Joint application of metagenomic next-generation sequencing and histopathological examination for the diagnosis of pulmonary infectious disease

ABSTRACT The diagnosis of some pulmonary infectious diseases and their pathogens is very difficult. We clarified the diagnostic value of the joint application of metagenomic next-generation sequencing (mNGS) and histopathological examination for the diagnosis of pulmonary infectious diseases in this study. We retrospectively enrolled 108 patients with infectious and noninfectious diseases who visited the West China Hospital of Sichuan University from January 2019 to June 2022. Pathogens in core needle biopsy tissue were detected using mNGS and traditional pathological examination. The diagnostic accuracy of the combined mNGS and histopathology protocol for pulmonary infectious diseases was contrasted with that of histopathological examination alone. The area under the receiver operating characteristic curve (ROC curve) of mNGS combined with histopathological examination was 0.840 for pulmonary infectious diseases, and the area under the ROC curve of histopathological examination alone was 0.644. The areas under the ROC curve of mNGS combined with histopathological examination for pulmonary fungal infection, pulmonary tuberculosis, and lung abscess were 0.876, 0.875, and 0.840, respectively. In pulmonary infectious diseases, the areas under the ROC curve of histopathological examination alone for the above three types of pulmonary infections were 0.760, 0.719, and 0.575, respectively. For pulmonary infectious diseases, a joint application of metagenomic next-generation sequencing and histopathological examination can not only be used for differential diagnosis but also increase the positive rate of pathogen identification compared with histopathological examination alone, especially for rare pathogen infections. IMPORTANCE The diagnosis of some pulmonary infectious diseases and their pathogens is very difficult. A more precise diagnosis of pulmonary infectious diseases can help clinicians use proper antibiotics as well as reduce the development of drug-resistant bacteria. In this study, we performed both mNGS and pathology on lung puncture biopsy tissue from patients and found that combined mNGS and histopathology testing was significantly more effective than histopathology testing alone in detecting infectious diseases and identifying infectious diseases. In addition, the combined approach improves the detection rate of pathogenic microorganisms in infectious diseases and can be used to guide precision clinical treatment.

results are timely and of interest, but the presentation of the result data does not provide clear insight into the clinical significance of the increased sensitivity provided by mNGS.I have specific suggestions for improvement.
Major suggestions: 1. Line 163; while the morphology of microorganisms on histopathologic examination affords some suggestion of genus, in my experience it is not typically possible to definitively identify the genus of a microorganism in a sample (for example, filamentous bacteria could potentially represent actinomyces or nocardia, while hyaline fungal hyphae could represent aspergillum or fusarium or many other fungi); correlative culture or molecular testing may resolve the issue, but in our practice we would not describe microorganisms at the genus level in most cases by morphology alone.It might be more appropriate to indicate the the general morphologies that were observed by histopathology.Extending from this thought, the data presented in table 2 illustrates the incremental improvement in genus level identification of microorganisms utilizing mNGS.There is little surprise (in my opinion) that additional organisms would be identified at the genus level; I am not sure detailing each organism in this way clarifies the value of the additional diagnostic sensitivity.I this this figure (or a simple table of these organisms) could be added to appendix material rather than presented here.2. Figure 3.I'm not certain that the performance differences between histopathologic examination alone, mNGS alone and the combination is best represented in this manner.I would suggest instead presenting the data in a simple tabular format wherein the performance differences for bacteria / fungi / mycobacteria by case are more easily appreciated.For instance, I cannot tell from this analysis if there were any cases where histopathology was successful but mNGS was not.Similarly, is the increased "sensitivity" of mNGS in this context a factor of detecting additional organisms per case or organisms in additional cases of both? 3. It would be helpful if the clinical significance of the additional sensitivity reported for mNGS were more clearly illustrated.The two provided case examples show lesions that were histopatholgically identified as fungal infections and yet were identified as bacterial infections by mNGS.Did mNGS also identify the fungal infections?Is there a rationale for why a polymicrobial bacterial infection (one assumes abscess) was not detected by histopathologic examination?Is sampling (different cores sent for pathology and molecular testing) a potential reason? 4. The low-sensitivity and poor turnaround time of culture is referenced in the paper, but comparative turnaround time for mNGS and histopathologic examination is not provided -it should be. 5. Additional details on the mNGS method should be provided.How was the nucleic acid extracted and how were libraries prepared?What sequencing platform was employed?What was the average depth of sequencing?If this has been published elsewhere, the method could be referenced.
