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: 3 Date: 27 April 2010
Rio de Janeiro, April 27, 2010.
Dr. Melissa Norton
Editor-in-Chief of the BMC Infectious Diseases
Dear Dr. Norton
Thank you very much for coordinating the re-review of our manuscript entitled “High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load”. We have made revisions to the paper according to the newest comments of the reviewers. Their suggestions were greatly appreciated and are discussed in detail below. Please find attached a file containing our revised manuscript (MS: 1737485794296556), which we are now resubmitting for editorial review. The modifications are incorporated into the manuscript. Attached to this letter are our direct responses to reviewer comments.
We hopefully anticipate that this new version is judged ready for publication in the BMC Infectious Diseases. However, we remain prepared to make any additional, recommended modifications to satisfy your high standards. Thank you, we look forward to your decision.
Yours sincerely,
Alda Maria Da-Cruz, MD, PhD
Corresponding author: Alda Maria Da-Cruz,
Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz – FIOCRUZ Av. Brasil 4365, Pav. Leonidas Deane 4o. andar
Manguinhos, Rio de Janeiro – Brasil, CEP 21040-900
EDITORIAL COMMENTS Dear Associated Editor,
We have addressed the latest suggestions from the Reviewers by making modification to the text and figures. All of their suggestions were carefully considered and, in our view, certainly enhanced the quality of the manuscript. We are grateful for your choice of reviewers. We are attaching our direct answers to the specific comments.
Dr. Benito was particularly concerned with the presentation of data supporting our conclusions. The way in which HIV-1 infected patients were paired was better explained and the range of the viral load levels was modified. The figures B and D now clearly show that AVL/HIV-AIDS patients with viral load under 400 copies/mL present higher activation levels than HIV-1 infected controls patients with the same range of viral load. This support our hypothesis that leishmaniasis can act as a co-factor for activating lymphocytes in HIV infected patients, which in turn can lead to severe depletion of CD4+ T cells, observed in AVL/HIV-AIDS cases.
Considering the reviewers’ comments, the following specific changes were made in the manuscript. Abstract: The sentence “Furthermore, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load” was modified to “Furthermore, five out of nine AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load.”
The conclusions were modified to: “Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.”
Text:
2. Results section. The following sentence was added: “HIV infected controls presented a positive correlation between the viral load and cellular activation (p=0.01, r=0.60).” Page 7, lines 22-23. 3. Discussion: The sentence was modified to “This fact was observed in leishmaniasis patients during the remission phase, i.e, after the end of anti-leishmanial therapy and under HAART use.” Page 9, Line 10.
Yours sincerely,
RESPONSES TO REVIEWER COMMENTS:
REVIEWER : Jose Benito
We agree with the Reviewer that the way in which the results are presented did not enable the reader to figure out that in our casuistic, there are AVL/HIV co-infected patients with high activation levels despite low or undetectable viral load (five out nine cases) and CD4+ T cells count lower than 200 cells/mm3. The figures B and D were modified in order to clarify the results regarding HIV viral load versus CD4+ T cell counts or percentage of CD38+ in CD8 T lymphocytes. The range of HIV viral load was modified to < 400, > 400 to 10,000 and >10,000 copies/mL.
In this new presentation, the figure shows that five out of nine AVL/HIV-AIDS patients presented viral loads < 400 copies per mL, one with between >400-10,000 copies per mL (5,810 copies/mL) and three with viral load > 10,000 copies/mL (42,240; 242,000; 316,000 copies/mL). All of these patients presented CD4+ T cells counts lower than 200 cells/mm3. In addition, the measured percentage of CD38+ in CD8+ T lymphocytes was greater than 85% in all of the patients. Even the five patients with undetectable or low viral load (< 400 copies/mL) presented cellular activation levels much higher than HIV-1 infected controls with low viral load. There was no correlation between the viral load and cellular activation in co-infected groups (see results section). On the other hand, HIV infected controls presented a positive correlation between the viral load and cellular activation (p=0.01, r=0.60). This last phrase was added to the manuscript text.
The comment “the high level of activation in AVL-HIV patients may be a consequence of the
low CD4 counts and not necessarily a consequence of the presence of Leishmania infection” is
fully applied for those co-infected patients with high viral load. However, how to explain the high activation levels and the low CD4+ T cell counts in co-infected patients with HIV viral load under control? Figure C shows that co-infected patients paired by HIV viral load with HIV-1 infected patients without leishmaniasis, presented significantly higher levels of activation. We had suggested that the persistence of Leishmania parasites can act as a co-factor, along with residual lymphnod replication of HIV as a mechanism to explain the increased cell activation.
Comment “Even more important is the issue of HIV load. The authors state that HIV groups
were matched for HIV load. This is not true.”
The range of viral load for these HIV-1 infected controls was the same range used for AVL/HIV or ATL/HIV infected patients. For each group of infected patients, AVL or ATL/ HIV, co-infected patients were matched according to the same range by viral load levels. HIV-1 co-infected controls presented HIV viral load levels ranging from undetectable to 255,000 copies/mL and CD4 T cells counts from 34 cells/mm3 to 635 cells/mm3. The HIV controls with HIV viral load under 400 copies per/mL had activation levels ranging from 30% to 85%, used here. However, we made changes to make this clearer in the text for future readers.
In conclusion, based on data in the literature, we believe that low CD4+ T cells counts found in co-infected patients are a consequence of high levels of activation. These activation levels were much higher in AVL/HIV-AIDS than in HIV-1 infected controls (Figure C). The points of discussion are: 1) which infectious agent contributed to this activation? HIV, Leishmania or both. 2) Why, despite the same low HIV viral load (<400 copies/mL), do AVL/HIV-AIDS patients present activation levels higher than HIV-1 patients without leishmaniasis? The hypothesis that besides HIV, Leishmania persistence could be a co-factor contributing to increased lymphocytes activation levels and consequently to a severe depletion of CD4+ T cells even under HAART, (Figure E) should not be ruled out.
REVIEWER : Luciano Nigro Abstract:
The comment “Conclusions are still not well written. The concept of higher proportion of CD8 T … can limit the use ….is not clear written”.
The sentence was modified as follows: “Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even
when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.”
Page 8, line 19. “I would change disruptions with depletion or similar word”.
We agree with the reviewer and the text was modified as follows: “Additionally, CD4 T cell depletion of bone marrow caused by both HIV infection [33] and AVL [9,11] was already demonstrated and may lead to deficiencies in the input of new lymphocytes into the periphery.
Page 9, line 10. “I would re-write the sentence….This phenomenon ….HAART use”.
The sentence was modified to “This fact was observed in leishmaniasis patients during the remission phase, i.e, after the end of anti-leishmanial therapy and under HAART use.”
REVIEWER : Guido Silvestri
