Method for the Identification of Clinical Cryptococcus Neoformans and Cryptococcus Gatti Isolates

The present invention relates to a method of detecting the presence or absence, and if present the identity of a Cryptococcus pathogen in a biological sample from a subject. The invention also provides for a kit for the detection and identification of a Cryptococcus pathogen in a biological sample.

Cryptococcosis is a life-threatening fungal disease that may present in either immunocompetent or immunocompromised persons (Day, 2004). Persons with an impaired cell-mediated immunity are more vulnerable to the disease. The advent of HIV has led to an increase in the number of cryptococcosis cases, and in Sub-Saharan Africa, it is estimated that cryptococcosis annually accounts for over 500,000 deaths of HIV-infected individuals (Park et al., 2009).

Although cryptococcosis can be managed, its clinical diagnosis may often be inaccurate. A diagnostic error could leave survivors with neurological problems or have fatal consequences (Saha et al., 2008). Moreover, inaccurate diagnosis undermines efforts to understand the extent and scope of this infectious disease, and by necessary implication, could have a negative impact on plans and management strategies by health authorities.

Over the years, Indian ink preparations have been used to diagnose cryptococcosis (Saha et al., 2008, 2009). However, Indian ink has poor sensitivity, and there have been reported cases of acapsular strains inducing pathogenesis (del Poeta, 2004). Although serological tests tend to be more specific, they are not always consistent in diagnosing cryptococcosis due to the generation of false positive or negative results (Saha et al., 2009). More importantly, traditional serological techniques cannot resolve the identity of the responsible etiological agent. This information is crucial in understanding the epidemiology, pathogenesis, clinical presentation and drug resistance of clinical isolates (Ito-Kuwa et al., 2007). In addition, the identity of the etiological agent can also provide information relating to the agent's oxylipin production pattern and, possibly, associated pathogenesis (Sebolai et al., 2007, 2012).

The availability of an extensive molecular database has allowed for easy identification and referencing of yeasts (Scozetti et al., 2002). Traditionally, Cryptococcus (Cr.) neoformans has been classified into three varieties viz. Cr. neoformans var. neoformans (serotype D), Cr. neoformans var. grubii (serotype A), Cr. neoformans var. gattii (serotype B and C) as well as a hybrid (AD) (Chen et al., 2010). Recently, it has been proposed that the species complex should be reclassified into two distinct species viz. Cr. neoformans (serotypes A, AD and D) and Cr. gattii (serotype B and C) (Kwon Chung & Varma, 2006). 

Based on this proposal, the inventors sought to examine the usefulness of sequencing the internal transcribed spacers (ITS) including the 5.85 gene, as a reliable method for the identification of etiological agents from clinical strains obtained from patients with cryptococcosis. Importantly, the inventors point out that the uncovering of nucleotide polymorphism in the ITS region, implies strain identification can be achieved without the need to sequence amplicons. In resource-limited settings, a simple molecular method would have a clear competitive advantage over more elegant typing techniques that are too expensive and laborious for routine use (Sidrum et al., 2010). We also sought to confirm cases of Cr. gattii, found to be less prevalent in temperate regions such as South Africa, by cultivation on carvanine-glycine-bromothymol blue media.