PONE-D-19-29100

Comparison of Alere q whole blood viral load with DBS and plasma viral load in the classification of HIV virological failure

PLOS ONE

Dear Dr Khan,

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.

Two reviewers have raised serious issues with the manuscript as written, and each of their concerns must be addressed. Careful attention should be paid to use of "specificity" and "sensitivity" in the correct context, and provide sufficient detail of how the samples were created and how they were tested.

We would appreciate receiving your revised manuscript by Feb 01 2020 11:59PM. When you are 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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PLOS ONE

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Reviewers' comments:

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

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Reviewer #1: Partly

Reviewer #2: No

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

Reviewer #1: No

Reviewer #2: No

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

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Khan and colleagues presents a laboratory evaluation of the new Alere q point of care (POC) machine to measure HIV viral load from whole blood as compared to plasma which is the gold standard; interestingly, they also compare its performances to those of DBS, again as compared to plasma. This kind of evaluation is essential to help scaling-up of HIV viral load monitoring in resource-limited settings which still is scarce in many settings. In that respect, I am favorable to accept this manuscript if the modifications are conducted.

1) In the method section, the authors mention that the samples originate from routine care. The plasma is intended for HIV viral load monitoring, while the whole blood sample was intended for CD4 viral load measurement. Due to the sampling strategy, HIV viral load on plasma needed to be performed first before DBS samples were prepared and before a sample dedicated to Alere q was saved. Some details about the delay between whole blood sampling and DBS preparation, about the condition of storage of the whole blood, and about the condition and duration of storage of DBS would be welcome. Could the authors also mention if the CD4 measurement was performed or not.

2) In table 1, the reportable HIV viral load range of each technique is indicated. It is surprising to see that the range is 2491 copies/mL to 10 million copies/mL for the Alere q POC, and 1000 copies/mL to 10 million copies/mL for the Abbott DBS technique. This is not correct for the Abbott DBS technique. Moreover, this does not make sense given the evaluation they conduct to identify virological failure at the threshold of 1000 copies/mL. It is also surprising to read that each spot is 75µL as the manufacturers recommend 70µL/spot; 70µL is also mentioned in the reference 14 chosen by the authors in table 1, I was unable to identify reference 13. Tang et al. in Journal of clinical virology 2017 is probably a better reference.

3) In the “analysis” subsection of the “methods” section, it should be clearly stated that correlations and Bland-Altman analysis aim at comparing the methodologies from a quantitative perspective. The ROC curve analysis is mentioned in this section but never presented in the result section and briefly mentioned in the discussion; this is however not of prime interest in this study. If the authors insisted to present the results from the ROC curve, it should come after mentioning that the main goal of the evaluation is a qualitative evaluation, and estimates the ability of the different techniques to identify patients in virological failure defined at the threshold of 1000 copies/mL, when this threshold is used for both the technique evaluated and the plasma. This evaluation relies on the estimation of sensitivity and specificity of each technique as compared to plasma. These terms never appear in the method section while they are much more important than the “proportions of correctly classified”; indeed, the WHO guidelines state that DBS techniques should present a sensitivity and specificity >85% in order to be an acceptable option for HIV viral load monitoring, it is clearly not a criteria based simply on the proportion of correctly classified. The “analysis” subsection must be written in a more intelligible way, and the “results” section should clearly state the sensitivities and specificities as this is not done.

4) In the “results” section, nearly all the data from the table 2 are already in the text. Consider removing the table. Figure 1 provides no informative data; consider removing. The section 3.2 alternates between quantitative comparisons and qualitative comparisons. This should be rewritten for better clarity.

5) In the “results” section, the authors mention indifferently specificity or upward misclassification on the one hand, and sensitivity or downward misclassification on the other hand; this makes the reading very complex and confusing. The standard statistical method to compare a test to a gold standard relies on estimation of sensitivity and specificity. The authors should stick to these terms for the sake of comparison. In table 3, it would be very valuable to present not only % but numbers as well. Two columns could also be added to present sensitivity and specificity with their confidence intervals. This section suffers from repetition.

6) The Bland-Altman is supposed to be a representation of the mean of two techniques on the x-axis against the difference between the two techniques on the y-axis. The caption on the x-axis seem to indicate that the Bland-Altman analysis is not correctly conducted. A legend for the x-axis should be reported. Moreover, this analysis requires to represent three horizontal lines: the mean difference and the 95% confidence interval of the difference. None of these appear in the figures. This must be addressed as it is difficult with the current figures to judge the quality of the techniques.

