Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Abstract

Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Current status of rapid bacterial detection methods for platelet components: A 20‐year review by the ISBT Transfusion‐Transmitted Infectious Diseases Working Party Subgroup on Bacteria

“…PR, pathogen reduction; WB, whole blood 99.8% (with a lower one-sided 95% CI of 99.0%) and an analytical sensitivity that ranges from approximately 10 3 CFU/ml for Escherichia coli, Klebsiella oxytoca, S. epidermidis and Bacillus cereus to 10 5 CFU/ml for Streptococcus agalactiae, P. aeruginosa and Serratia marcescens [24]. The BacTx test was evaluated in spiking studies, showing high specificity and reproducibility with limits of detection ranging from 10 2 to 10 4 CFU/ml for clinically relevant PC contaminants, including slowgrowing and biofilm-forming species [25,26]. There are no published reports on the routine use of the BacTx assay for PC screening.…”

“…In general, low PC volumes (approximately 0.5 ml) are used for these assays, and nucleic acids are extracted on the basis of the test used and manufacturer's instructions (manually or using automated platforms). Universal bacterial detection can be performed targeting the 16s [25,26] or 23s [28] ribosomal genes or other genes such as rpoB or groEL [28]. Quality control of these tests is very important and is usually performed using culturenegative PC.…”

“…They concluded that NAT assays have great potential to screen blood components but require stringent method standardization and comparability to other PC screening systems. More recently, a Brazilian team reported the optimization of an RT‐PCR method to detect bacterial contamination in PCs [26]. In this protocol, a bacterial ribosomal 16s gene sequence representing common PC contaminants, including Cutibacterium acnes , was targeted, and the method was optimized to avoid non‐specific background noise by treating the PCR master mix with ethidium monoazide [31].…”