Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens

Braz J Infect Dis. Nov-Dec 2015;19(6):563-70. doi: 10.1016/j.bjid.2015.07.006. Epub 2015 Sep 26.

Abstract

The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA.

Keywords: Cryptococcosis; Cryptococcus neoformans/Cryptococcus gattii complex; Molecular diagnosis; Nested PCR.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Cryptococcosis / diagnosis*
  • Cryptococcosis / microbiology
  • Cryptococcus gattii / genetics*
  • Cryptococcus gattii / isolation & purification
  • Cryptococcus neoformans / genetics*
  • Cryptococcus neoformans / isolation & purification
  • DNA, Fungal / analysis*
  • DNA, Ribosomal Spacer
  • Humans
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Analysis, DNA

Substances

  • DNA, Fungal
  • DNA, Ribosomal Spacer