Novel fast pathogen diagnosis method for severe pneumonia patients in the intensive care unit: randomized clinical trial

Abstract

Background: Severe pneumonia is one of the common acute diseases caused by pathogenic microorganism infection, especially by pathogenic bacteria, leading to sepsis with a high morbidity and mortality rate. However, the existing bacteria cultivation method cannot satisfy current clinical needs requiring rapid identification of bacteria strain for antibiotic selection. Therefore, developing a sensitive liquid biopsy system demonstrates the enormous value of detecting pathogenic bacterium species in pneumonia patients.

Methods: In this study, we developed a tool named Species-Specific Bacterial Detector (SSBD, pronounce as 'speed') for detecting selected bacterium. Newly designed diagnostic tools combining specific DNA-tag screened by our algorithm and CRISPR/Cas12a, which were first tested in the lab to confirm the accuracy, followed by validating its specificity and sensitivity via applying on bronchoalveolar lavage fluid (BALF) from pneumonia patients. In the validation I stage, we compared the SSBD results with traditional cultivation results. In the validation II stage, a randomized and controlled clinical trial was completed at the ICU of Nanjing Drum Tower Hospital to evaluate the benefit SSBD brought to the treatment.

Results: In the validation stage I, 77 BALF samples were tested, and SSBD could identify designated organisms in 4 hr with almost 100% sensitivity and over 87% specific rate. In validation stage II, the SSBD results were obtained in 4 hr, leading to better APACHE II scores (p=0.0035, ANOVA test). Based on the results acquired by SSBD, cultivation results could deviate from the real pathogenic situation with polymicrobial infections. In addition, nosocomial infections were found widely in ICU, which should deserve more attention.

Conclusions: SSBD was confirmed to be a powerful tool for severe pneumonia diagnosis in ICU with high accuracy.

Funding: National Natural Science Foundation of China. The National Key Scientific Instrument and Equipment Development Project. Project number: 81927808.

Clinical trial number: This study was registered at https://ichgcp.net/clinical-trials-registry/NCT04178382" title="See in ClinicalTrials.gov">NCT04178382).

Keywords: randomized clinical trial; CRISPR/Cas12a; DNA tag; infectious disease; medicine; microbiology; pathogen detection; severe pneumonia.

Conflict of interest statement

YW, XL, YJ, DD, CZ, TS, MC, YY, HL, MG, HD, FX, WZ, JC, QW, QC, WY No competing interests declared

© 2022, Wang, Liang et al.

Figures

Figure 1.. Study design.

This study contained four stages: discovery stage, training stage, validation stage I and validation stage II. All patients were from the Department of Critical Care Medicine, Nanjing Drum Tower Hospital. Patients were randomly divided into two groups for the clinical trial.

Figure 2.. Screening workflow and statistics of species-specific DNA tags.

(A) Schematic diagram of screening species-specific DNA tags. (B) Genomic distribution of species-specific DNA tags in 10 bacteria. (C) Genomic proportion of species-specific DNA tags in 10 bacteria.

Figure 3.. SSBD development and effectiveness validation.

(A) SSBD workflow for clinical validation stages. (B) Cas12a and Cas12a-after-PCR detection of different concentrations and reaction times including 30 min (left) and 60 min (right). Blue bars indicated the Cas12a-after-PCR test. Brown bars indicated Cas12a test only. The concentration gradient of pGL3 plasmid from 10–17 M-10–7M was established as the test group. NC stood for the fluorescence values of PCR products of using DEPC-H2O as input. Each group had three repeats. Error bars indicated mean ± SEM of fluorescence value. ** indicated p-value <0.01 and *** indicated p-value <0.001 of unpaired t-test. (C) SSBD results of 10 pathogenic bacteria. Every test panel for each of 10 bacteria was used to detect genome DNA samples of 10 bacteria by SSBD. NC stood for the fluorescence values of PCR products of using DEPC-H2O as input. Each group had three repeats. Error bars indicated mean ± SEM of fluorescence value. *** indicated p-value <0.001 of unpaired t-test.

