PONE-D-20-29217

Xpert® MTB/RIF diagnostic test for pulmonary and extrapulmonary tuberculosis in immunocompetent and immunocompromised patients: benefits and experiences over 2 years in different contexts

PLOS ONE

Dear Dr. TOMAZ,

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.

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The findings of the study has important implications in TB diagnostics and control. But the manuscript needs work and improvements in the clarity, especially distinction in the results between Xpert and and Xpert ultra. I hope the authors consider the reviewers comments useful in improving the manuscript and I look forward for your revised version.

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We look forward to receiving your revised manuscript.

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Padmapriya P Banada, PhD

Academic Editor

PLOS ONE

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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

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

Reviewer #2: 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: Yes

Reviewer #2: Yes

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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: Summary: This prospective observational study aimed to evaluate the performance of Xpert MTB/RIF in different contexts as listed below

(i) laboratory and clinical/epidemiological diagnosis;

(ii) HIV-positive and -negative populations; and

(iii) type of specimens: pulmonary and extrapulmonary.

The Xpert MTB/RIF results were compared with those of Ziehl–Neelsen stained smears and BAAR cultures on solid L.J medium for specimens belonging to clinically suspected TB cases during a two-year period. Total, 924 specimens (pulmonary 514/55.7% and extrapulmonary 410/44.3%) from 743 patients were evaluated. Among these, according to clinical criteria, 101 (13.6%) were classified as confirmed TB cases, 36 (4.8%) probable TB and 606 (81.6%) improbable TB cases. Overall sensitivity, specificity, positive predictive value, and negative predictive value results of Xpert MTB/RIF compared to those of the L.J cultures were 96%, 94%, 57%, and 99%, respectively. The Xpert MTB/RIF test showed an increase in the detection of 13

(35.1%) of 37 confirmed cases of TB in patients with advanced immunosuppression showing negative culture.

Recommendation: Accept with major revisions as described below

Comments:

1. Phenotypic culture confirmatory method used in this study (L.J culture) is conventional and very suboptimal reference standard for detection of extra-pulmonary and paucibacillary TB. In HIV-positive patients, the diagnosis of pulmonary tuberculosis (PTB) is a challenging task due to the paucibacillary nature of the infection, which greatly reduces the effectiveness of smear microscopy and conventional L.J. culture techniques that are used as a phenotypic reference standard to confirm TB in this study. So, I would recommend authors to use composite reference standard (CRS) as a gold standard to evaluate the true diagnostic potential of Xpert MTB/RIF assay and other diagnostic tests for detection extra-pulmonary and paucibacillary TB. Calculation of sensitivity, specificity, PPV and NPV of all laboratory tests against CRS+ and CRS - patients would give more realistic picture.

2. Clarity in writing and flow need to be improved especially in abstract, methods, results and discussion sections.

Reviewer #2: This study by Tomaz A and colleagues compares Xpert MTB/RIF with smear and culture, and against a clinical/laboratory classification for pulmonary TB, extrapulmonary TB, and TB in persons living with HIV over a 2 year period. The study adds to a large body of literature describing experience with Xpert in these populations. The authors conclude that Xpert leads to increased diagnosis of active TB cases compared to phenotypic tests.

Suggest that the comparators and reference standard for each cohort are explicitly outlined in the methods section.

Lines 142, 149-150 mentions outcomes –how long were the participants followed?

Differences in time to diagnosis may be an opportunity to look at operational advantages of Xpert versus culture – was this captured?

Did the local providers or study investigators make the determination of confirmed, probable and improbable TB for each patient? Were these reference determinations made blinded to the study Xpert, culture, and smear results?

Along these lines, were Xpert MTB/RIF results shared with providers? If so, would Xpert have played a role in provider classification of improbable, probable, and confirmed TB?

Consider including a baseline characteristics table to elucidate proportions and overlap of characteristics such as pediatric, extrapulmonary, HIV, demographic, recent TB treatment, smear positivity, confirmed/probable/improbable

The study procedures are not entirely clear. How did obtaining TB case records (line 84) fit into the prospective enrollment? Were these case records used for recruitment or follow-up? Please describe what procedures took place during the study encounter (how many samples were collected, etc).

Samples can be heterogenous and majority of patients had smear, culture, and Xpert performed on separate samples which can lead to increased discordancy between these tests. Please comment how far apart the sample collections took place particularly in cases where Xpert was compared with culture on separate samples. Please confirm no interval treatment took place between those samples.

Line 164-65: Please clarify how Xpert MTB/RIF showed the best accuracy for confirmed TB when final classification of confirmed and improbable TB were based on phenotypic tests culture and/or sputum smear (line 96-97)?

Lines 139-142: Along these same lines, please clarify how could 36 of 42 culture-negative patients be classified as confirmed TB if the SINAN definition was used?

