Abstract
Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.
Highlights
Cryptococcosis is the leading systemic fungal infection worldwide with an estimated number of cases per year close to 1 million [1]
The cryptococcal meningitis caused by Cryptococcus neoformans and Cryptococcus gattii are clinically indistinguishable, some authors suggest that meningitis due to C. gattii appears to be more aggressive than that caused by C. neoformans, and may require extended periods of treatment [4,5]
Eleven cerebral spinal fluid (CSF) were positive for Cryptococcus from nine patients, 15 samples were positive for bacteria, and 107 CSF samples were negative for fungi, bacteria, and mycobacteria (Table 1)
Summary
Cryptococcosis is the leading systemic fungal infection worldwide with an estimated number of cases per year close to 1 million [1]. The microbiological identification by automated systems and phenotypic methods applied in the differentiation of Cryptococcus species requires confirmation by molecular methods. Immunoassays for the diagnosis of cryptococcosis have been applied in clinical practice to detect cryptococcal capsular antigen in body fluids, such as the Cryptococcus antigen lateral flow assay (CrAg LFA). The CrAg LFA assay identifies the four main serotypes of Cryptococcus (A, B, C, and D) [7], but not to Molecular diagnoses of Cryptococcus spp species level. Cerebral lesions and hydrocephalus are more common in infections caused by C. gattii [8,9]. In these cases, the accurate species identification would be useful in guiding the appropriate therapy scheme. Because the CrAg LFA assay is not available here, other techniques, including molecular ones, are necessary to improve the diagnosis of cryptococcosis
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