Use of polymerase chain reaction for Cryptococcus neoformans genome detection in cerebrospinal fluid for neurocryptococcosis diagnosis

Página do item simplificado
dc.creatorCidiane Graciele-Melo
dc.creatorFabiana Rocha-Silva
dc.creatorChristo Paulo Pereira
dc.creatorCaligiorne Rachel Basques
dc.date.accessioned2024-11-25T21:10:23Z
dc.date.accessioned2025-09-08T22:50:58Z
dc.date.available2024-11-25T21:10:23Z
dc.date.issued2016
dc.description.sponsorshipCNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
dc.description.sponsorshipFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
dc.format.mimetypepdf
dc.identifier.doi10.4172/2471-8521.100013
dc.identifier.issn2471-8521
dc.identifier.urihttps://hdl.handle.net/1843/78267
dc.languageeng
dc.publisherUniversidade Federal de Minas Gerais
dc.relation.ispartofMedical Mycology: Open Access
dc.rightsAcesso Aberto
dc.subjectCryptococcosis
dc.subjectHIV
dc.subjectReação em Cadeia da Polimerase
dc.subjectLíquido Cefalorraquidiano
dc.titleUse of polymerase chain reaction for Cryptococcus neoformans genome detection in cerebrospinal fluid for neurocryptococcosis diagnosis
dc.typeArtigo de periódico
local.citation.epage7
local.citation.issue2
local.citation.spage1
local.citation.volume2
local.description.resumoCryptococcosis is a systemic infection caused by encapsulated yeast of Cryptococcus genus, has a worldwide distribution, affecting about 1/3 of human immunodeficiency virus (HIV) carriers and thus, is considered an opportunistic infection. Rapid and specific diagnostic tests for fungal infections are extremely important to effective treatment of infected patients. With the progress of molecular biology has been possible to develop new techniques for diagnosis and Cryptococcus spp identification. This study aimed standardizes a Cryptococcus spp DNA extraction method; determine the detection limit of fungus genome by PCR (Polymerase Chain Reaction) technique and determine the sensitivity and specificity of this technique in biological samples from infected patients. To standardize the DNA extraction were compared two ways for breaking fungus cell wall: the first one, using only glass beads, and the second one, using CTAB reagent. For standardization of conventional PCR and real time PCR (q-PCR), primers previously described were used, specific for Cryptococcus genus (CN4-CN5). A culture of C. neoformans (ATCC 24067) was used as a positive control. For q-PCR standardization for Cryptococcus spp genome detection and quantification in cerebrospinal fluid (CSF) were used 56 samples collected from positive neurocryptococcosis patients, which diagnostic was confirmed by other tests such as India ink, culture, latex and clinical findings. As negative control were used 44 CSF samples collected from negative neurocryptococcosis patients, according the same tests mentioned above. To confirm the specificity of the primers, q-PCR was tested in genomes of other fungal species. According to the results, no significant difference was observed between two DNA extraction methods, suggesting that for DNA extraction in CSF samples it is not necessary to use CTAB to lyse the fungus cell wall. The CTAB residues may inhibit the q-PCR, which is highly sensitive and requires a high degree of purity of the DNA. With respect to the detection limit, it was observed that the lowest DNA concentration detected by q-PCR was 47.0 femtograms (10-5) DNA/µL. The specificity test showed that primers were 100% specific, because it was not aligned in the other fungi species genomes tested. All 56 CSF samples of confirmed neurocryptococcosis patients were positive in q-PCR, while 18 (18.8%) CSF samples among negative neurocryptococcosis patients group were positive in q-PCR. This divergence of results can be explained by the fact that q-PCR has higher sensitivity than other tests used in cryptococcal meningitis diagnosis. Therefore, the q-PCR technique proved to be feasible for neurocryptococcosis diagnosis support, after it is well standardized, as in this study.
local.publisher.countryBrasil
local.publisher.departmentMED - DEPARTAMENTO DE CLÍNICA MÉDICA
local.publisher.initialsUFMG
local.url.externahttps://mycology.imedpub.com/articles/use-of-polymerase-chain-reaction-for-cryptococcus-neoformans-genomedetection-in-cerebrospinal-fluid-for-neurocryptococcosis-diagno.php?aid=9738

Arquivos

Pacote original

Agora exibindo 1 - 1 de 1
Imagem de Miniatura
Nome:
Use of Polymerase chain Reaction for Cryptococcus neoformans Genome Detection in Cerebrospinal Fluid for Neurocryptococcosis Diagnosis.pdf
Tamanho:
1.01 MB
Formato:
Adobe Portable Document Format
Baixar

Licença do pacote

Agora exibindo 1 - 1 de 1
Imagem de Miniatura
Nome:
License.txt
Tamanho:
1.99 KB
Formato:
Plain Text
Descrição:
Baixar

Coleções

Artigo de Periódico

Logo UFMG