Response to Reviewer
Reviewer:jen layden
Reviewer's report:
Major Compulsory Revisions:
1.) While the aim is stated in the background, the background section does not
provide cohesive support of why this is an important aim.
In developing countries, patients monitoring including viral load measurement is rarely
accessible. If only one measurement is available, it may not reflect the real viral activity.
Possible fluctuations may affect the indication of treatment. We have now addressed this
point in the introduction section page 3 line 65 to 69.
2.) Laboratory assays are expected to have normal fluctuations, especially for
quantitative values such as HBsAg and HBV DNA levels. Since the objective was
to examine the kinetics of these parameters, it is important that these are
accounted for, or at least addressed. What is the expected fluctuation level of
HBV DNA levels with the used assay?
We perfectly agree that it is important to determine the role of the method variability on
results as done previously and now reported in our paper.
Reliability of Cobas
AmpliPrep/Cobas Taqman HBV test, v1.0 assay (Roche Diagnostics, Meylan, France) was
assessed by comparing HBV DNA levels measured at two different locations using the
same sample. The first analysis was performed in our laboratory in Saint-Louis Hospital,
while the second was conducted by an independent laboratory (Biomnis, Lyon, France). Of
the 26 subjects concerned, 24 had a viral load measurement in the sample 1 and 2. The
results were initially expressed in the same unit (log UI / mL) to compare the variability of
themethod.
The concordance between the two tests was represented graphically by the method of
Bland & Altman and the degree of concordance was estimated by the intra-class
correlation coefficient and the relative error.
Analysis of viral loads performed in our laboratory and that done by Biomnis laboratory
were significantly correlated (r = 0.9969, p <0.001). The mean viral load in our laboratory
is 4.83 Log UI / ml and 4.86 in Biomnis. The bias between the two analyzes (mean
difference) is 0.027 log / IU [95% CI: -0.056; 0.11]. The difference between the two analyzes
is located between -0.383 to 0.437 log / UI. The comparison between the two analyzes by
the Bland-Altman diagram shows good agreement. The diagram shows that the amplitude
of the deviation is not related to the level of viral load. These results were confirmed by an
intra-class coefficient of 0.998 and a relative error of 2.4%.
1
3.) I am not certain I appreciate the importance of the HBVsAg/HBV DNA ratio.
There is no reference for this. Why is this ratio looked at?
The HBsAg/HBV DNA ratio represents the HBsAg synthesis and the relationship between
HBs production and HBV DNA replication.
Recent reports have demonstrated that serum HBsAg levels vary among different stages in
the natural history of Chronic Hepatitis B. Moreover, HBsAg production does not change in
parallel with HBV DNA across the natural history of CHB. Serum HBsAg/HBV DNA ratio is
higher in the low-replicative phase compared to immune-tolerant, immune-clearance and
HBeAg negative hepatitis phase. Dissociation between HBV DNA and HBsAg levels may be
caused by HBsAg production from integrated viral genome in low-level HBV replication
stage or preferential immune control of HBV replication. In either case, high HBsAg/HBV
DNA ratio may indicate enhanced host immunity which preferentially suppresses HBV
replication pathway (transcription of pregenomic RNA), relatively sparing HBsAg
transcription. Moreover, HBsAg levels are genotype-dependent while the HBsAg/HBV DNA
ratios of immune clearance (HBeAg-positive) and HBeAg-negative CHB seems to be a
better marker which is less influenced by genotypes.
We now included some references in this way in the text.“Pretreatment serum HBsAg-to-HBV DNA ratio predicts a virologic response to entecavir in
chronic
hepatitis
B.
Korean
J
Hepatol.
2011
Dec;17(4):268-73.
doi:
2
10.3350/kjhep.2011.17.4.268. Song JC1, Min BY, Kim JW, Kim JY, Kim YM, Shin CM, Lee SH,
Hwang JH, Jeong SH, Kim N, Lee DH
Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: a
perspective on Asia. J Hepatol 2010;52:508-513.Nguyen T, Thompson AJ, Bowden S, Croagh
C, Bell S, Desmond PV, et al.
Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus
(HBV)-infection: a European perspective. J Hepatol 2010;52:514-522Jaroszewicz J, Calle
Serrano B, Wursthorn K, Deterding K, Schlue J, Raupach R, et al.
4.) Why was the used HBV DNA classes used? There is no reference for this.
We chose to do a descriptive approach creating subjectively four classes based on the
change in the viral load of 0.5 log that is considered as a clinically significant change
Here we addressed some references .
Panel on Antiretroviral Guidelines for Adults and Adolescents. Guideline for the use of
antiretroviral agents in HIV-1-infected adults anadolescents. Department of Health and
Human Services. December 1,2009. Available at:
http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. Accessed July 12,
2010.11.
Morlat P. Prise en charge médicale des personnes vivant avec le VIH—Recommandations
du groupe d’experts. Paris: La documentation Française, 2013.
5.) HBV Genotype E in West African countries, especially in older populations,
has been reported to have low frequencies of detectable viral load and eAg. At
baseline, what % of this population had detectable viral load and what % had a
positive eAg?
90% of patients had detectable viral load at baseline (M0) and among them 2 patients
(5%) had a positive Ag HBe. 94% of patients had at least one detectable viral load on the
three samples (M0, M2 and M4) and 6% were indetectable (< 1.1 log) on the three
samples). We included this point in the results section page 7 lines 160 to 163.