7) In the discussion, the authors mention the DBS protocols had the tendency to lower viral load quantification. I disagree with this statement. It might be very true for the Roche protocol with a sensitivity to detect failure of 76%; this is also obvious on the scatter plot (especially if the authors add the line with equation y=x). This is less true with the Abbott protocol with sensitivity and specificity of 95% and 92%, respectively. The under-estimation of the viral load level with the Roche technique has already been described in the literature, and this should be reported here. The low sensitivity of the Roche DBS technique is particularly worrying as about 25% of those in failure would not be identified as such. The authors state that Alere q has a very good sensitivity, which is indeed 100%. However, the specificity was particularly bad (61%) which should be discussed. It looks like they attempt to discuss it by mentioning that in case of “false virological failure”, the patients would undergo confirmatory plasma VL testing at a laboratory of further POC VL monitoring. But this is not totally clear. The discussion needs clarification and should be put in perspectives with other similar studies as well as with the WHO recommendations.

Minor comments:

Throughout the manuscript, ul is written instead of µL.

IQR should be written in full at the first appearance

ART should be written in full at the first appearance, and not at its second appearance

Mention of “Alere q” is not homogeneous throughout the manuscript

Reviewer #2: The present study aims to address a cotemporary, globally important issue for HIV treatment and care; however, the manuscript in its current format needs significant improvement in order to reach publication standard. Following are some comments hopefully can help

Introduction

- The argument that “quality of evidence is low due to insufficient published data….” (Lines 45-47 page 3) is not well supported by existing evidence: According to a recent WHO report there were 40 technical evaluations of DBS across 25 countries examining 6 different commercially available VL technologies resulted in more than 10,000 paired DBS-plasma points. It suggested “sufficient evidence has been generated on the performance of DBS specimens for viral load testing to support the initiation of scale-up” (HIV molecular diagnostics toolkit to improve access to viral load testing and infant diagnostics, July 2019). The rationale for the present study need to be revised and strengthen

- Detailed descriptions of the POC VL test under investigation are needed. The Alere q POC viral load had been on the market (?) for a while (previous study published in 2016 – reference 19) why there is still “prototype” whole blood protocol? More details on operational requirements of the technologies are also needed

- Clear statement of the study objectives: primary and secondary objectives are recommended: which was the index test, which was the reference, and comparators

Methods

- Lines 60-61: … Lower than detection limit (LDL) of which technology? There were 3 different tech/protocols mentioned. Please explain why these specific technologies/protocol were selected? Was Sample size calculation performed?

- As this is a diagnostic accuracy study (as it appears in the introduction of the manuscript) , I’m not quite sure why was it needed/authors wanted) to collect data to “determine potential predictors of VL method discrepancies” ??? How did it come even before knowing there were “discrepancies” across measures in this study? As paired samples were used/tested

- VL threshold of 1000 copies/ml was used so detailed analysis for diagnostic accuracy of this cut-off needed to be described (sensitivity, specificity…). Same as for Bland-Altman analysis for quantitative measures

Results

- Lines 98-100 page 5 CD4 count, ART regimen/history not directly relevant/necessary for the main purpose of this study

- Lines 105-118 page 6: presentation of the results is not clear and somewhat repetitive. Suggest to separate out caparisons for primary and secondary objectives Alere q vs. plasma and Alere q vs DBS

- Lines 121-133 page 7 repeated results (with different expressions). Lines 146-154 page 8 not necessary/relevant

Discussion

- Lines 159-161 “the tendency of Alere q… most likely due to cell associated HIV RNA…” this is also true for DBS (which is also dried whole blood sample). Should/can the Alere q run on fresh plasma sample processing at site using mini centrifuge?

- Discussions of clinical factors influencing the variability in VL measures across technologies are not necessary. Potential reasons for reduced accuracy of Roche DBS/FVE has been extensively discussed in literature

- The recommendation of increasing correction factor or using more spots are too general and prompt the question of why this challenge has not been addressed even though is has been identified previously (low specificity)?

