PONE-D-21-10435

Combination of Xpert®MTB/RIF and DetermineTMTB LAM assayimproves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, Ethiopia

PLOS ONE

Dear Dr. Tadesse,

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 Aug 02 2021 11:59PM. 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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We look forward to receiving your revised manuscript.

Kind regards,

Shampa Anupurba, MD

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The manuscript has been reviewed thoroughly. I have very minor additional comments although they might be repetition of the reviewer's comments:

1. Line 188- 'Forty-nine (33.3%) of the presumptive EPTB cases had the classification of colitis'-On what basis was this classification made and what was the sample collected from these patients?

2. Although 4 of the 23 HIV positive patients were bacteriologically confirmed, it is not clear how many were positive by Xpert MTB/RIF. What is meant by 50% in Line 211? Similarly for Determine TB LAM assay

3. Line 320- How was the sensitivity of LJ culture derived to be 62.2%? What was the gold standard?I think that was not within the scope of the study

4. Fig 1- The flow chart could start with inclusion of 147 patients suspected of presumptive TB instead of 160 patients

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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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: In the current study the authors aim to investigate the diagnostic performance of combined use of Xpert MTB/RIF and TB LAM Ag test for detection of M. tuberculosis complex among EPTB patients in a medical center of Ethiopia. Considering the high prevalence of EPTB and association of HIV in Ethiopia, the current diagnostic approach provides the important insight for increased detection of EPTB cases in clinical settings. However, the current manuscript needs to address several issues before approval.

Major comments:

1. According to the manufacturer (Abbot Laboratories, Chicago, US), the test name is ‘Determine™ TB-LAM Ag’. It is suggested to use the exact test name in the Title of the manuscript. Throughout the text, authors should be consistent mentioning the test name. Examples of some discrepancies are: ‘Determine TB LAM test’ in Line-31, ‘TB LAM’ in Line 41, ‘Determine TB-LAM test’ in line 136, ‘TB-LAM assay’ in line 140 and so on. It is suggested to use ‘Determine™ TB-LAM Ag (TB-LAM)’ when appears first in the text, followed by ‘TB-LAM’ all through the text.

2. In the introduction section, please add one paragraph describing the other published data on the performances of the Xpert MTB/RIF assay and Determine TB-LAM assay for detection of EPTB cases.

3. One of the EPTB samples as has been described inconsistently throughout the text. For example, in Table-1 as ‘pus (abscess)’, Table-2 and throughout the text as ‘Abscess’. According to Line-191, it appears that samples were obtained from ‘Lymph node TB’. It would be easier for the readers to understand if the term ‘Lymph node aspirate’ is used all through the manuscript instead of ‘Abscess’.

4. Gastric aspirate is considered as an alternative specimen for detection of pulmonary TB when patients cannot expectorate the sputum. As this study aims to detect the EPTB cases, authors should exclude the two ‘gastric aspirate’ samples from the study and re-analyze the results and correct the statistics accordingly in different relevant Tables, Figures and throughout the text.

5. For Table-2 and Table-3: Authors should check carefully that the statistics are presented correctly. It seems that the value of sensitivity, specificity, PPV and NPV and their corresponding 95% CI are not accurate. As for example, in Table-2 (based on the calculation of Fig-1), for ‘Determine TB LAM test’: the sensitivity would be 34.8% with 95% CI ranging from 16.4-57.3, specificity would be 92.7% (86.7-96.6), PPV would be 47.1% (27.7-67.4), and NPV would be 88.5% (85.0-91.2). Please check both table 2 and 3, and correct accordingly. Please also add the 95% CI for specificity of Xpert MTB/RIF test in both tables. Should use the value of 95% CI in a consistent format like 0.0% (0.0-0.0) instead of 0.0% (0.0%-0.0%) in the tables and text.

6. For clarity and better understanding authors should include the number of samples detected or not detected by the index tests compared to the reference tests along with the value of sensitivity and specificity. As for example, in Table-2, for ‘Determine TB LAM test’ please write the Sensitivity: 34.8% (34.1-35.5); 8/23 instead of 34.8% (34.1-35.5), and for specificity: 92.7% (86.7-96.6); 115/124 instead of 92.7% (86.7-96.6). Please add the numbers in Table-2 and Table-3 and describe accordingly in the text.

7. Figure 2: please describe in the result section (by mentioning the numbers of samples) how the percentages of different tests were obtained between HIV positive and negative patients.

8. Line 215-217: It is not clear how the sensitivities of 44.4% for Xpert and 30.6% for TB LAM were obtained. Please clarify. Please also mention the statistical methods in ‘Methods and Material’ section that was used to obtain the p value.

9. Recently, Xpert MTB/RIF Ultra and Fujifilm SILVAMP TB-LAM have been appeared with higher sensitivities for detection of tuberculosis. In the discussion section authors should add one paragraph about the future applicability of these two assays compared with the current findings of Xpert MTB/RIF assay and Determine TB LAM assay.

10. Line 344: Please clarify the statement ‘equivalent to culture to diagnose EPTB’. It is not clear how the sensitivity of combined use of Xpert MTB/RIF and TB LAM was equivalent to ‘culture’ as the authors did not mention anything about the sensitivity of ‘culture’ in the manuscript.

11. While the text is readable, there are some grammar mistakes. Please correct the text to improve the comprehensibility.

Minor comments:

Line 37-The authors should mention the name of ‘reference’ test that is the ‘CRS’ to whom the results of Xpert MTB/RIF were compared.

Line 42: Please write ‘EPTB cases’ instead of ‘TB cases’.

