Avaliação de métodos moleculares para detecção de Acinetobacter baumannii multidroga resistentes recuperados de pacientes com suspeita de pneumonia associada à ventilação mecânica

Abstract

Health Care Related Infections (IRAS) are considered one of the biggest public health problems in the world. The genus Acinetobacter represents an important pathogen related to hospital infections, mainly in ventilator-associated pneumonia (VAP). The objective of this study was to evaluate the performance of molecular methods in the laboratory diagnosis of Acinetobacter baumannii isolated from bronchoalveolar lavage (mini-BAL), obtained from adult patients with suspected VAP in an intensive care unit of the hospital of the clinics of Minas Gerais. The quantitative culture of the mini-BAL samples was used to compare the results obtained by evaluating the results of the molecular techniques: FISH, LAMP, conventional PCR, qPCR (SYBR® Green, TaqMan®) and ddPCR. A total of 44 mini-BAL samples were collected and eight of them were culture positive for A. baumannii all of them multidrug-resistant. The FISH technique showed no hybridization in agreement when compared to the controls used. LAMP the technique is sensitive and versatile, there was success in the standardization in the blaNDM-1 research presenting one agreeing in 100% with the results of conventional PCR, however for the other initiators researched as; ITS region, blaOXA-51 and blaKPC-2, false positives were detected when compared to PCR. In the comparison of the three generations of the PCR against the tested target, the conventional PCR showed good specificity and sensitivity, there was a correlation of 75% of the amplified products to the results of quantitative culture. SYBR® Green demonstrated low specificity to the bacterial panel tested, making quantification of questionable miniBAL samples possible. TaqMan® showed good sensitivity and specificity with detection limit equal to that obtained with SYBR® Green, 1.02 pg / ìl, but there was a discrepancy in the quantification of the 44 mini-BAL samples compared to the quantitative culture. The sensitivity and specificity of the ddPCR technique proved to be excellent, there was a perfect correlation of the results of the absolute quantification of the mini-BAL samples, to the quantitative culture results proving to be a precise and extremely efficient technique. Therefore, the results obtained with conventional PCR proved to be a good technique for detection and ddPCR proved to be more accurate and sensitive and could be used as a tool for monitoring the infectious process and for descaling the antimicrobial therapy used.