Author's response to reviews

Transcription

1 Author's response to reviews Title: Stable Hepatitis C Virus RNA Detection by RT-PCR During Four Days Storage Authors: Anne-Isabelle de Moreau de Gerbehaye (ng.deradigues@yucom.be) Monique Bodeus (bodeus@mblg.ucl.ac.be) Annie Robert (annie.robert@epid.ucl.ac.be) Yves Horsmans (horsmans@gaen.ucl.ac.be) Patrick Goubau (goubau@mblg.ucl.ac.be) Version: 3 Date: 10 Aug 2002 PDF covering letter

2 Title of paper: Stable hepatitis C virus RNA detection by RT-PCR during four days storage. Authors: A.- I. De Moreau de Gerbehaye et al. Reviewer: Dr Harald Kessler Point to point reply to the reviewer's report. General: the English language has been improved and smalle errors corrected. Major problems Abstract: suggested changes have been followed. "Whole blood" has been replaced by "clotted blood". "HCV-infected" has been replaced by "HCV RNA positive". Background: suggested changes have been followed. "Whole blood" has been replaced by "clotted blood". Materials and methods: a number of suggested changes have been followed. Details are given on patients (age and sex). Centrifugation was done "within one hour", meaning that it was done as soon as possible (running from the patient to the lab and processing the sample does not always take exactly the same time). We have dropped the details about the in-house nested RT-PCR which is no more in use. Doing the test "on the same tube" does not mean it had to be thawed again, because all aliquots were taken when necessary and all tests were performed for a single patient in one run. The statement from this reviewer that Roche has moved to Pleasanton, California may be correct, but our present day kits still state that they are manufactured in Branchburg, NJ. So this is left as it is. We did use the controls as requested by the manufacturer including a negative control. We added however two independent high and low positive controls to check for interrun variability. We adapt this in M & M and in the results section to make it clear. Results: we have moved where necessary the sentences to other sections. Precision and SE of log values have been added where asked for. We did not repeat the outlier value. We have tested all samples from the same patients in one run and did not think it was proper to correct values afterwards. Discussion and conclusion:we have largely rewritten this section to answer to the request of clarity and some precisions have been added. However we did not delete the sentence on freeze-thaw cycles nor the well-known inhibitory effect of heparin. In our view the discussion may comment more widely than the strict bounderies of the

3 study on the topic of sample preparation and storage. But we specify that freeze/thawing was not the subject of this study and that the heparin knowledge is 10 year old. We apologize for having missed a paper by this reviewer and add it to our discussion. Figures:They have been adapted as requested.

4 Title of paper: Stable hepatitis C virus RNA detection by RT -PCR during four days storage. Authors: A.- I. De Moreau de Gerbehaye et al. Reviewer: Dr Bernadette Ramirez Point to point reply to the reviewer's report. General The text has been adapted, errors corrected and conclusions modified. Specific Background: We restate the importance of viral load and genotyping at the beginning of the discussion. We do not agree with the reviewetr that our study would show that suboptimal storage temperatures affect HCV RNA. Indeed it shows mainly the stability of HCV RNA. Further we state other conditions which possibly affect RNA results. We did not observe higher values at day 4 as suggested by this reviewer. In two patients the results are somewhat wider spread, but stay within 0.3 log limits. "whole blood" has been replaced by "clotted blood". The terminology "routine conditions" is avoided. Room temperature has in fact been monitored and the observed limit have now been added. Materials and methods: We change "samples" to "blood samples". The use of serum separator was used when separation of serum was done immediately or at 6 hours. Tubes without separator were used when several hours of contact between serum and clot was investigated. "Plasma" was changed to "serum". We have revised the section on the different tubes and times and added a table, which we hope will clarify things. A type error was corrected ( C). We have added shortly which data-analysis was done with the PRISM software. Results: All results showed an evolution of values starting with a first value on an immediately frozen sample (at -80 C), which may be taken as an optimal reference. This is compared to the interrun variability of the multiple retesting of two positive samples. Therefore it is unclear to us what the reviewer means by "what are the controls

5 (reference) which were used to compare the experimental data with?" This question is not asked by the other two reviewers. Discussion: this section has been rewritten and in this the sentences "no significant difference." and " The distribution of the viral load values " are modified. We hope this answers to this and the other reviewers. Conclusions: included in the discussion and modified.

6 Title of paper: Stable hepatitis C virus RNA detection by RT-PCR during four days storage. Authors: A.- I. De Moreau de Gerbehaye et al. Reviewer: Dr Paul Grant Point to point reply to the reviewer's report. General: This reviewer presents another way to look to the data. We have looked to them in different directions and groupings without finding differences. Therefore we think that visually it is most convenient to present the evolution of values in the individual patients as we did. Spelling and language have been further checked.