PONE-D-19-27597

Pre-treatment Drug Resistance and HIV-1 Genetic Diversity in the Rural and Urban Settings of Northwest-Cameroon

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: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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?

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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: The manuscript provides information about the prevalence of HIV drug resistance in a small population of Cameroonian drug-naïve patients enrolled just in 4 months. In addition, the authors tried to find potential correlations between drug resistance and viral load or CD4 cell count, but due to the low number of enrolled individuals, no evidence for that was found. Thus, valuating the clinical impact of drug resistance in this kind of population remains challenging. In order to have a real overview of drug resistance in urban and rural area, and to really evaluate its clinical implication, more patients should be tested in a longer time period. This is the most important limitation for this study, that should be accurately stressed along all the manuscript.

Below are major comments:

1) The authors asserted to have considered for their study pre-treatment HIV drug resistance (PDR) mutations, defined like transmitted or acquired drug resistance. According to what the authors wrote, these mutations may have been transmitted at the time of infection (TDR), or it may be acquired by prior ARV drug exposure. However, which mutations the authors used for their analysis remain unclear. A list with all the NRTI, NNRTI, and PI resistance mutations used for this study should be added in the supplementary material. For example, were the revertant mutations at RT position 215 considered in this list? If not, these mutations should be added for their role in drug resistance. These revertants might easily develop in T215Y/F and their presence by standard sequencing may indicate the presence of T215Y/F as a minority variant.

2) Results, “Prevalence of HIV-1 Drug Resistance” paragraph. The authors stated that the PDR prevalence in rural and urban setting is 12.9% and 6.7%, respectively. It is not clear if this prevalence reflects the number of patients carrying at least one drug resistance. Please, clarify. The authors should also define clearly the number of patients carrying at least one NRTI and NNRTI mutation.

3) In order to better evaluate drug resistance mutation patterns, the authors should add a table that summarizes for each one of the 6 patients carrying drug resistance, viral load, CD4, drug resistance mutations, subtype, and location of ART facility where the diagnosis was done (urban or rural).

4) Discussion section. The authors stated that their study “showed a majority of mutations to NNRTIs (52.94%) than NRTIs (47.06%)”. The prevalence of NRTI and NNRTI resistance should be reported on the overall population, and not, as done, on the 6 patients infected by drug resistant virus.

5) Discussion section. The authors should compare their results with the most recent literature published on this field in Africa. In a recent paper based on NGS, Hassan et al defined the prevalence of drug resistance mutations in HIV-1 infected drug naïve patients from rural area of Kenya (Hassan et al., PloS One 2019). The authors reported a 24.0% of participants with at least one drug resistance, 12% against PI, 8.0% against NRTI and 6% against NNRTI. Despite the use of NGS, the prevalence of NRTI and NNRTI resistance in this paper is quite similar to those reported by Fokam et al. Differently, the prevalence of PI resistance that is zero in Fokam et al. is similar to that reported by another Cameroonian paper using NGS technology (Koizumi, JAC 2006).

Minor revision:

1) Abstract and Results: Please, report drug resistance mutation without quasispecies (i.e. no M41ML but M41L).

2) Results, Subtypes of HIV-1 Protease-Reverse Transcriptase sequences paragraph. Instead of A1/F2 subtype, define the exact recombinant form of this sequence.

3) Results, Subtypes of HIV-1 Protease-Reverse Transcriptase sequences paragraph. Please, revise the sentence “the rural setting showed more two more genetic variant [8 subtypes; A1 (3), G (1), F2 (1), CRF02_AG (21), CRF06_cpx (1), CRF11_cpx (1), CRF18_cpx (1) and A1/F2 (1)], compared to the Urban setting [5 subtypes A1 (3), G (3), F2 (1), CRF02_AG (23) and CRF18_cpx (1)].” What does “more two more genetic variant” mean? Add the prevalence of subtypes in rural and urban settings, and p-value.

4) Figure 1, resolution should be improved

5) Please, revise reference 10. The HIV drug resistance report 2017 is available at the following link: https://www.who.int/hiv/pub/drugresistance/hivdr-report-2017/en/

6) Authors used PDR or DRM interchangeably. Please, use an uniform terminology along all the manuscript.

Reviewer #2: This work quantifies the prevalence of pre-treatment drug resistance (PDR) in both rural and urban settings in Cameroon. A total of 61 sequences from 61 patients were analysed for NNRTIs and NRTIs resistance mutations. Fifteen resistance mutations were found in 4 patients from the urban setting compared with only two mutations (NNRTIs only) in two patients from the rural setting. Comparison between rural versus urban setting or level of CD4 are limited by the small sample size. The manuscript needs careful reading to correct few typos or English wording. I have only few minor comments

1. There are only few mutations observed in few patients so the complete list of mutations for the four patients from the urban setting should be given.

2. It is somewhat over-interpreted to consider that the difference in CD4 between urban and rural of 184 vs. 161 indicate a delay in diagnosis. Both IQR are very large likely as the range.

3. Comparing the level of CD4 and the prevalence of mutations shows no statistical difference likely due to the small sample size. I’m surprised that the level of HIV-1 RNA was not available in the study to compare the presence of mutations according to the level of viral load at genotyping time. I guess that the CD4 count is a proxy of the level of viral load based on the correlation between HIV-1 RNA and CD4 cell count. This point should be discussed.

4. In the discussion Section, I’m not convinced that discrepencies between studies can be explained by the different algorithms used. I understood that the percent gives for the studies are % of resistance mutations not resistance to drugs. The difference in the list of mutations used in the different algorithms is marginal.

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Reviewer #1: No

Reviewer #2: No

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Pre-treatment Drug Resistance and HIV-1 Genetic Diversity in the Rural and Urban Settings of Northwest-Cameroon

PONE-D-19-27597R1

Dear Dr. Fokam,

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.

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Kind regards,

Francesca Ceccherini-Silberstein, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

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: All the comments have been apropriately adressed.

The modifications of the manuscript are apropriate.

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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: No