Author's response to reviews Title: High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load Authors: Joanna R Santos-Oliveira (joannars@ioc.fiocruz.br) Carmem BW Giacoia-Gripp (carmembg@ioc.fiocruz.br) Priscilla A. de Oliveira (priscillaalexandrino@terra.com.br) Valdir S Amato (valdirsa@netpoint.com.br) José Angelo L Lindoso (jlindoso@usp.br) Hiro Goto (hgoto@usp.br) Manoel P Oliveira-Neto (manoel.paes@ipec.fiocruz.br) Marise S Mattos (marise.smattos@gmail.com) Beatriz Grinsztejn (beatriz.grinsztejn@gmail.com) Mariza G Morgado (mmorgado@ioc.fiocruz.br) Alda M Da-Cruz (alda@ioc.fiocruz.br) Version: 5 Date: 11 October 2010 Author's response to reviews: see over
Ministério da Saúde Rio de Janeiro, October 11, 2010. Dr. Melissa Norton Editor-in-Chief of the BMC Infectious Diseases Dear Dr. Norton Thank you for continuing to consider publishing our manuscript entitled, High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load. I am attaching two files of our newly revised manuscript (MS: 1737485794296556). One has modifications underlined and strikethrough characters to indicate deleted text. The other does not track changes. The latest revisions are direct responses to the reviewer s comments and suggestions. A major issue concerning the presence of Leishmania infection as a cofactor for increasing T cell activation was finally accepted by Dr. Benito. Indeed we included all his last suggestions. The Dr. Lombard s requests were also answered, as possible. Some requests were not addressed because they were already questioned and solved in previous reviews. We believe that we have attended to all queries. Indeed, all of the suggestions were quite relevant. We hope that this new version may be judged ready for publication in the BMC Infectious Diseases. We look forward to your decision. Yours sincerely, Alda Maria Da-Cruz, MD, PhD Corresponding author: Alda Maria Da-Cruz,, FIOCRUZ Av. Brasil 4365, Pav. Leonidas Deane 4o. andar Manguinhos, Rio de Janeiro Brasil, CEP 21040-900 Fax number: 00.55-21-2290-0479 Phone number: 00.55-21-3865-8102 Electronic mail: alda@ioc.fiocruz.br, IOC/. Av. Brasil, 4365 Pav. Leônidas Deane 4º
REVIEWER COMMENTS: REVIEWER : Jose Benito Comment: The table reporting the individual values for HIV viremia and CD4 count must be included in the manuscript apart from the table summarising clinical and laboratory characteristics of the different groups. Answer: The table was included as Table 2 in the Results section. Comment: Authors must include in table 1 (in the actual version, ie table showing clinical and laboratory characteristics of study groups) the p values for the different comparisons between groups. Also in this table, proportion of patients with undetectable HIV viremia in each group, as well as median HIV viremia in the subgroup of subjects presenting detectable viremia in each group must be given. Answer: The p values were included as recommended in table 1. The proportion of patients with undetectable HIV viremia in each group, as well as median HIV viremia in the subgroup of subjects presenting detectable viremia in each group was given. Comment: Given the small sample size of each group, only median (interquartile range) must be used to describe each parameter analysed (no means+sd). Answer: The means+sd values were excluded. The interquartile ranges were added in the Results section. Comment: In the last sentence of results section ( A multivariate statistical analysis showed..), the authors must add:..independently of CD4 counts and HIV viremia). Answer: The sentence was added as recommended., IOC/. Av. Brasil, 4365 Pav. Leônidas Deane 4 º
REVIEWER : Carl J Lombard Comment: I am not familiar with epidemiology of the emergence of leishmaniasis as an opportunistic infection in HIV patients but I find the explanation to my question on why the sample size for the two exposed groups was 9 and 8 respectively " in Brazil this association is rare, justifying this low number" versus the statement in the Abstract " have emerged as important opportunistic infections in HIV-AIDS patients"(lines 4&5) and the sentence in lines 10 and 11 contradictory. Answer: A disease is considered important and emergent because of this severity and not only because the number of cases. As an example we can cite the Virus Ebola infection, that is an important emergent disease but affect relatively few people around the world. Comment: The multiple regression analysis of CD38 to adjust for cd4 counts is a relevant approach. As the reviewer (Benito) stated the comparison between the groups should be matched for cd4 level. This can be done through an analysis of covariance. Analysis of covariance will allow one to compare the cd38 levels of the groups at the same cd4 level. The analysis of covariance can be formulated as a multiple regression model. Answer: We have considered that multivariate analysis was an adequate strategy to address our main question that was to show that leishmaniasis is likely a cofactor increasing T cell activation. According to our experience and also to Dr Benito s previous comments matching patients by CD4 is a difficult approach, considering the clinical characteristics of patients and the recruitment criteria. Comment: In the current analysis presented the AVL/HIV and ATL/HIV groups have been pooled. It is my opinion that the AVL and ATL groups should be separate groups compared to the HIV only group. One also has to compared whether the association between CD38 and cd4 is the same across the three groups i.e. the slopes of the cd38 on cd4 is parallel. This can be achieved by looking at an interaction between cd4 and group. If this is not significiant the contrasts between the groups as presented in Figure, IOC/. Av. Brasil, 4365 Pav. Leônidas Deane 4 º
C should hold. With paralell slopes the mean difference is constant over the observed cd4 range and can be estimated. Answer: The AVL/HIV and ATL/HIV groups were separated throughout the paper. They were pooled only for determination of leishmaniasis as a cofactor for increasing T cell activation in the multivariate analysis. Comment: The scatterplot of CD38 vs cd4 with the three groups indicated will be informative and should be presented. Answer: As the results relate to these two variables that were not significant in multivariate analysis it was recommended to not include these data. Comment: The authors should state the limitation of the study. Observational with small numbers in the ATL and AVL which will limited the power to do a proper analysis of covariance. Answer: The sentence A covariance was not performed because of the low number of ATL and AVL was added in results section., IOC/. Av. Brasil, 4365 Pav. Leônidas Deane 4 º