Rapid Point-of-Care Detection of Pathogens for Lower Respiratory Tract Infections in ICU Patients

Summary

This protocol describes a fully integrated microfluidic chip-based LAMP method enabling point-of-care detection of 15 pathogens causing lower respiratory tract infections in ICU patients within one hour.

Abstract

Lower respiratory tract infections (LRTIs) are among the leading causes of clinical deterioration and death in critically ill patients. In the intensive care unit (ICU), timely identification of the causative pathogen is essential for appropriate antimicrobial therapy; however, conventional culture methods take 3 to 5 days and have suboptimal positivity rates, particularly for fungi. Molecular approaches such as PCR shorten turnaround time but still depend on manual nucleic acid extraction and thermocycling, limiting their suitability for bedside use. This protocol describes a point-of-care workflow built around a fully integrated, disc-shaped microfluidic chip (CD chip) that couples magnetic bead-based nucleic acid purification with loop-mediated isothermal amplification (LAMP). After a simplified manual liquefaction and lysis step, the processed specimen is loaded onto the chip together with two prepackaged reagent vials. The instrument then autonomously performs extraction, amplification at 65 °C, and real-time fluorescence readout, returning qualitative results for 15 targets, namely 11 bacteria, 3 fungi, and 1 atypical pathogen, within 45 min of instrument run time (approximately 1 h for the complete sample-to-answer workflow, including the manual preprocessing step). We present detection data from 10 ICU specimens, including sputum, bronchial aspirates, and bronchoalveolar lavage fluid, encompassing both single-pathogen and polymicrobial co-infections involving up to 7 organisms on a single chip. The closed cartridge design minimizes the risk of aerosol contamination, and the single loading step requires no specialized training, positioning this system as a practical bedside diagnostic tool for ICU teams.

Introduction

Among critically ill patients in the ICU, lower respiratory tract infections, with ventilator-associated pneumonia (VAP) being chief among them, remain a persistent driver of morbidity, prolonged mechanical ventilation, and excess mortality^1,2. The microbiological landscape of ICU-acquired lower respiratory tract infections (LRTIs) differs markedly from that of community-acquired disease. Multidrug-resistant Gram-negatives (Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) predominate, often in polymicrobial constellations that confound empirical regimens^3,4. Opportunistic fungi add a further layer of complexity. Invasive pulmonary aspergillosis, once considered a hallmark of profound immunosuppression, is now recognized with increasing frequency in non-neutropenic ICU patients, most notably after severe influenza and COVID-19⁵. Existing diagnostic algorithms for aspergillosis couple low-yield culture with galactomannan (GM) antigen testing, yet the GM assay is constrained by well-documented diagnostic windows and variable sensitivity in non-neutropenic hosts⁶. Each hour of delay in initiating appropriate antimicrobial therapy for sepsis is associated with a measurable increase in mortality⁷. Taken together, the speed and complexity of ICU infections demand diagnostic tools that can match clinical urgency.

Standard microbiological culture, while indispensable for susceptibility testing, is fundamentally rate-limited: turnaround spans 3 to 5 days, fastidious organisms are missed, and fungal culture positivity remains low⁸. Metagenomic next-generation sequencing (mNGS) offers an unbiased alternative, yet its clinical adoption is tempered by cost, processing times of 24 to 48 hours, reliance on bioinformatics pipelines, and limited point-of-care feasibility⁹. PCR-based panels have narrowed the time gap, but they still require multi-step nucleic acid extraction, thermal cycling instrumentation, and, for conventional formats, post-amplification electrophoresis¹⁰. None of these platforms fully satisfies the convergent demands of the ICU: speed of less than one hour, minimal hands-on time, multiplexed pathogen coverage including fungi, and operational simplicity sufficient for non-laboratory personnel.