Fig 1. Consider providing the Venn diagram in context of confirmed, probable, and improbable TB.

Fig 2 and Fig 3. Please define which is element in each comparison is the reference standard.

Fig. 2 legend: "TB confirmed (sick) as probable and improbable (not sick)." Unclear what this means.

Almost half of the patients evaluated were treated for TB (line 134) – how recent were these treatment courses? Even treatment over 6 months prior [Dorman et al] (and even years ago along some lines of evidence) are implicated to result in remnant nucleic acids which are picked up by Xpert but not culture leading to discordancy in favor of Xpert (Xpert-positive, culture-negative).

Lines 297-300: Both authors have pointed out…its [Ultra’s] performance is comparable with that of the Xpert MTB/RIF…

On the contrary - Ultra’s sensitivity was found to be superior to that of Xpert in patients with smear-negative TB and HIV, which was the target population of Ultra that Xpert MTB/RIF too frequently missed [Dorman et al, Lancet 2017]. There is generally little difficulty for nucleic acid amplification tests to pick up smear-positive TB.

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Reviewer #1: Yes: Dr Shubhada Vasudeo Shenai

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewers comments_181020.docx

The Xpert® MTB/RIF diagnostic test for pulmonary and extrapulmonary tuberculosis in immunocompetent and immunocompromised patients: benefits and experiences over 2 years in different clinical contexts

PONE-D-20-29217R1

Dear Dr. TOMAZ,

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.

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Kind regards,

Padmapriya P Banada, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your resubmission and addressing the concerns raised by the reviewers. I am happy to recommend this for publication.

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 #3: (No Response)

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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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

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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 #3: Yes

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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 #3: Yes

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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: Many of the reviewer’s comments have been addressed in the revised version, however, few minor corrections are recommended below for further improvement,

1. Table 1: Actual numbers of confirmed, probable, possible, and non-TB patients can be included in brackets (n=…) along with patient’s number for AFB smear, culture, Radiology and other tests data.

2. Line 160 & 162: Among the confirmed TB cases, Xpert detected 32 of the 33 positive samples in culture and AFB smear, 21 of the 23 positive samples in culture, and all positive samples in AFB smear. Kindly reframe this statement to simplify for better understanding.

Recommendation: Among 33 smear and culture positive cases one was not detected by Xpert, and among 23 smear negative culture positive samples 2 were not detected by Xpert. Authors can describe possible causes of these 3-culture positive Xpert negative (false negatives) controversial cases in discussion.

3. Figure 2: Kindly add total no of pulmonary and extra-pulmonary samples in brackets e.g. A) Pulmonary (n= ), and B) Extra Pulmonary (n= ). Further, as described in Fig 2, there were 3 culture positive Xpert negative cases among pulmonary samples and one culture positive Xpert negative case among extra-pulmonary samples. So total 4 culture positive Xpert negative cases. However, in the text (line 160-162) authors have mentioned only three false negative (culture positive Xpert negative) from pulmonary cases instead of total four (pulmonary + extra-pulmonary) cases. Kindly recheck and make corrections wherever required.

4. Confirmed and Probable TB cases data can be separated in table 2, and can be discussed separately in the text.

5. References: All references should be written properly as per PLOSOne guidelines. Please recheck all references and make necessary corrections.

Reviewer #3: This manuscript describes the performance characteristics of Xpert MTB/RIF (Xpert) using a composite reference standard and solid culture (Lowenstein Jensen) in HIV positive and HIV negative individuals. Most of the previous comments have been addressed, or, reasonable justification has been provided by authors to support current presentation of findings. Please find additional comments / recommendations for your consideration.

Specific comments:

Line 65-66: Syntax issue noted, kindly consider rephrasing.

Line 83: Please confirm this is in reference to individuals without a valid (definitive positive or negative) culture result.

Line 94: Reviewer #1 raised questions regarding sample processing for Xpert, however it is still not clear if samples used for Xpert testing were processed sediments or if the samples were split before processing for Xpert and culture (all sample types that were tested by both methods). Please confirm.

Line 234: Typographical error noted, please remove "a"

Line 266: Typographical error noted, please change "tree" to "three"

Line 315: Syntax issue noted, kindly consider rephrasing

Line 334-338: The detection of nonviable mycobacterium could also result in Xpert (+) / culture (-) results, please consider adding.

Line 361-363: Please confirm CRS category for patients described (quoted from manuscript: "five patients who did not have active TB but had previously undergone treatment, and one patient was under treatment at the time of the molecular test")

Line 374-382: The increase in EPTB in HIV negative individuals as compared to HIV positive individuals is an interesting observation, this could be further explored in this section of the discussion.

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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: Yes: Dr Shubhada Shenai

Reviewer #3: No