Establishment of Cryptococcus neoformans and C. gattii as certified reference materials according to the requirements of ISO 17034:2016

Abstract

The use of certified reference materials (CRMs) is essential for laboratories that want to achieve reliable results because these materials have well-defined property values that enable the traceability of certain parameters in several areas. The production of CRMs for microbiological tests is complex, as there is a natural instability of microorganisms. The objective of this study was to establish Cryptococcus neoformans and Cryptococcus gattii as CRM, according to the requirements of ISO 17034. The availability of these materials will assist in the cryptococcosis diagnosis and support the academic community, since cryptococcosis is among the most lethal mycoses and affects more than one million people worldwide each year. C. neoformans CFRVS CRM 40,323 and C. gattii CFRVS CRM 40,324 batches, in skim milk at 20 %, were homogeneous and stable for 1 year. The materials were also stable at 4 °C, 25 °C and 35 °C, temperatures that simulate transportation conditions, for 7 days. In addition to the quantitative characterization, qualitative characterization of materials was conducted by observing melanin production, l-Canavanine Glycine Bromothymol Blue (CGB) Agar test and by RFLP-URA5 analyses. Certified property values and their associated uncertainties were assigned for each CRM. For C. neoformans CFRVS CRM 40,323, expanded uncertainty (U_CRM) was 13 CFU mL⁻¹ for 33 CFU mL⁻¹ property value. For C. gattii CFRVS CRM 40,324, U_CRM was 9.2 CFU mL⁻¹ for 15 CFU mL⁻¹ property value. Our study contributes to the development of CRM production in the microbiological field, whose supply is still very limited worldwide.