Rapid and precise identification of bloodstream infections using a pre-treatment protocol combined with high-throughput multiplex genetic detection system

Abstract

Background Bloodstream infection (BSI) is a life-threatening condition with high morbidity and mortality rates worldwide. Early diagnosis of BSI is critical to avoid the unnecessary application of antimicrobial agents and for proper treatment. However, the current standard methods based on blood culture are time-consuming, thus failing to provide a timely etiological diagnosis of BSI, and common PCR-based detection might be inhibited by matrix components. Methods The current study explored an integrated pre-analytical treatment protocol for whole blood samples, wherein pathogens are enriched and purified by incubation and concentration, and inhibitors are inactivated and removed. Further, this study developed and evaluated a novel high-throughput multiplex genetic detection system (HMGS) to detect 24 of the most clinically prevalent BSI pathogens in blood culture samples and pre-treated whole blood samples. The specificity and sensitivity were evaluated using related reference strains and quantified bacterial/fungal suspensions. The clinical utility of BSI-HMGS combined with the pre-analytical treatment protocol was verified using blood cultures and whole blood samples. Results The combined pre-treatment protocol and BSI-HMGS was highly specific for target pathogens and possessed a low detection limit for clinical whole blood samples. The pre-treatment protocol could deplete the PCR inhibitors effectively. For blood culture samples, the current method showed 100.0% negative percent agreements and > 87.5% positive percent agreements compared to the reference results based on blood culture findings. For whole blood samples, the current method showed 100.0% negative percent agreements and > 80.0% positive percent agreements compared to the reference results for most pathogens. The turnaround time was ≤ 8 h, and all the procedures could be conducted in a general clinical laboratory. Conclusion The BSI-HMGS combined with the pre-treatment protocol was a practical and promising method for early and precise detection of BSIs, especially for areas without access to advanced medical facilities.

Evaluation of complete blood count, c-reactive protein, and lactate dehydrogenase in culturable and unculturable bacteremia for early diagnosis of sepsis

“…One of the simplest and most frequently employed investigations to determine the origin of bloodstream infections is the laboratory detection of bacteremia using blood cultures (Schwarzenbacher et al, 2019). A precise diagnosis of the bacteria causing bloodstream infections gives essential clinical data to identify and treat sepsis (Zhang et al, 2022). However, a blood culture alone is not sensitive enough to detect bacterial sepsis, especially when the patient has already taken antibiotics or there is fastidious organisms present that cannot grow under normal conditions (culture gives false negatives).…”

Culture‐free biphasic approach for early sepsis detection: A true game changer

“…2 Particularly, in the event of septic shock, a 7.6% increase in mortality is observed for every hour that passes without proper antimicrobial therapy. 3 It is also worth noting that those that survive sepsis may be prone to long-standing adversities that involve physical, psychological, and cognitive impairments. 2 Thus, early diagnosis of the condition, followed by timely treatment, is crucial.…”

“…Moreover, when the infection is initially detected, even a mild degree of organ failure is linked to an in‐hospital death rate of more than 10% 2 . Particularly, in the event of septic shock, a 7.6% increase in mortality is observed for every hour that passes without proper antimicrobial therapy 3 . It is also worth noting that those that survive sepsis may be prone to long‐standing adversities that involve physical, psychological, and cognitive impairments 2 .…”

Evaluation of the Performance of a Multiplex Real-Time PCR Assay for the Identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii Simultaneously from Sputum in Multicenter [Response to Letter]

“…In contrast, multiplex PCR, with simple primer and probe design, allows for the the detection of three to six pathogens in general and even dozens of pathogens simultaneously. 4 , 5 …”

“…In contrast, multiplex PCR, with simple primer and probe design, allows for the the detection of three to six pathogens in general and even dozens of pathogens simultaneously. 4,5 Then, the entire process of MCDA-LFB can be controlled within 60 minutes, if the technique is used for highthroughput detection. However, it needs a lot of manual work or complex equipment.…”