Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice

Darunee Chotiprasitsakul; Pataraporn Pewloungsawat; Chavachol Setthaudom; Pitak Santanirand; Prapaporn Pornsuriyasak

doi:10.1371/journal.pone.0244023

Peer Review History

Original SubmissionSeptember 1, 2020
28 Oct 2020 Decision Letter - Giuseppe Vittorio De Socio, Editor PONE-D-20-27498 Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice PLOS ONE Dear Dr. Chotiprasitsakul, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by November 30 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This article is about the comparison of PCR and IFA for the diagnosis of Pneumocystis jirovecii pneumonia (PJP). This an interesting article, however some points need to be clarified. Introduction: Line 61 66: Authors should better define the link they are doing between colonization and asymptomatic carriage. Materials and Methods: Line 78: I’m surprised that the authors described only two types of samples: BAL and sputum. Are all “BAL” real “BAL”? How many cycle are performed for the amplification Line 115 118: What was the purpose of defining CT. In this article, were the CT used for the diagnosis of PJP? It is confusing because in the text there is no mention of the CT to define PJP and authors classified the patients as definite PJP and potential PJP. In addition, as there is no standard for the PCR, it is difficult to compare the CT from on study to another. Results: Table 1: Authors should separate HIV and non HIV patients. In additions, when added all the other categories of patients (Hematologic malignancy, solid malignancy…) plus the HIV patients, there are more than 86 patients. Could the authors clarify this classification? This is an important point because immunocompromised HIV patients and immunocompromised non HIV patients are different in terms of diagnosis of PJP. Were all the data described in table 1 available for all the patients? Line 173 175: The authors mentioned cytology and pathology examination. Could they describe the technique in the materials and methods section? Figure 2: I don’t understand the classification “testing did not change the management” For example in the first case, for the 83 patients who were treated before the test results, does the treatment was stopped for those not diagnosed PJP? If so, testing did change the management. In the second case, 6 patients were treated for PJP after the test result but only 4 were diagnosed PJP. Does this mean that the 2 missing patients had colonization? Table 3. I would have changed the title as it is not a comparison. Discussion Line 212: In the discussion, authors should precise that in the ref 16, the tested population was HIV positive. Line 219 220: It look like this sentence is a result, but I didn’t find in the result section where it was mentioned. Could the authors clarified this point? Line 225: PCP? Is it PJP? Line 246: What does the sentence “Interpretation of PCR…. Is challenging” mean? Line 255: The sentence “A higher CT cut off…” is in contradiction with the sentence line 117 “In accordance with…”. Could the authors better explain how they consider if it is a PJP or a colonization according to the CT? Line 281-282: When did the authors differentiate infection from colonization. In the results section, it is only mentioned PJP. In the light of the sentence line 286 – 288, I would have completed the conclusion in the abstract in order to make it more attractive. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. 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Revision 1
14 Nov 2020 Author Response Dear Editor-in-Chief and Reviewers: We would like to thank the editors and reviewers for the very helpful and constructive comments, which we indeed appreciate. We have revised the format of the manuscript, provided more details of IFA and the dates which we accessed the database in the method section. Our ethics committee allows sharing a de-identified data set. We have uploaded this data set on Open Science Forum (https://osf.io/3cgh4). We have addressed all the comments as shown in the revised manuscript with track changes and believe that they have greatly improved the quality of the manuscript. Please find the summary of responses below. Thank you again. Best regards, Darunee Chotiprasitsakul Reviewer #1 evaluation Introduction: Line 61 66: Authors should better define the link they are doing between colonization and asymptomatic carriage. Thank you for this comment. The notion of colonization or asymptomatic carriage of P. jirovecii has been described when P. jirovecii DNA is detected in patients without any symptoms. (N A Maskell, et al. Thorax. 2003 Jul; 58(7): 594–597.) We have revised the word Line 63: “asymptomatic carriage” to “colonization” Materials and Methods: Line 78: I’m surprised that the authors described only two types of samples: BAL and sputum. Are all “BAL” real “BAL”? How many cycle are performed for the amplification Types of respiratory specimen for PJP diagnosis include BAL and sputum sample. (Cruciani M, et al. Eur Respir J. 2002 Oct;20(4):982-9) Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) was performed for diagnostic indications, according to the standard guidelines. The induction of sputum was not performed because of no available negative-pressure room for this procedure at our institution and concern for risk of P. jirovecii airborne transmission. (Valade S, et al. Intensive Care Med. 2015) Sputum was obtained by expectoration, or tracheal suction, which has been shown to provide a comparable quality. (Novaseb V, et al. J Infect Dev Ctries. 2014 Mar 13;8(3):349-57, Choe PG, et al. Med Mycol. 2014 Apr;52(3):326-30) We add more details Line 97-99: “Sputum was obtained by expectoration, or tracheal suction. BAL fluid was collected via bronchoscopy, as previously described [11]. Instillation of normal saline 50 ml, suction with low pressure, and expulsion into glass bottles was repeatedly done 2-4 times.” Based on the LOD determinations (Ct ≥40), the PCR amplification war performed until no longer expected to be detected at 45 cycles. Line 121-122: “A CT value of 45 was the limit of the detection of this assay.” Line 115 118: What was the purpose of defining CT. In this article, were the CT used for the diagnosis of PJP? It is confusing because in the text there is no mention of the CT to define PJP and authors classified the patients as definite PJP and potential PJP. In addition, as there is no standard for the PCR, it is difficult to compare the CT from on study to another. Thank you for this comment. We would like to demonstrate the available reference CT cut-off value. We agree that there is no standard cycle