Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

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.

Cryptococcal Immune Reconstitution Inflammatory Syndrome: From Blood and Cerebrospinal Fluid Biomarkers to Treatment Approaches

“…The LAMP technique is based on the principle of isothermal loop amplification to identify the species of Cryptococci from the CSF culture isolates targeting the internal transcribed spacer (ITS) region and CAP59 gene. The LAMP assay has high specificity for molecular genotypes VNI, VNII, and VNIII of Cryptococcus neoformans , and is able to differentiate from C. gattii and other fungal species [ 48 ]. LAMP does not require expensive laboratory instrumentation to perform, thus in the future it can be introduced as a point-of-care assay.…”

Síndrome inflamatória da reconstituição imune associada à meningite criptococócica: fatores de risco e biomarcadores

“…A técnica de LAMP é baseada no princípio da amplificação isotérmica de ácido nucléico simples para identificar espécies de Cryptococcus dos isolados de cultura de LCR, visando a região do espaçador transcrito interno (ITS) e o gene CAP59. O ensaio LAMP tem alta especificidade para os genótipos moleculares VNI, VNII e VNIII do C. neoformans, e é capaz de diferenciar do C. gattii e outras espécies fúngicas 28 . LAMP não requer instrumentação dispendiosa de laboratório para ser executado podendo assim, no futuro, ser introduzido como um ensaio de point-of-care.…”

Molecular Diagnostics of Cryptococcus spp. and Immunomics of Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome

“…Loop-mediated isothermal amplification (LAMP) is a point-of-care isothermal molecular testing method that has significantly improved the diagnosis of cryptococcal disease. LAMP is designed to identify the species of Cryptococci from clinical isolates targeting genotypes VNI, VNII, and VNIII of C. neoformans [28].…”