This experimental study reports on the development of a sensitive, specific and inexpensive high-resolution melting (HRM) test that detects fungi and differentiates filamentous fungi from yeasts directly from clinical specimens within 2.30 hours of DNA extraction.
The authors performed this work because first-line therapy for keratitis patients is different for bacteria, filamentous fungi and yeasts, and the timely onset of treatments depends on rapid and accurate diagnosis. However, fungal cultures produce high rates of false-negative results. Nucleic acid amplification techniques (polymerase chain reaction [PCR]) improve fungal diagnosis performance but require complex postamplification procedures to differentiate filamentous fungi from yeasts or identify the agent.
In the current study, the authors assessed HRM detection limits and specificity with isolated strains, agents (other than fungi) producing keratitis, corneal scrapings from fungal keratitis (culture positive and negative), and corneal scrapings from bacterial, viral or Acanthamoeba keratitis.
Their results indicate that HRM consistently detects the equivalent of 0.1 colony-forming units/ml of yeasts and filamentous fungi, differentiates filamentous fungi from yeasts and discriminates among relevant species of yeasts. Sensitivity and specificity were 100 percent for culture-positive samples, detecting and characterizing fungi in seven of 10 culture-negative suspected fungal keratitis samples.
The authors say that compared with classic PCR (amplification followed by electrophoreses, hybridization or sequencing), this new HRM test has the advantage of minimizing risks for false-positive results due to cross-contamination because the targeted sequence amplification, signal detection and DNA melting analyses are carried out in closed tubes. In addition, HRM minimizes risks for false-negative conclusions because the yields of extraction of DNA from corneal scrapings and the potential interference of PCR inhibitors are systematically monitored in each run and for all the samples.
The authors note that HRM does not require probes, gels, restriction enzymes or any additional equipment for postamplification procedures, and the cost for reactants to test one DNA extract could be reduced to less than $2.