Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Abstract

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

The potential of whole genome NGS for infectious disease diagnosis

“…[11] Using a similar test with improved sensitivity in 331 specimens collected from patients with suspected bloodstream infections demonstrated 35 positive specimens compared with 18 positive by culture. [12] The most probable cause of this discrepancy is likely that in most infections, the load of cultivable bacteria is several order of magnitude lower than the genome copy number, [12] and this is amplified in patients under antimicrobial therapy. [13] What could be the indication of such a wide-range test in immunocompromised patients suspected of infection?…”

Diagnosis of bacterial infection

“…In a recent study of 331 blood samples it was able to detect twice as many organisms as culture. [21] …”

In vitro diagnosis of sepsis: a review

“…130 This approach shows a sensitivity of 83% and a specificity of 94% compared to BC. 131 A major strength of PLEX-ID is the possibility to evaluate both sterile and nonsterile blood samples. IRIDICA is able to detect an extra 80 microorganisms that did not grow in BC.…”