Loop-mediated isothermal amplification (LAMP) addresses several of these constraints. First described by Notomi et al., the method amplifies target DNA at a constant 65 °C using a set of four to six primers that recognize six distinct regions, driven by a strand-displacing polymerase¹¹. The reaction generates characteristic stem-loop and cauliflower-like amplicon structures detectable via real-time fluorescence, yielding S-shaped curves within minutes of threshold crossing. Sensitivity at low template concentrations is high, and the multi-primer design confers selectivity that limits false positives^12,13. When coupled with microfluidic chip platforms, LAMP can be parallelized across multiple targets from a single specimen load¹⁴. Earlier work demonstrated that LAMP chip systems detect respiratory pathogens with turnaround well under three hours¹⁵. A key limitation of those systems, however, is their dependence on separate, manual DNA extraction, which adds time, operational complexity, and contamination risk.

The system described here eliminates that bottleneck. Built around a single-use, disc-shaped microfluidic chip (86 mm in diameter), it integrates sample loading, magnetic-bead-based nucleic acid purification, and 16-plex LAMP amplification in a single sealed cartridge (Figure 1). The operator performs only a brief manual liquefaction-lysis step using a purpose-designed processing tube, then loads the lysate and two reagent vials onto the chip (Figure 2). From that point forward, the instrument handles extraction, isothermal amplification at 65 °C, and fluorescence acquisition autonomously, delivering qualitative calls for 15 clinically relevant targets, namely 11 bacteria, 3 fungi (Aspergillus spp., Candida albicans, Pneumocystis jirovecii), and 1 atypical pathogen (Mycoplasma pneumoniae), within 45 min. A neuroblastoma breakpoint family (NBPF) gene used as an internal control validates specimen adequacy and reaction integrity in each assay. This protocol provides step-by-step instructions for the complete workflow, from ICU specimen collection through preprocessing, chip loading, automated detection, and result interpretation, intended for both clinical laboratory staff and ICU bedside teams.

Protocol

All samples were collected under a protocol approved by the Ethics Review Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (Approval No. I-24PJ1778). Written informed consent was obtained from each participant or their legally authorized representative prior to specimen collection. The target pathogen panel and reaction-well assignments are listed in Table 1, and the consumables used during sample preprocessing are listed in Table 2.

1. Collection of clinical samples from the lower respiratory tract in the ICU

1. Sputum collection
   1. Rinse the oral cavity with clean water or sterile physiological saline three times to clear food residue and commensal oral flora.
   2. Instruct the patient to take two to three deep breaths, then cough forcefully to expel deep respiratory sputum directly into a sterile collection cup.
      NOTE: An acceptable specimen consists of viscous, purulent, or blood-streaked material from below the trachea; clear saliva or postnasal drip is inadequate and may compromise downstream detection.
2. Bronchial aspirate (BAS) collection
   1. For non-intubated patients, insert a sterile suction catheter (a 10–12 Fr catheter, with the suction source set to a pressure of −0.0267 MPa, equivalent to −200 mmHg) through the nasal or oral route to a depth of approximately 30 cm.
   2. Apply suction while rotating the catheter and withdraw the catheter slowly. Collect the aspirated secretions in a sterile specimen trap.
      NOTE: If secretions are scant, instill approximately 5 mL of sterile saline via the catheter before suctioning.
   3. For mechanically ventilated patients, select the suction catheter according to the endotracheal tube size: use a 10 Fr catheter for a 7.0–7.5 mm tube and a 12 Fr catheter for an 8.0 mm tube.
   4. Pre-oxygenate the patient with 100% oxygen for 30–60 s before suctioning. Disconnect the ventilator circuit and insert the selected catheter through the artificial airway until resistance is encountered.
   5. Apply a negative pressure of −0.0267 MPa (equivalent to −200 mmHg), limiting each suction pass, and thus the ventilator-disconnection time, to no more than 15 s to minimize derecruitment and hypoxemia, and re-oxygenate the patient with 100% oxygen for 30–60 s after suctioning.
   6. Instill 1–2 mL sterile saline through the catheter and wait for 3 s.
   7. Apply suction and withdraw the catheter. Collect the aspirated secretions in a sterile specimen trap. Reconnect the ventilator circuit immediately after specimen collection.
      NOTE: Strict aseptic technique is essential throughout to avoid nosocomial contamination of the specimen.
3. Bronchoalveolar lavage fluid (BALF) collection
   1. Identify the target segment on chest imaging. Advance the bronchoscope and wedge it into the appropriate segmental or subsegmental bronchus.
   2. Instill 20–50 mL aliquots of sterile saline rapidly, for 3–5 aliquots (60–120 mL total). Apply gentle suction after each aliquot, then collect the recovered fluid in a sterile, sealed container.
      CAUTION: Continuous hemodynamic and ventilatory monitoring is mandatory during BAL in ICU patients. Adjust ventilator settings as needed for mechanically ventilated subjects.
      NOTE: Typical recovery is 40–60% of the instilled volume. Poor return may necessitate repositioning the bronchoscope tip.

