on the scraped samples. Results: Out of 79 corneal scrapings of 79 eyes subjected to Gram stain, microbial culture and PCR, 61 corneal scraping samples (61 eyes) had positive results with direct PCR, giving a positive detection rate of 73.4% (61/79), 45 scrapings positive (61 eyes) for culture and 19 scrapings positive (61 eyes) for Gram staining. The sensitivities for the diagnosis of bacterial keratitis with direct PCR, culture and Gram’s staining were 98.0%, 49.0%, 42.2% respectively whereas the specificities were 90.9%, 100%, 100% respectively for sample suspicion of bacterial keratitis. Conclusion: The direct PCR assay without template DNA extraction yielded high sensitivity and specificity for the diagnosis of bacterial keratitis. Beyond its high sensitivity and specificity, the direct PCR assay yielded more isolates compare to CDC. Direct PCR is rapid, requiring 3 hours for the entire process while culture required about 24-48 hours for the process.