Validação de protocolo de extração de DNA de amostras biológicas para a identificação molecular de Aspergillus spp

Abstract

Fungi are eukaryotic microorganisms that have important ecological functions in the environment, but they are also responsible for causing diseases in humans, such as Invasive Fungal Infections (IFIs). The increase in the number of cases is associated with various factors that compromise the patient's immunity, including low socioeconomic standards. The gold standard diagnosis for Aspergillus is performed through histopathological and cytopathological examinations, collected through lung biopsy or thoracoscopy. With the aim of proposing less invasive methods for patients, this study sought to validate methods for extracting deoxyribonucleic acid (DNA) from samples of different respiratory system fluids from patients suspected of Aspergillus. For this purpose, two mucolytic buffers were tested to reduce the amount of mucus present in the samples, aiding in the diagnosis by Real-time Polymerase Chain Reaction (qPCR). The obtained results were compared with the galactomannan test. The data from the 98 samples were statistically analyzed using Cohen's Kappa statistic and Pearson's chi-squared test, both operated in the R software. The results showed a 66% agreement (Kappa = 0.158) and 79% agreement (Kappa = 0.412) for the Lophomonas and N-acetylcysteine buffers, respectively. This study did not show statistically significant results to distinguish between the two evaluated mucolytic solutions (p> 0.05), suggesting the need to improve existing techniques, search for new tests, and explore different combinations of diagnosis in order to obtain a less invasive and more accurate analysis of the patient.