Minor suggestions: 1. Line 30; the phrase "punctured lesion tissue" may not be familiar to all readers, I might suggest "core needle biopsy tissue" or some other surrogate descriptor.2. Line 55; it may be helpful to quantify the positive rate of culture (described as 'low') and provide a reference to help contextualize the results for the reader 3. Line 79; The construction of the sentence beginning "The preliminary clinical diagnosis..." Is somewhat awkward, I would suggest revising the sentence to make the key points more clear.4. Line 93; A 'lyophilized tube containing 10% formalin" is referenced; this may simply be out of my experience, but I have not encountered 10% formalin in a non-liquid preparation (in our practice is typically buffered formalin in water).Is the descriptor lyophilized appropriate here? 5. Line 104; the stain descriptors "hexamine silver staining, and antacid staining" are less familiar to me and may not be readily understood by all readers.Would these stains be equivalent to Gomori-Grocott methenamine silver stain (GMS) and/or Ziehl-Neelsen acid-fast bacilli staining (AFB)? 6. Microorganism genus and species names should be italicized 7. Line 125; the descriptor "bidirectional fungi" may be less familiar than "dimorphic fungi" 8. Line 125; "Penicillium marneffei" was renamed "Talaromyces marneffei" in 2015; it may be helpful to employ the updated name.9. Line 151; the expanded form of G/GM tests should be written before the abbreviations are employed 10.Line 159; I assume the P-value does not actually equal 0.000, perhaps a less than symbol be employed?11.Line 197; was a beta-lactamase inhibitor used in combination with penicillin as stated, or another beta-lactam antibiotic (such as ampicillin)?
Reviewer #3 (Comments for the Author): Major issues: -Culture is often not ordered when obtaining a lung biopsy and an infectious process is only suspected after pathology has been performed -comparison of the culture data with the mNGS results would be useful to assess performance.Culture remains the diagnostic standard, from the patients enrolled, how many specimens were actually processed for culture?-This manuscript is either proposing mNGS as a diagnostic tool or a case report -see Case studies section -consider publishing those separately.This manuscript could be more focused -Pathogen abundance in the mNGS data is missing, how significance was assessed in the case of polymicrobial findings -mNGS detailed procedure or reference to a detailed procedure is missing

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Review manuscript: The joint application of metagenomic next-generation sequencing and histopathological examination for the diagnosis of pulmonary infectious disease Authors: Yang L, et al Summary: The authors present the comparison between mNGS and traditional pathological examination for the diagnosis of pulmonary infectious diseases.Metagenomics has been proven to be a powerful tool for the diagnosis of occult infections were other techniques may have limitations in sensitivity.Lung infectious are particularly difficult to diagnose for reasons the authors stated and often also because an infectious process is not suspected until after pathology has been performed.

Major issues:
-Culture is often not ordered when obtaining a lung biopsy and an infectious process is only suspected after pathology has been performed -comparison of the culture data with the mNGS results would be useful to assess performance.Culture remains the diagnostic standard, from the patients enrolled, how many specimens were actually processed for culture?-This manuscript is either proposing mNGS as a diagnostic tool or a case report -see Case studies section -consider publishing those separately.This manuscript could be more focused -Pathogen abundance in the mNGS data is missing, how significance was assessed in the case of polymicrobial findings -mNGS detailed procedure or reference to a detailed procedure is missing Microbiology Spectrum.We appreciate the constructive comments/suggestions provided by the reviewers.We have revised this manuscript to address the concerns raised by the reviewers, and all changes in the revision are highlighted in red font.Our point-by-point replies are listed below.We hope that the revised version of the manuscript is now suitable for publication in Microbiology Spectrum.We are willing to provide further information if necessary.
None of the authors have any conflicts of interests or financial disclosures to declare.
Thank you again for considering our revised manuscript!If you have any queries, please don't hesitate to contact me at the address by liudan10965@wchscu.cn.

