We have changed the manuscript in accordance with the reviewer s suggestions, and have addressed the reviewer s comments as follows:

Transcription

1 Author s response to reviews Title: Widespread hepatitis B virus genotype G (HBV-G) infection during the early years of the HIV epidemic in the Netherlands among men who have sex with men Authors: Marion Cornelissen (m.i.cornelissen@amc.uva.nl) Fokla Zorgdrager (f.zorgdrager@amc.uva.nl) Sylvia Bruisten (sbruisten@ggd.amsterdam.nl) Margreet Bakker (m.e.bakker@amc.uva.nl) Ben Berkhout (b.berkhout@amc.uva.nl) Antoinette Van Der Kuyl (a.c.vanderkuyl@amc.uva.nl) Version: 1 Date: 18 Apr 2016 Author s response to reviews: Amsterdam, April 18th, 2016 Dear Editor, Hereby we submit our revised manuscript entitled Widespread hepatitis B virus genotype G (HBV-G) infection during the early years of the HIV epidemic in the Netherlands among men who have sex with men, by Marion Cornelissen, Fokla Zorgdrager, Sylvia M. Bruisten, Margreet Bakker, Ben Berkhout, and Antoinette C. van der Kuyl to be considered for publication in BMC Infectious Diseases. We have changed the manuscript in accordance with the reviewer s suggestions, and have addressed the reviewer s comments as follows: Reviewer #1: Comment 1, page 5. The characteristics (sensitivity, specificity, lower limit of detection) of the genotype specific PCR should be described in more detail.

2 A supplementary table 1 with all primer sequences used has been added to the manuscript. A more detailed description of the genotype-specific PCR assays has been added to the Methods sections dealing with PCR-assays. Comment 2. Authors should describe in a figure how they tested exactly the HBsAg(+) samples for the presence of dual- or mono-infection with HBV. The lower limit of detection should be reported for each step. We do not understand how this should be included in a figure as we just tested all samples with the HBV genotype A and HBV genotype G specific real-time PCR assays. So, there were no steps, only a confirmatory PCR was performed on an aliquot of the same sample. If the confirmatory PCR was negative, the sample was counted as negative. This was the case for fourteen samples (6%). Comment 3 (Table 1). The numbers of HIV(+) and HIV(-) do not equal the total number of samples Corrected, N should have been 74 for the HIV-negative samples. Comment 4 page 8, lines How authors estimated the incidence of HBV infection (Fig. 2B)? The incidence is not determined here, just the number of HBV-DNA positive samples per year is counted. Terminology has been changed accordingly. The cited study by van Houdt et al did measure incidence. Comment 5 page 9, lines 2-7. Sample age and storage condition seem to have an effect on the assay. Authors should comment on the effect of these conditions on the validity of their study. Added to page 9:, limiting its predictability. Also added to Discussion on page 13: It is possible that the age of the samples could have influenced our study, as some samples have been stored for almost thirty years. However, we believe that sample age did not influence our results, firstly because most samples that were HBV DNA positive were from 1985, and secondly because the

