title: Method for identification of Cryptococcus neoformans and Cryptococcus gattii useful in resource-limited settings.
abstract: OBJECTIVE
  The high HIV/AIDS burden in Sub-Saharan Africa has led to cryptococcosis becoming a public health concern. In this resource-limited setting, conventional identification methods are mainly used to diagnose cryptococcal infections. However, these methods are often inconsistent, and importantly, cannot discriminate between the aetiological agents, Cryptococcus neoformans and C. gattii. Therefore, there is a need for an alternative reliable method to identify these species.
  
  
  METHODS
  We examined the usefulness of a PCR method, including a restriction digest, in identifying clinical C. neoformans and C. gattii isolates. In addition, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) was performed for validation purposes.
  
  
  RESULTS
  The intraspecific variation between tested strains allowed for their delineation into three traditional varieties of C. neoformans, that is, varietal forms: neoformans, grubii and gattii. Furthermore, we uncovered a restriction site (signature sequence: 5'-AATATT-3') that is present only in the distinct species C. neoformans (varietal forms neoformans and grubii), and is, importantly, absent in the distinct species C. gattii (C. neoformans var. gattii). Thus, we were able to discriminate the distinct species by directly digesting the PCR amplicons using the endonuclease SspI. It was also possible to delineate some tested isolates as either C. neoformans or C. gattii using our MALDI-ToF MS data.
  
  
  CONCLUSIONS
  The possibility of performing only a restriction digest makes the outlined method, similar to conventional techniques, economical and easy to optimise for routine use in resource-limited settings.
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa.
authors.forename: Adepemi O
authors.initials: AO
authors.lastname: Ogundeji
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa.
authors.forename: Jacobus
authors.initials: J
authors.lastname: Albertyn
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa.
authors.forename: Carolina H
authors.initials: CH
authors.lastname: Pohl
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa.
authors.forename: Olihile M
authors.initials: OM
authors.lastname: Sebolai
mesh_terms: D001483:Base Sequence; D003453:Cryptococcosis / Q000175:diagnosis* / Q000382:microbiology*; D056285:Cryptococcus gattii / Q000302:isolation & purification*; D003455:Cryptococcus neoformans / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
keywords: CRYPTOCOCCUS; DIAGNOSIS; MOLECULAR BIOLOGY