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  1. Evaluation Summary:

    This paper is of broad interest to clinicians focused on improving sepsis outcomes. The method developed by the authors can identify 10 common pathogens with species-specificity in 4 hours, thus significantly reducing the turnaround time compared to conventional diagnostic methods. Using their method to identify sepsis-causing pathogens early to guide antibiotic treatment, the authors demonstrate high clinical sensitivity and specificity, and some clinical benefit in a real-world scenario.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Wang et al., developed a CRISPR/Cas 9 based protocol with the aim to accurately and quickly detect bacteria in ICU patients with severe pneumonia.

    The development of such a tool is important as quick and reliable identification of pathogens is extremely important. This study is innovative and aims to address an important clinical problem. The authors de novo designed an algorithm to screen species-specific . Then they used the species specific DNA tags to identify 10 pathogens.

    1. It is not very clear on which epidemiological data these pathogens were selected on. Moreover, the selected pathogens are only bacteria.

    2. Page 9. It is not very clear on how the primers' specificity was evaluated.

    3. Page 9. Were patients on antibiotics before getting into the trial?

    4. Page 10 At which timepoint the patients received different treatment based on the results of the culture or SSBD? Was this consistent?

    5. Page 11. The second sentence of 3.1 section in results is not clear.

    6. How were patients allocated to groups? Randomised?

    7. The table describing the patient cohort is in supplementary. This shall be in the main manuscript. It seems that the control and experimental groups were not balanced.

    8. The exact protocol of the study needs to be in the supplementary.

    9. Were any samples poly-microbial?

    10. Which was the threshold level of fluorescence (Figure 3) which was considered important?

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  3. Reviewer #2 (Public Review):

    Current culture-based, gold standard methods used for diagnosing the cause of sepsis provide results in 48-96 hours slowing antibiotic treatment initiation leading to poor patient recovery. This work provides a new tool for identifying sepsis- and pneumonia-causing pathogens in less than 4 hours with species-specificity with the hope that the fast turnaround time leads to early treatment and improved clinical outcomes. Using an optimized PCR+CRISPR-Cas12a DNA detection method, the assay demonstrates good analytical sensitivity and specificity for 10 common bacterial pathogens that cause pneumonia. The method is validated in a clinical cohort and the clinical benefit analyzed using a second cohort that is an intervention study used to guide clinicians on treatment choice.

    Strengths:

    Good clinical validation in two cohorts, including a blinded intervention study, indicating high clinical sensitivity and specificity when compared to conventional culture-based diagnostic methods and NGS.

    Combining PCR+CRISPR/ Cas12a assays increases specificity and allows for species-specific identification in less than 4 hours even if pathogen DNA sequences are highly similar. Due to the high specificity, many pathogens could be added to the panel in the future without cross-reactivity. The ability to detect 10 different pathogens separately using this method improves polymicrobial pneumonia detection and can identify and inform treatment choices in these problematic cases.

    The authors describe a workflow for targeting DNA regions with high similarity that could be used in future work outside of sepsis and pneumonia. For many diseases, there is an urgent need for multiplex analysis of various targets at once and these biomarkers may have high sequence similarity that could be targeted using similar methods explained in this work.

    Weaknesses:

    The authors imply that the implementation of their method has a clear clinical benefit but the data supporting this claim is sparse. Although the method seemed to improve disease severity, no improvement was indicated for important clinical measures including organ function, time of ventilation, and mortality. Testing more samples may provide a better understanding of the clinical benefit of the method but the data presented provide limited evidence.

    BALF is the primary sample used for diagnosis in this work but can be difficult to obtain in patients with severe symptoms and young children so may not be as useful in these patient populations reducing the application of this method.

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  4. Reviewer #3 (Public Review):

    In the manuscript, the authors provided the development of a sensitive and rapid diagnostic tool for detection of pathogenic bacteria in respiratory infections given the limitations of traditional cultures in the clinical settings. Rapid identification and treatment of bacterial infections can impact the prognosis in sepsis. This work highlights how a new rapid diagnostic tool may be beneficial in the treatment of patients with bacterial pneumonia given the time-consuming nature and low sensitivity of traditional culture methods.

    Strengths:

    The manuscript authors created a diagnostic tool using CRISPR-Cas12 with bacterial species-specific DNA-tags to 10 epidemic bacteria at their local intensive care unit (ICU). The appendix data provided detailed reports of the reaction conditions, sample preparations and reaction incubation time.

    A 2-stage validation process was used. The initial validation stage compared the use of the novel diagnostic tool to traditional cultures from bronchoalveolar lavage samples from ICU patients. Once the accuracy of the diagnostic tool was evaluated, the second validation stage was pursued in the form of a randomized controlled trial at the ICU of the study. The second validation stage demonstrated that the proposed novel diagnostic tool had faster results and correlated with improved APACHE II scores and more effective antibiotic coverage rates in the experimental group.

    The use of the novel diagnostic test highlighted limitations traditional culture modalities may have in identifying polymicrobial infections which were identified more frequently in the two validation stages

    Weaknesses:

    Although the study has many strengths, a potential weakness could lie in the unclear use of next-generation sequence (NGS) testing where samples were reported to be sent at random. However, similar to the novel diagnostic tool proposed in this manuscript, NGS testing has been noted to have high sensitivity and specificity and both had similar results in the manuscript.

    Additionally, the novel diagnostic testing demonstrated increased detection of polymicrobial infection when compared to traditional cultures; however, clinical evaluation will remain important to help decipher potential "false positive" results or identification of non-pathogenic colonization.

    Based on the author's proposed aims to develop a rapid and sensitive diagnostic tool for bacterial pathogens in pneumonia; the authors demonstrated a highly sensitive and specific test when compared to gold-standard testing. Random samples were assessed against NGS testing technology with similar reported results. The development of this rapid, sensitive diagnostic tool can have wide-spread clinical implications to guide management in patient care where earlier time to effective treatment can have important impacts on prognosis.

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