3 real-time PCR amplifies very short DNA fragments, and in general only amplification of longer fragments is affected by DNA degradation. Reviewer #2: Cornelissen and co-authors have retrospectively analyzed HBV/G infection in 226 MSM with and without HIV infection from the years by using genotype-specific PCR assays. A total of 149/226 patients were HBV/A- and/or HBV/G-DNA positive. Of these, 27% were infected with both genotypes, while 3% with only HBV/G. HBV-DNA viral loads were significantly lower in HIV-positive than in HIV-negative patients. This is an interesting study and the manuscript is well written. Major points 1) In the "HBV-G mono-infection and HBeAg expression" subsection, in Results part (page 8), the authors analyzed the HBeAg status of the five apparently HBV/G mono-infected patients and five other HBV/A and HBV/G co-infected ones. HBeAg is not a good marker to investigate the occurrence of HBV/G mono-infection, since dual infections with HBV/G and other genotype can be also HBeAg negative, as showed by the authors. Thus, what are the conclusions to be drawn from this analysis? I consider that this subsection should be shortened and focus only in the five apparently HBV/G mono-infected patients. Additionally, the authors should inform if they tried to amplify a helper genotype in all five HBV/G mono-infected patients or only in the HBeAg positive patient (ACS ). We have re-aligned Table 2 to show first the results for the mono-infected patients, and then the dual infected patients. We agree that HBeAg negativity is not a good marker for HBV-G monoinfection, but the contrary, HBeAG positivity can only be interpreted as due to the presence of a helper virus in HBV-G infection. So, testing for HBeAg is not conclusive for mono-infection, but it can be helpful when determining if an helper virus could be present. Indeed, all apparent HBV-G mono-infection were tested for helper viruses. All HBeAg-negative patients were tested for a putative helper genotype. We have changed the wording on page 9 to: Repeated attempts to amplify a helper genotype from samples ACS , ACS , ACS , ACS that were HBeAg negative and sample ACS that was HBeAgpositive, failed. Using other samples from patient ACS collected 14 days before and 29

4 days after sample ACS was obtained also did not result in the amplification of another HBV genotype (result not shown). 2) In the "HBV-G and HBV-A serum viral load in HIV-positive and HIV-negative individuals" subsection, in Results part (page 10), the authors analyzed MSM patients from , and a significant reduction on HBV-DNA viral load in HIV-positive patients in comparison to HIVnegative ones was observed. Did the authors perform such analysis encompassing all patients from the whole period? The authors should justify why viral load comparisons were restricted to that period since the other analyses involved the whole period ( ). On page 10 we have already explained why we have restricted our analysis here to : For HIV-negative MSM, only samples from yielded HBV DNA, therefore the HBV- G viral load comparisons were restricted to that period (Table 3).So, because HIV-negative MSM ceased to be HBV-DNA positive over time, and only for we had large enough groups to compare. We have done comparisons for all years, with approximately the same outcomes, but feel that the analysis is not justifiable, as in the later years, many HIV-positive men are progressing towards AIDS, which could affect HBV DNA viral load, while in at the start of the cohort, we are certain that no participant was showing AIDS symptoms, as one of the inclusion criteria was to be disease-free. 3) The authors have sequenced a fragment of the HBV/G genome and found a unique sequence variation G1776A in 15/29 samples. Is G1776A frequently found in HBV/G genomes from other geographical regions? The authors should discuss this point in the Discussion part. Added to the discussion: ; it has not yet been reported in HBV-G sequences obtained from other geographic regions than the Netherlands. on page ) In "Genotype-specific PCR Assays" subsection, in Methods part (page 5), the authors should provide the sequences of the primers used in the three nested-pcr assays, or indicate a previous reference. See comment 1, reviewer 1. An additional Table 1 with all primer sequences used has been added to the manuscript.

5 Minor points 1) In Background, page 4, line 14/15, please include "and Brazil" before [11]. Added 2) All percentages should be standardized (e.g. 20.8% or 21%). Changed were appropriate 3) In Table 1, replace the percentage "62%" with "63%". Done 4) In Table 1, replace the percentage "73%" with "75%". This correction should also be done in the text (page 7, line 53/54). We have corrected the N for the HIV-negative samples, should have been N=74 (see comment reviewer 1) 5) Table 3 should be formatted to present the results more clearly. Reviewer 1 has no problems with Table 3, and we feel changing it will not improve its clarity. And, frankly, We do not know how it could be improved, as it is only a small table. 6) In Figure 1 legend, replace "HBsAg and/or HBcAb positive" with "HBsAg and preferably HBcAb positive" Replaced Yours sincerely, Dr. Antoinette van der Kuyl

6 Laboratory of Experimental Virology Department of Medical Microbiology Center for Infection and Immunity (CINIMA) Academic Medical Center of the University of Amsterdam Meibergdreef AZ Amsterdam The Netherlands tel.: