Full text
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| Author(s): |
Cleison Ledesma Taira
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2013-09-06 |
| Examining board members: |
Gilda Maria Barbaro Del Negro;
Arnaldo Lopes Colombo;
José Eduardo Levi
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| Advisor: | Gilda Maria Barbaro Del Negro |
| Abstract | |
The incidence of candidemia among intensive care unit patients is associated with high mortality rates. The non-specific nature of the signs and symptoms of this infection difficult the diagnosis. Development of methods that allow early identification of the agent in clinical samples still represents a challenge. Classical methodologies (blood cultures and phenotypic identification) and serological tests (beta-D-glucan and mannan detection; anti-mannan antibodies) to diagnose candidemia lack sensitivity and specificity. Molecular methods are an alternative approach to the diagnosis of candidemia. In this work we developed a nested PCR multiplex assay able to identify seven Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae and C. pelliculosa) directly in blood samples of patients at high risk of candidemia. In the first stage of amplification universal fungal primers (ITS1 and ITS4) were used and in the second stage, the species-specific primers were divided into two amplification systems, one with primers for C. albicans, C. glabrata, C. tropicalis and C. lusitaniae, and another with primers for C. parapsilosis, C. krusei and C. pelliculosa. The multiplex nested PCR was able to detect approximately 4 CFU/mL of the seven Candida species, and showed no cross reactivity when evaluated with samples of DNA from other fungi and bacteria causing hematogenous infection episodes. We analyzed 55 samples obtained from patients admitted to pediatric intensive care units of the Instituto da Criança do HCFMUSP and Hospital de Base of Medical School from São José do Rio Preto - UNESP, with ages ranging from six days to 16 years, presenting the Systemic Inflammatory Response Syndrome and/or two or more risk factors of fungal infection. A control group of 28 children without risk of fungemia, to check for occurrence of nonspecific reactions in the molecular technique, was also evaluated. Blood cultures were positive in eight patients (14.8%), five were identified as C. albicans, and the remaining as C. tropicalis, C. parapsilosis and C. krusei. The nested multiplex PCR assay detected Candida DNA in 13 patients (23.6%), and there was concordance in species identification in 100% of the cases. In five patients with negative blood cultures, the molecular technique was capable of detecting dual candidemia in three patients\' samples, identifying C. tropicalis and C. parapsilosis simultaneously. However, there was no statistically significant disagreement (p = 0.063) between the two methods. In these five cases for whom only the PCR was positive, the amplified samples were sequenced, showing similarities from 99 to 100% when compared with sequences of Candida strains deposited in GenBank. Time for execution of the technique and release of results was about 24 hours. The cost of PCR assay was compared with the cost of the conventional blood culture tests and was lower. In conclusion, we demonstrated that the nested PCR multiplex developed in our laboratory is an alternative technique for the detection and identification of Candida species in patients at risk of candidemia, showing appropriate detection threshold and the ability to identify simultaneously more than one Candida species in clinical samples. The technique is also rapid, giving results in 24 h and exhibiting lower cost when compared with the conventional culture methodology (AU) | |
| FAPESP's process: | 10/02626-6 - Detection and identification of Candida spp. from bloodstream infections of critically ill pediatric patients: validation of a multiplex PCR |
| Grantee: | Cleison Ledesma Taira |
| Support Opportunities: | Scholarships in Brazil - Master |