2. Liquefaction and pathogen lysis

1. Using the #2 disposable pipette, aspirate the specimen to the marked line (approximately 1 mL) and dispense it into the liquefaction tube. Squeeze the pipette bulb repeatedly to transfer all viscous material, then cap the tube.
   NOTE: For BALF or watery sputum, load two pipette volumes to ensure sufficient pathogen input.
2. Shake the liquefaction tube vigorously by hand for 1 min. Inspect the sample visually and continue shaking until no visible mucus clumps remain and the specimen flows freely through the #1 disposable pipette without resistance (a uniform, water-like consistency). Shake for at least 1 min, extending the time as needed for highly viscous specimens until these criteria are met.
3. Open the sealing cap of the lysis tube.
4. Aspirate approximately 0.5 mL of the homogenized sample using the #1 disposable pipette and dispense the sample into the lysis tube.
5. Close the sealing cap and verify that the tip cap is securely tightened.
6. Seat the lysis tube in the Sample Preprocessing device (SP device), pressing down until it is secure. Press Start.
7. Allow the SP device to complete the automated liquefaction-lysis cycle (parameters in Table 3) lasting approximately 15 min.
   NOTE: Do not open or remove the tube during processing.

3. Chip loading and automated detection

1. Reagent and sample loading
   1. Remove the CD chip from 4 °C storage and equilibrate it at room temperature (approximately 25 °C) for 10 min.
      ​NOTE: Reagent vials O and B and the preprocessed lysate are used at room temperature; the on-chip LAMP amplification is performed at 65 °C (see step 3.2.3 and Table 3).
   2. Open the foil pouch and place the chip on a flat surface.
   3. Rotate the cap counterclockwise to open the chip.
   4. Flick the vial O to collect the liquid at the bottom of the vial.
   5. Uncap the vial O, insert the nozzle into the O port, and dispense the entire contents into the port.
   6. Discard vial O. Repeat the procedure using vial B and the B port.
   7. Remove the processed lysis tube from the SP device and remove the tip cap.
   8. Align the tube tip with the S port and dispense the entire lysate volume into the chip.
   9. Discard the used lysis tube.
   10. Twist the chip cap clockwise until two audible clicks confirm a complete seal.
       NOTE: Incomplete loading of any port may cause individual channel failures. Confirm full dispensing before sealing. To avoid incomplete sealing and contamination risks, do not wear PE gloves when sealing the cap; latex or nitrile gloves are recommended instead.
2. Instrument operation
   1. Power on the automated nucleic acid analyzer. Tap Detect, scan the barcode on the chip foil pouch, and enter patient demographics and sample ID.
   2. Mount the chip on the rotational spindle and press down until a click confirms engagement.
   3. Tap Start. The instrument autonomously performs centrifugal reagent distribution, magnetic bead extraction, LAMP amplification at 65 °C, and fluorescence acquisition. Run time is approximately 45 min (the reagent/sample loading and instrument workflow are summarized in Figure 3).
      NOTE: Do not open the lid during the run.
3. Result interpretation
   1. The software identifies the inflection point of each S-shaped amplification curve by second derivative maximum analysis and reports the corresponding TP value (time to positivity).
   2. Compare each TP value with the corresponding threshold listed in Table 4. Classify channels with TP values at or below the threshold as positive and channels with TP values above the threshold as negative.
   3. Verify that the internal control channel (well 16) exhibits an S-shaped amplification curve with a TP value below 12 min.
   4. Reject the run if the internal control fails. Repeat testing using a fresh chip or recollect the specimen.