- The “excellent sensitivity” note (of Alere q whole blood) is somewhat misleading without an equal highlighted note/discussion of “low specificity” of <50%, which is consistent with previous research and suggested that the technology has not been improved over last few years??? This has an important implication for field implementation as it indicates very high/unacceptable over estimation of HIV VL leading to unnecessary retesting/confirmatory testing of treatment failure and switching regimen

- Lines 199-200 please check the references

Conclusion

- Not clear on key messages. Again highlighting the “excellent sensitivity” without noting low specificity is misleading and based on results of this study the use of “prototype” whole blood protocol Alere q for HIV VL testing should not be “warranted” without further technology improvement and validation

Figures 3a/b BA plots should be revised clearly shown 95%CI of the mean deferences & LoAs

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Reviewer #1: No

Reviewer #2: Yes: Minh D Pham

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PONE-D-19-29100R1

Comparison of Alere q whole blood viral load with DBS and plasma viral load in the classification of HIV virological failure

PLOS ONE

Dear Dr Khan,

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.

Remaining issues to address include those of Reviewer 1 below. In particular, the study needs to include data from after 2015. While it is true that the data presented are from the earlier time, more studies have been published and the pooled results must be addressed to make this manuscript relevant to 2020. 

The authors must also address the following issues. The samples included in the testing were all from HIV-infected individuals, although some were not detectable by the Abbott gold standard assay. The Abbott assay has both <40cp/mL detected and <40cp/mL not detected as result options, so it must be addressed if these two categories were combined as not detected or if they were split, putting the detected ones with the 40-1000cp/mL group. Overall, there must be a better description of how the data were compared. There is a description of the VF level, but it is unclear how that was applied to the data from samples with <1000cp/mL as the plasma VL. The authors must be very specific: how were the data from each of the assays treated to decide if they had the correct classification? In particular, Table 2 is confusing for this reason: for the <40 sample category, was it that 22% were correctly not detected by Alere (and the others were incorrectly detected) or that 22% were correctly detected by Alere (and the other were incorrectly not detected)? For the other groups, what was considered "correct classification", detected at any number or detected at a number in the same range (such as 1000-10,000)? Or were they correctly classified if they were above 1000cp/mL as the VF threshold? There are no figure legends to help describe the figures, and there are no differences in markers between the groups to see how the data in the table fit with the graphs. The authors must add more transparency to their results and calculations to make their data useful to the wider research community.

We would appreciate receiving your revised manuscript by Apr 10 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Julie AE Nelson, PhD

Academic Editor

PLOS ONE

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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)

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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

**********

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

Reviewer #1: 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

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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

**********

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: The authors have made great efforts to answer to the comments, and the article has improved considerably. I still have some minor comments however before the manuscript is deemed suitable for publication.

1. In the method section, the very first sentence is in contradiction with the following. Indeed, the authors state that the goals are to compare HIV viral load results obtained with Alere q POC with plasma HIV viral load results on the one hand, and with DBS viral load results on the other hand. However, two sentences later thy mention that secondary objectives are to compare DBS viral load results to plasma viral load results.

I guess the first sentence can be removed.

2. Line 73, when the authors say “the remaining paired CD4 sample was used for whole blood Alere q test…” I think that the terminlogy “CD4 sample was used” is not correct. Wouldn’t it be more accurate to say something like “the remainis of the blood sample for CD4 measurement”?

3. In current reference 14 (as cited in Table 1) , the limit of detection of the Abbott technique was found to be 839 copies/mL. This is also the threshold mentioned in the WHO pre-qualification document (1) This is also the technique specified in Taieb et al (PLOS one 2018) following the manufacturer’s information.

4. In the statistical method section, reference to ROC curve appears but no such result is presented. Consider removing the sentence referring to ROC curves.

5. In the results section, lines 140-144 are strangely placed. Consider moving them before the paragraph currently starting line 132.

6. In the discussion, I do not understand why the authors specify that they stopped their literature search in June 2015. This does not make sense in 2020. Moreover, some articles evaluating the DBS techniques of interest have been published since. These articles should also be regarded especially since some articles fou,d similar results with the Roche technique (e.g. Taieb et al. OFID 2016).

7. I still have issues with the Bland-Altman analysis. The figure should include the 95% confidence limits of the differences. I doubt the two horizontal grey lines correspond to these limits. The figures must be amended.

(1) https://www.who.int/diagnostics_laboratory/evaluations/pq-list/hiv-vrl/170830_amended_final_pr_0145_027_00.pdf

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Reviewer #1: No

[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 to be viewed.]

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Comparison of Alere q whole blood viral load with DBS and plasma viral load in the classification of HIV virological failure

PONE-D-19-29100R2

Dear Dr. Khan,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Julie AE Nelson, PhD

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

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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

**********

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

Reviewer #1: 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

**********

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

**********

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)

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Reviewer #1: No