threshold and could cause confusion. We delete Line 122-123 “A CT value > 35 could exclude Pneumocystis colonization and pneumonia with a specificity of 80% and a sensitivity of 80% [11].” Results: Table 1: Authors should separate HIV and non HIV patients. In additions, when added all the other categories of patients (Hematologic malignancy, solid malignancy…) plus the HIV patients, there are more than 86 patients. Could the authors clarify this classification? This is an important point because immunocompromised HIV patients and immunocompromised non HIV patients are different in terms of diagnosis of PJP. Were all the data described in table 1 available for all the patients? Thank you for this suggestion and double check the number of co-morbidities. We have revised table 1, divided the total, HIV and non-HIV patients. We have included our de-identified data on Open Science Forum (https://osf.io/3cgh4). Some patients had more than one active pre-existing conditions as shown in the table below. Number of pre-existing medical conditions PJP patients (N=86) non-PJP patients (N=136) 0 2 patients 5 patients 1 35 patients 58 patients 2 30 patients 41 patients 3 19 patients 21 patients 4 0 patients 11 patients Total 86 patients 136 patients Line 173 175: The authors mentioned cytology and pathology examination. Could they describe the technique in the materials and methods section? Following your suggestions, we have added the description of cytology and pathology Line 127- 139: “Cytology BAL fluid was processed using liquid-based slide systems for ThinPrep (Hologic Inc., Mariborough, MA, USA). The slides were prepared following the manufacturer’s instructions. If granular cast or foamy material or honeycomb appearance was seen in the Papanicolaou-stained smear, then a GMS stain was performed to confirm the presence of cyst forms of Pneumocystis. Pathology Lung biopsy sample was taken via bronchoscopy [11]. A total of 4-6 samples were placed into a glass bottle containing 10% formalin solution, and processed as previously described [12]. All samples were evaluated for the presence of lung tissue, and pathological changes. If clinical, radiological, or histological findings of routine haematoxylin and eosin staining was suspicious for PJP, then GMS stain was done to detect Pneumocystis in tissue sections.” Figure 2: I don’t understand the classification “testing did not change the management” For example in the first case, for the 83 patients who were treated before the test results, does the treatment was stopped for those not diagnosed PJP? If so, testing did change the management. Thank you for this comment. All 83 patients continued and completed PJP treatment after knowing positive test results. All were diagnosed PJP by attending physicians during admission, but 4 patients did not meet the specified criteria of this study (clinical course, imaging, and treatment response) for diagnosis of PJP. In the second case, 6 patients were treated for PJP after the test result but only 4 were diagnosed PJP. Does this mean that the 2 missing patients had colonization? Yes, the 2 missing patients had colonization. We have revised and add more details on Figure 2. Table 3. I would have changed the title as it is not a comparison. Thank you for this suggestion. We have revised the title of Table 3 “Sensitivity and specificity of IFA and PCR for diagnosis of PJP.” Discussion Line 212: In the discussion, authors should precise that in the ref 16, the tested population was HIV positive. We have revised accordingly. Line 242: “In a previous study of 305 induced sputum specimens among HIV-positive patients” Line 219 220: It look like this sentence is a result, but I didn’t find in the result section where it was mentioned. Could the authors clarified this point? Thank you for this comment. We have revised the result and discussion section. Result Line 218-219 “Seventy-one of 91 discordant PCR-positive and IFA-negative samples (78.0%) in our study were collected following PJP treatment (Table 4).” Discussion Line 252-253 “The proportion of patients who received treatment prior to sample collection was highest among the discordant PCR-positive IFA-negative group (78.0%).” Line 225: PCP? Is it PJP? We have corrected the word Line 258: “PJP” Line 246: What does the sentence “Interpretation of PCR…. Is challenging” mean? Thank you for this suggestion. We changed the word “challenging” to “difficult” Line 280. Line 255: The sentence “A higher CT cut off…” is in contradiction with the sentence line 117 “In accordance with…”. Could the authors better explain how they consider if it is a PJP or a colonization according to the CT? Thank you for this comment. The higher CT allows the amplification of DNA at more cycles than lower CT. Therefore, higher CT cut-off increases the chance of detecting pneumocystis. As the cut-off increases, sensitivity, as well as the false-positives would increase, while false-negatives would decrease. Positive detection by CT could be 1. PJP, 2. colonization, 3. Artifacts/cross contaminations. We have add more explanation Line 290-295: “The higher CT cut-off value would increase the chance of detecting pneumocystis, as well as false-positives from artifact or cross contamination. Although a strongly positive PCR (CT <31.5) was highly suggestive of PJP, a weakly positive PCR (CT 31.5-45) could be colonization or PJP undergoing treatment.” Line 281-282: When did the authors differentiate infection from colonization. In the results section, it is only mentioned PJP. We have revised Line 319-320: “to ascertain true pneumocystis infection.” In the light of the sentence line 286 – 288, I would have completed the conclusion in the abstract in order to make it more attractive. Thank you for this suggestion. We have revised the abstract Line 38-40: “Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.” Attachments Attachment Submitted filename: Response to reviewer.docx https://doi.org/10.1371/journal.pone.0244023.r002
2 Dec 2020 Decision Letter - Giuseppe Vittorio De Socio, Editor Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice PONE-D-20-27498R1 Dear Dr. Chotiprasitsakul, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Giuseppe Vittorio De Socio, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0244023.r003
Formally Accepted
10 Dec 2020 Acceptance Letter - Giuseppe Vittorio De Socio, Editor PONE-D-20-27498R1 Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice Dear Dr. Chotiprasitsakul: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Giuseppe Vittorio De Socio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244023.r004

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