Only, we kept the 36 patients who had at least one viral load below 3.2 log copies/ml on
one of the 3 samples.
6.) Tables: In general I found the tables difficult to read. For example, in table 1,
what does ALT (xN) mean? Can any of the dynamic changes in HBV DNA be
better captured in figures?
We agree that tables are difficult to read and had to be clarified. ALT (xN) means upper
limit of normal ULN and we changed it in Table 1.
7.) It mentions that lamuvidine was proposed to patients. Did any receive this
anti-viral during the measured time points?
3
No patient received antiviral therapy during the follow-up. To be included in this study, the
patients had to be naïve of any anti HBV treatment.
8.) I am concerned that there is limited power to make some of the statements
that are made. For example, is there adequate power to state that the differences
are, or may be due to the high frequency of genotype E?
We agree there is too few samples belonging to genotype A, so we propose to gather
data from both genotypes A and E. We revise figures (fig. 2 and 3) showing viral load
fluctuation between the 2nd and 3rd sample when viral load fluctuation between the 1st
and 2nd was >0.5 log10 IU/mL and < 0.5 log10 IU/mL
Figure 2: Viral load fluctuation between the 2nd and 3rd sample when viral load
fluctuation between the 1st and 2nd sample was >0.5 log10 IU/mL
3
(log10 IU/mL)
2
1
0
0
.5
1
1.5
2
2.5
Viral load fluctuation between the 1st and 2nd sample
(log10 IU/mL)
3
Figure 3: Viral load fluctuation between the 2nd and 3rd sample when viral load
fluctuation between the 1st and 2nd sample was ≤0.5 log10 IU/mL
4
3
(log10 IU/mL)
2
1
0
0
.5
1
1.5
2
2.5
Viral load fluctuation between the 1st and 2nd sample
(log10 IU/mL)
3
Minor essential revisions:
1.) There are periodic spelling errors that need to be fixed.
2.) There should be consistency with abbreviations. For example both HBsAg
and AgHBs is used.
This has been corrected and the text has been reviewed by a native English speaker.
Reviewer: jeremie guedj
Reviewer's report:
1- having a figure with the longitudinal evolutions of HBV DNA (spaghettis plots)
for all patients and/or medians. This would help get a sense of the data.
We included a figure with the longitudinal evolutions of HBV DNA of the patients who had
at least one viral load below 3.2 log copies/ml on one of the 3 samples. This figure shows
that the fluctuations are spontaneous; We added this figure number 1 page 7 line 168
Figure 1 : Evolutions of HBV DNA in patients with one or two samples with HBV
DNA greater than 3.2 log10 IU/mL (n=36).
5
5
4.5
4
3.5
3.2
3
2.5
2
1.5
1
0
2
Month
4
- giving better justifications in the cutoffs (class for HBV DNA). In particular the
reason
why a cutoff at the median for BMI and age is used in the statistical analysis in
unclear. Why not analyzing it as a continuous variable or using a clinically
relevant cutoff ? In Table 2, I can't find a justification for the cutoff used for HBV
DNA ?
We chose to do a descriptive approach creating subjectively four classes based on the
change in the viral load of 0.5 log that is considered as a clinically significant change
Here we addressed some references .
Panel on Antiretroviral Guidelines for Adults and Adolescents. Guideline for the use of
antiretroviral agents in HIV-1-infected adults anadolescents. Department of Health and
Human
Services.
December
1,2009.
Available
at:
http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. Accessed July 12,
2010.11.
Morlat P. Prise en charge médicale des personnes vivant avec le VIH—Recommandations
du groupe d’experts. Paris: La documentation Française, 2013.
A BMI value of 21 was chosen as cutoff because it was the median.
- improving the table editing. The samples sizes in Tables are difficult to follow.
For instance it is written in
Table 1 N=63 for HBV genotypes and in Table 4 it is N=43.
Genotype is known for 63 patients and among them 43 patients had HBsAg known.
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HBsAg was performed in 43 patients only, because this test was done later on remaining
sera. The remaining quantity of serum was too small for 20 patients.
- improving the quality and the descriptions of the figures. Figure legends should
be improved such that the figures are self consistent.
We are agree and we improved the figure legends
Minor Essential Revisions
- How did you manage data below the limit of quantification
- Can you confirm that no patients initiated a treatment during the follow up
period ?
We confirm that no patient received any treatment during the follow up because it was a
inclusion criteria for this study.
- Please provide P-values when describing viral load fluctuations
The p values were respectively of 0.85 between the 1 st and 2nd sample, 0.49 between
the 2nd and 3rd sample and 0.17 between the 1st and 3rd sample (using the Wilcoxon
matched-pairs signed-ranks test).
- It is stated p7 "such significant fluctuations were only observed for genotypes
E". However from figure 2 there are only 3 Genotypes A. Is this really
necessary ?
We agree with the reviewer. There is too few samples belonging to genotype A, so
we propose to gather data from both genotypes A and E.
Patients in whom HBV DNA fluctuated >0.5 log10 IU/mL between M0-M2, had also
significant fluctuation between M2-M4 (positive correlation r=0.34, p=0.005) (Figure 2),
while patients with stable HBV DNA between M0-M2 also showed a stable viral load
between M2-M4 (Figure 2). The largest variation observed between two samples was
between the 2nd and 3rd samples for 45% of patients.
Figure 2: Viral load fluctuation between the 2nd and 3rd sample when viral load
fluctuation between the 1st and 2nd sample was >0.5 log10 IU/mL
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