Reviewer comments:
Reviewer #2: The authors' manuscript "The joint application of metagenomic next-generation sequencing and histopathological examination for the diagnosis of pulmonary infectious disease" describes the combination of molecular sequencing and histopathology to diagnose infections in the pulmonary setting.The authors conclude that metagenomic NGS testing is superior to histopathologic examination and that combined metagenomic testing and histopathologic examination has the highest degree of sensitivity.The results are timely and of interest, but the presentation of the result data does not provide clear insight into the clinical significance of the increased sensitivity provided by mNGS.I have specific suggestions for improvement.
Answer: Thank you for your suggestions and recommendations.
Major suggestions: 1. Line 163; while the morphology of microorganisms on histopathologic examination affords some suggestion of genus, in my experience it is not typically possible to definitively identify the genus of a microorganism in a sample (for example, filamentous bacteria could potentially represent actinomyces or nocardia, while hyaline fungal hyphae could represent aspergillum or fusarium or many other fungi); correlative culture or molecular testing may resolve the issue, but in our practice we would not describe microorganisms at the genus level in most cases by morphology alone.It might be more appropriate to indicate the the general morphologies that were observed by histopathology.Extending from this thought, the data presented in table 2 illustrates the incremental improvement in genus level identification of microorganisms utilizing mNGS.There is little surprise (in my opinion) that additional organisms would be identified at the genus level; I am not sure detailing each organism in this way clarifies the value of the additional diagnostic sensitivity.I this this figure (or a simple table of these organisms) could be added to appendix material rather than presented here.
Answer: Thank you for your suggestions and recommendations.As suggested, molecular nucleic acid testing will be added after finding pathogenic bacteria in pathological tissue, such as TB-DNA for Mycobacterium, and molecular PCR for Aspergillus and Nocardia to identify pathogens.Therefore, "histopathological testing alone" may confuse the reader, and we changed "histopathological testing alone" to "pathological tissue + molecular nucleic acid testing (PCR)" (line 181).In addition, we modified the means of histopathological examination into the sentence "In addition, molecular nucleic acid testing will be added after the discovery of suspected pathogenic bacteria, such as TB-DNA and polymerase chain reaction (PCR) for Mycobacterium spp., and PCR for Aspergillus spp.and Nocardia spp." (line 120).Besides, we have removed Figure 2 in response to your comments and replaced it with a more informative Table S1.Thanks for your professional consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.
2. Figure 3.I'm not certain that the performance differences between histopathologic examination alone, mNGS alone and the combination is best represented in this manner.I would suggest instead presenting the data in a simple tabular format wherein the performance differences for bacteria / fungi / mycobacteria by case are more easily appreciated.For instance, I cannot tell from this analysis if there were any cases where histopathology was successful but mNGS was not.Similarly, is the increased "sensitivity" of mNGS in this context a factor of detecting additional organisms per case or organisms in additional cases of both?
Answer: Thank you for your suggestions and recommendations.After careful consideration, we thought that the ROC curve can be used to compare the diagnostic efficacy of the two protocols.The main roles of ROC curves are (1) making it easy to find out the ability of a classifier to recognize a sample at a certain threshold value.(2) selecting the best diagnostic threshold value of a diagnostic method.[2] The closer the ROC curve is to the upper left corner, the higher the sensitivity and the lower the false positive rate, the better the performance of the diagnostic method.(3) comparing the ability of two or more different diagnostic methods to identify a disease [2].When comparing two or more diagnostic methods for the same disease, the ROC curves of each diagnostic method can be drawn in the same ROC space, so that the advantages and disadvantages of each diagnostic method can be visually identified.The ROC curve near the top left corner represents the better performance of the diagnostic method.Besides, the increased sensitivity of mNGS is due to the ability to detect more pathogenic microorganisms in each case.In addition, the test results for each patient have been added in Table S1.Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.3. It would be helpful if the clinical significance of the additional sensitivity reported for mNGS were more clearly illustrated.The two provided case examples show lesions that were histopatholgically identified as fungal infections and yet were identified as bacterial infections by mNGS.Did mNGS also identify the fungal infections?Is there a rationale for why a polymicrobial bacterial infection (one assumes abscess) was not detected by histopathologic examination?Is sampling (different cores sent for pathology and molecular testing) a potential reason?Answer: Thank you for your suggestions and recommendations.Both methods used core needle biopsy tissue (which we have described in detail in Line 102).As suggested, we carefully compared the histopathological findings with the mNGS findings in both patients and found that mNGS detected the presence of a mixture of microorganisms (Table S1).