Figure 4.. Statistical analysis of test results and clinical outcomes in the two validation stages.

(A) Cross-tables for 5 of 10 bacteria by both SSBD and CCT in the validation stage I. (B) Cross-tables for 5 of 10 bacteria by both SSBD and CCT in the validation stage II. (C) Antibiotics coverage rate of each test in the two groups. Exp meant the experimental group, and Con meant the control group. Test 1: Day 1. Test 2: Day 3–5. Test 3: Day 7+. Raw antibiotics coverage results of each patient were available in Appendix 1—figure 4B. Detailed judging guidelines were shown in Appendix 1. (D and E) Line charts for APACHE II and SOFA scores, respectively. Error bars indicated mean ± SEM of scores of all the recorded patients. * indicated a significant difference between the two groups using two-way ANOVA.

Figure 5.. Statistical analysis of polymicrobial infection and nosocomial infection in the two validation stages.

(A) Statistics of pathogenic infection status of BALF samples in the two validation stages. (B) Verification from NGS results for 6 samples identified as polymicrobial infection by SSBD but not CCT or missed pathogens by CCT. (C) Case study of polymicrobial infection detected by SSBD and CCT. (D) Statistics of pathogens involved in polymicrobial infections in the two stages. (E) Case study of nosocomial infection identified by SSBD. (F) Case study of nosocomial infection identified by CCT. (G) Percentage of nosocomial infection identified by SSBD and CCT.

Appendix 1—figure 1.. Diagram of core principles for screening species-specific DNA-tags.

(A) Optimizing the algorithm of sequence alignment. Abbreviations: SA, sequence alignment; N, number of double sequence alignment; n, number of sequences. (B) Schematic map of screening intra-species conserved DNA fragments. (C) Schematic map of screening species-specific DNA tags.

Appendix 1—figure 2.. Epidemic data of pathogens in the Nanjing Drum Tower Hospital ICU in 2017.

10 targeted bacteria were indicated with the box.

Appendix 1—figure 3.. SSBD development and effectiveness validation.

(A) SSBD results of purified and unpurified DNA. NC, namely the fluorescence values of PCR products of using DEPC-H2O as input. Each group had three repeats. Error bars indicated mean ± SEM of fluorescence value. *** indicated p-value <0.001 of unpaired t-test. (B) SSBD results of reaction time gradient with Cas12a. Fluorescence values of K. pneumoniae and E. faecium by Cas12a through different incubation times after PCR. Gray represented NC, namely the fluorescence values of PCR products of using DEPC-H2O as input. Green and blue represented the fluorescence values of bacteria strains from different patients. Each group had three repeats. Error bars indicated mean ± SEM of fluorescence value. ** indicated p-value <0.01 and *** indicated p-value <0.001 of unpaired t-test. (C) SSBD results of 10 pathogenic bacteria with Cas12a. Gray represented NC, namely the fluorescence values of PCR products of using DEPC-H2O as input. Green and blue represented the fluorescence values of bacteria strains from different patients. Each group had three repeats. Error bars indicated mean ± SEM of fluorescence value. *** indicated p-value <0.001 of unpaired t-test.

Appendix 1—figure 4.. Judgment process and results of antibiotics coverage.

(A) Judgment process of antibiotics coverage. (B) The raw results of antibiotics coverage in two groups. Exp meant the experimental group, and Con meant the control group.

Appendix 1—figure 5.. Analysis of false-positive samples.

Numbers and fractions of different strength levels among all false-positive samples of each bacteria species in the validation stage I (A) and II (B). Strength could be seen roughly as bacterial amounts (level I-level III, the definition could be seen in the Appendix 1). False-positive situations meant pathogenic bacteria detected by SSBD but not by CCT in a given BALF sample.

Author response image 1.

Author response image 2.