A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix

Abstract

Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

Keywords: biphasic; blood stream infection (BSI); isothermal amplification; porous dried blood matrix; sepsis diagnosis.

Fig. 1.

Biphasic reaction for pathogen identification. (A and B) Blood culture–based PCR methods as current gold standard. (A) Diagnosis time is governed by blood culture time. (B) If the blood culture is positive, PCR is performed. (C–H) Protocol workflow of our blood-processing module and following biphasic LAMP reaction for culture-free pathogen identification. (C) RBC lysis using ACK lysis buffer. (D) Mechanical vortex for bacteria lysis and DNA extraction. (E) Direct drying of whole blood without purification to create dried blood matrix while inactivating the inhibitory elements in whole blood. Thermal lysis improves the porosity of microfluidic and nanofluidic networks within the dried blood matrix. (F–H) Biphasic LAMP reaction (F) where the solid phase (G) of dried blood matrix acts as a substrate. The enzyme initiates LAMP amplification at a single temperature (65 °C). The amplicons diffuse out to the liquid phase (H) and bind to the fluorescent dye in clear supernatant, increasing the signal-to-noise ratio (SNR). Total turnaround time is 2.5 h, including 1.5 h of sample processing and 1 h of LAMP reaction.

Fig. 2.

Biphasic reaction schematic and analysis of blood before and after thermal lysis. (A) Process flow schematic of biphasic reaction. After drying, LAMP buffer reagents are added, and thermal lysis is conducted. Finally, primers and polymerase are added for the final reaction. Micro/nanofluidic channels are created during the thermal lysis heating step, so primers and polymerase may enter the blood matrix and find target DNA. (B) SEM images of the blood cake before thermal lysis. Image segmentation data show that the porosity of the blood cake is 5.2%. (C) SEM images of the blood cake after thermal lysis. Highest porosity is seen at 95 °C (66.8%). (D) Bars graph of the dried blood cake porosity versus thermal lysis temperature (n = 3 samples).

Fig. 3.

Characterization of biphasic LAMP reaction with and without thermal lysis and biphasic LAMP reaction with pathogen lysis in whole blood. (A) Simulation of porosity differences before (∼5%) and after (∼67%) thermal lysis at 95 °C. (B and C) Single-molecule detection of MRSA and E. coli DNA in no thermal lysis control (low-porosity) reactions from whole blood. Amplification threshold timings for detecting MRSA (B) and E. coli (C). (D and E) Amplification threshold times for MRSA (D) and E. coli (E) DNA detection in the biphasic reaction (high-porosity reactions). For one-copy amplifications, an expected three out of eight amplifications are seen within 60 min of reaction time due to Poisson sampling statistics. (F and G) Characterization of pathogen lysis in whole blood. Amplification threshold times of biphasic reactions with MRSA (F) and E. coli (G) pathogens in 4 µL of whole blood. The bar graphs show mean and SD data from n = 8 replicates of amplification.

Fig. 4.

Biphasic reaction coupled with mechanical pathogen lysis by bead beating for a detection limit of ∼1 CFU/mL for MRSA, MSSA, E. coli, and C. albicans. (A) Process flow schematic consisting of RBC lysis, mechanical bead lysis, drying, and biphasic reaction from whole blood. (B–E) Amplification threshold data for the detection of MRSA (B), MSSA (C), E. coli (D), and C. albicans (E) pathogens in 800 µL of whole blood (eight curves for the eight tubes per 800 µL of starting blood sample). If not all eight tubes amplified for a sample, the number of tubes that amplified is indicated above. One bar represents one sample of 800 µL of whole blood spiked with a specific CFU count (1 × 10⁴ to 1 or 0).

Fig. 5.

Evaluation of the biphasic approach using clinical samples. (A) Threshold times of biphasic reactions for 14 amplified positive samples (13 E. coli and 1 MSSA) and average time (42.5 ± 10.1 min, with green bar) and not amplified samples for negative samples. (B) Table summarizing sensitivity and specificity of the biphasic approach against blood culture and identification using PCR. (C) Overall time-to-result comparison between the biphasic and blood culture and identification. (D) Species-specific time-to-result comparison between biphasic (circle) and blood culture and identification (square) for E. coli (blue) and MSSA (red). A statistical comparison was performed; 95% CI, 95% confidence interval (CI).