While diagnosis of pathogenic bacteria by morphology has some errors.In both cases, the pathologists misidentified the pathogen as a fungus and antifungal treatment was ineffective.
Resorption of the lesion after antibacterial treatment suggested the absence of fungal infection and confirmed the diagnosis of mNGS.Therefore, we explain it accordingly at line 206.
Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.4. The low-sensitivity and poor turnaround time of culture is referenced in the paper, but comparative turnaround time for mNGS and histopathologic examination is not provided -it should be.
Answer: Thank you for your suggestions and recommendations.As suggested, we provided comparative turnaround time for mNGS and histopathologic examination in line 267 "in our cases, it takes 24 hours for mNGS getting results, 7-10 days for histopathology section preparation and special staining, and about 3 days for common bacteria, 5-7 days for Nocardia, and 21 days for Mycobacterium in tissue culture".In addition, we added the culture positive rate of these cases in line 266 " in our cases, the culture positivity rate was only 16.7%".Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion. 5. Additional details on the mNGS method should be provided.How was the nucleic acid extracted and how were libraries prepared?What sequencing platform was employed?What was the average depth of sequencing?If this has been published elsewhere, the method could be referenced.
Answer: Thank you for your suggestions and recommendations.As suggested, we have added specific processes and information on nucleic acid extraction, library production, sequencing depth and sequencing platforms: nucleic acid extraction: DNA was isolated using magnetic bead adsorption after lung tissue samples were forcibly fractured with a wall breaker and then treated with lysozyme for 10 min.The BGI metagenomics library kit was used to prepare the library after lung tissue samples were split up by enzymatic digestion, end repair, primer adding, and PCR amplification.In this investigation, the sequencing depth was >20M reads using the MGIseq2000 sequencing technology with SE50 (Line 106).
Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.
Minor suggestions: 1. Line 30; the phrase "punctured lesion tissue" may not be familiar to all readers, I might suggest "core needle biopsy tissue" or some other surrogate descriptor.
Answer: Thank you for your suggestions and recommendations.After careful consideration, we thought it was more appropriate to use " core needle biopsy tissue " in Line 30, and we have corrected it in the revised manuscript.By reviewing the literature, we found a number of high-quality articles that also used the terminology you suggested [1,2].Of course, we have corrected similar statements in the other parts of revised manuscript.Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.o Line 197 -what is Clostridium forcestorum spp?Answer: Thank you for your suggestions and recommendations.We rechecked the patient's mNGS results and reviewed the literature on the bacterium.The correct expression of this bacterium should be " Tannerella forsythia" [1,2] (Line 219).Tannerella forsythia, a gram-negative anaerobic bacillus, is one of the important causative agents of periodontal disease and is closely associated with the development of periodontal disease [3].Of course, we have corrected similar statements in the other parts of revised manuscript.Thanks for your consideration and suggestions, and we believe the revised manuscript would be more improved according to your suggestion.Thank you for submitting the revised manuscript and the revisions have strengthened the study.If possible, please consider submitting the original mNGS data to NCBI to allow for broad availability.
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Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred Minor issues: -Line 88 -disease progression?Instead of regression -Scientific names need to be reviewed o Line 125 Penicillum marneffei is now known as Talaromyces marneffei o Line 125 ?Podocystis -do not know of a clinically relevant fungi named Podocystics histolytica -please review and correct o Line 158 Klebsiella penumoniae o Line 197 -what is Clostridium forcestorum spp?? o Line 208 -what is Microsomonas -Line 146 -imaging findings?Instead of performance -Review paragraph line 163 -special stains in histopathology can direct clinician to potential diagnostics but not detect pathogens: pathology would find acid fast bacilli resembling Mycobacterium or fungal elements/hyphae resembling Aspergillus, but cannot name the pathogens as implied on this statement -Line 174 -abbreviations should be defined first time used -ROC -Line 238 -Penicillium spp. is usually a contaminant not a rare pathogen considering our revised manuscript " The joint application of metagenomic next-generation sequencing and histopathological examination for the diagnosis of pulmonary infectious disease" (Manuscript ID: Spectrum00586-23) for publication in
would find acid fast bacilli resembling Mycobacterium or fungal elements/hyphae resembling Aspergillus, but cannot name the pathogens as implied on this statement -Line 174 -abbreviations should be defined first time used -ROC -Line 238 -Penicillium spp. is usually a contaminant not a rare pathogen