Results

On completion of a valid run, the instrument displayed real-time fluorescence traces for all 16 channels. Positive channels exhibited the expected S-shaped amplification kinetics with automatically annotated TP values, whereas negative channels remained at baseline. The IC channel (well 16) yielded an S-shaped curve with TP < 12 min in all valid runs.

As a pilot, proof-of-concept demonstration of the protocol, 10 representative specimens were selected to illustrate the range of clinical sce...

Discussion

Rapid pathogen identification is critical in ICU infection management. The system described here reduces the time to pathogen identification from the 3 to 5 days typically required for culture to less than 1 hour, a timeframe during which first-line antimicrobial decisions are often made⁷. By integrating nucleic acid extraction, purification, and 16-plex LAMP amplification within a single sealed chip, this workflow eliminates the manual DNA extraction step, which has been the principal operational...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated Nucleic Acid Analyzer	Beijing Taihao Biotechnology Co., Ltd	THE-T80-01	TurboFlow T; performs centrifugal reagent distribution, magnetic-bead extraction, LAMP at 65 °C, and real-time fluorescence detection (~45 min run)
#1 Disposable Pasteur pipette	Zhongtai Experimental Equipment Factory (Haimen)	Custom-made	Disposable consumable; 0.5 mL; transfers homogenized specimen into lysis tube
#2 Disposable Pasteur pipette	Zhongtai Experimental Equipment Factory (Haimen)	Custom-made	Disposable consumable; 2 mL; aspirates specimen to marked line into liquefaction tube
CD chip	Beijing Hongyang Chensheng Technology Co., Ltd	Custom-made	Disposable consumable; 86 mm disc with central loading zone (ports O, B, S), magnetic-bead purification zone, and outer ring with 16 reaction wells (15 pathogen targets + NBPF internal control)
LAMP reaction reagents	Yeasen Biotechnology (Shanghai) Co., Ltd	16730ES80	LAMP reaction reagents pre-stored in CD chip as lyophilized beads
Liquefaction tube	Beijing Taihao Biotechnology Co., Ltd	Custom-made	Disposable consumable; 2 mL/tube; receives specimen for manual homogenization
Lysis tube	Beijing Taihao Biotechnology Co., Ltd	Custom-made	Disposable consumable; 0.5 mL/tube; compressible silicone body with 200 µm filter membrane; preloaded lysis reagents
Primers	Sangon Biotech (Shanghai) Co., Ltd	Custom-made	Primer mixture embedded in reaction chamber of CD chip
Sample Preprocessing device	Beijing Taihao Biotechnology Co., Ltd	THE-R10-02	TS-2000; automated liquefaction–lysis cycle (~15 min)
Single-dose vial “B”	Ruifu Biotechnology (Guangzhou) Co., Ltd	Custom-made	Disposable consumable; pre-packaged on-chip reagent; one per test; stored at 4 °C and equilibrated to room temperature (~25 °C) before loading
Single-dose vial “O”	Ruifu Biotechnology (Guangzhou) Co., Ltd	Custom-made	Disposable consumable; pre-packaged on-chip reagent; one per test; stored at 4 °C and equilibrated to room temperature (~25 °C) before loading