Line 41-42: Please correct and re-phrase the sentence ‘The combination of Xpert MTB/RIF and TB LAM detected 61.1% of all EPTB participants and 83.3% of HIV co-infected TB cases’ by mentioning the sensitivity instead of detection.

Line 98: Should write Lowenstein Jensen (L-J) as it appears first in the text.

Line 135: Please mention the volume of urine used for centrifugation.

Authors should specify why the LAM test was performed on refrigerated urine samples instead of freshly collected samples. Should discuss the point in the discussion section whether the sensitivity of LAM test varies between refrigerated versus fresh urine sample.

Line172: Please write as 35 years (IQR, 22-45).

Line-187: ‘Forty-nine (33.3%) of the presumptive EPTB cases had the classification of colitis’. Please clarify whether all of the 49 cases from where peritoneal fluid were collected had ‘colitis’.

Line-195 and 202: Authors should mention ‘detection rate’ instead of ‘prevalence’.

Authors have mentioned in Table-1 that one sample among ‘Others’ group (2 gastric aspirates and 2 synovial fluids) was diagnosed as ‘Confirmed TB’. Please, specifically mention the sample type (either the gastric aspirate or synovial fluid) that was positive for TB in the table and add the description in line-196.

Line 228-229: please write as (0.0%; 95% CI, 0.0-0.0) instead of ‘(30.6% (95%CI, 30%-

229 31%))’ and ‘(93.3% (95%CI, 93.2%-93.5%))’.

Line 230 and 238: Authors should include the ‘p value’ of the significant difference, and the test used.

Line 235-236: Please rephrase the sentence ‘Adding Determine TB LAM test to Xpert MTB/RIF test increases the sensitivity of Xpert MTB/RIF test from 43.2% to 61.1%’ as it does not increase the sensitivity of Xpert MTB/RIF test, but increases the sensitivity of combined use TB LAM and Xpert assay.

Line 307-308: It is not clear how the values of 2 (33%) and 1 (17%) were obtained. Please add in the result section to clearly state how these values were obtained.

Line 320: It is not clear how the sensitivity of L-J culture (62.2%) was obtained. Please specify.

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Reviewer #1: No

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PONE-D-21-10435R1Combination of Xpert® MTB/RIF and DetermineTM TB-LAM Ag improves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, EthiopiaPLOS ONE

Dear Dr. Tadesse,

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 Jan 15 2022 11:59PM. 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Shampa Anupurba, MD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. 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: It is appreciated that the authors have responded well the comments. However, it would be easier for the reviewer to go through the text if the specific line numbers are mentioned against the comments (where appropriate) in the cleaned version.

From Table 2 it is evident that TB-LAM test was compared against 36 and Xpert MTB/RIF test against 37 positive samples found by CRS. Would be easier for the reader if it is explained in short in the result section how these CRS have been found.

Line 227: the author should write ‘sensitivity’ instead of ‘pooled sensitivity’.

Line 179-180: ‘MedCalc Software Ltd (Comparison of proportions calculator) was used……….’ this sentence can be written as ‘Comparison of proportion between the methods was done by Chi square test using the ‘MedCalc Software’ (please include the weblink).

Reviewer #2: 1. The referencing style need to be uniform throughout the manuscript in accordance with Journal’s requirement. For example, please note the discrepancy in citing reference no. 1, 7, 13, 20, 24.

2. In ABSTRACT

Line 29 of the revised manuscript: Replace “examined for tuberculosis (TB)” with “tested for Mycobaterium tuberculosis complex (MTBC)”.

Line 40 of the revised manuscript: Write “and 100% specificity” instead of “with 100% specificity”.

3. In BACKGROUND

Line 60 of the revised manuscript: Replace “category III biosafety level” with “bio-safety level III facilties”.

4. In MATERIALS AND METHODS

Line 106-107 of the revised manuscript: There is no mention about collection of ‘gastric aspirate’ and ‘synovial fluid’.

Line 121-122 of the revised manuscript: Was decontamination also carried out for gastric aspirate specimens?

Line 179-180 of the revised manuscript: Earlier minor comment no. 10 by the reviewer is not addressed adequately. The tool used in determining the statistical difference haS been mentioned but the statistical test used for the same has not been mentioned in the revised version of the manuscript.

5. In RESULTS

Line 206-107 of the revised manuscript: With respect to addition editor’s question no. 1 and the reply by the authors, will it be better if the authors mention the ‘colitis’ cases as ‘suspected abdominal TB’ cases because abdominal pain and discomfort could be due to causes other than colitis?

Line 215-216 of the revised manuscript: “1 disseminated TB from gastric aspirate”- It remains dubious how detecting MTBC from gastric aspirate only can label the case as ‘disseminated TB’. Clarification is needed.

Line 242-243 of the revised manuscript: It is not clear whether culture confirmation (23 patients) could only be done only among 126 presumptive EPTB cases who submitted urine samples for TB-LAM. In other words, was there no culture confirmed cases among the other 21 presumptive EPTB cases who did not submit urine sample for TB-LAM?

This needs to be clarified.

6. In DISCUSSION

Line 316 of the revised manuscript: Replace ‘Additionally’ with ‘However’.

Line 355 of the revised manuscript: Write ‘End TB strategy’ instead of ‘end TB strategy’.

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7. 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.

Reviewer #1: No

Reviewer #2: Yes: Arghya Das, MD

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Combination of Xpert® MTB/RIF and DetermineTM TB-LAM Ag improves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, Ethiopia

PONE-D-21-10435R2

Dear Dr. Tadesse,

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,

Shampa Anupurba, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. 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: (No Response)

Reviewer #2: (No Response)

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7. 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.

Reviewer #1: No

Reviewer #2: Yes: Arghya Das, MD