Author's response to reviews

Author's response to reviews Title: Contribution of Interferon Gamma Release Assays testing to the Diagnosis of Latent Tuberculosis Infection in HIV-Infected Patients: A comparison of QuantiFERON Gold in Tube, T-SPOT.TB and Tuberculin Skin Test Authors: José M. Ramos (jramosrincon@yahoo.es) Catalina Robledano (catirobledano@yahoo.es) Mar Masiá (marmasia@ya.com) Sofia Belda (sbelda_gas@hotmail.com) Sergio Padilla (sergiopadurr@gmail.com) Juan C. Rodríguez (jcrd65@gmail.com) Félix Gutiérrez (gutierrez_fel@gva.es) Version: 2 Date: 20 May 2012 Author's response to reviews: see over

Reviewer: Graham H Bothamley Major compulsory revisions 1. This paper assumes that a positive interferon-gamma release assay and latent tuberculosis (TB) are the same. However, the probability of latent tuberculosis is higher in subjects who have a positive test and have been exposed to TB. The paper should therefore be titled A comparison of QuantiFERON Gold in Tube, T-SPOT.TB and tuberculin in a cohort of HIV-infected subjects. The reviewer is right in his reflection, and we agree with him in the uncertainty of a positive IGRA in an HIV-infected patient with no other evidence of tuberculosis exposure (in the absence of a positive tuberculin test), as we state in the last paragraph of the Discussion. However, the limited available information suggests that these patients may have a higher risk of ulterior tuberculosis development. We have taken on board the reviewer suggestion and amended the title accordingly. The paper is now titled: Contribution of Interferon Gamma Release Assays testing to the Diagnosis of Latent Tuberculosis Infection in HIV-Infected Patients: A comparison of QuantiFERON Gold in Tube, T-SPOT.TB and Tuberculin Skin Test. 2. A Venn diagram would therefore be most useful to show how often the various tests coincide in terms of positives as defined by the manufacturers. The definition of a positive tuberculin skin test (5 mm) must be included in the abstract. According to the reviewer recommendation, we have included the Venn diagram (new Figure 1, and we have added a new sentence: TST induration > 5 mm was considered positive in the Methods section of the abstract. 3. The ultimate reason for doing these tests is to use preventive treatment to ensure that subjects do not develop TB and that sub-clinical TB is detected. The action taken for a positive test should therefore be included in the description of the study. We thank the reviewer for his valuable suggestion. We have included the following new paragraph in the Methods section (page 6, first paragraph): All patients with a positive tuberculin test who had not been previously treated, received chemoprophylaxis with isoniazide for 6-9 months. Patients with a positive IGRA plus a positive tuberculin also received isoniazide chemoprophylaxis. Patients with a positive IGRA but negative tuberculin test were individually managed by their doctors in charge, who decided to start chemoprophylaxis or not according to additional accompanying factors. 4. Those with contact with active TB should be examined separately. The degree of exposure should also be given.

Following the recommendation of the reviewer, we have examined separately the results of the test of the 302 patients with no past or current TB not positive TST according contact with active TB. For instance now there is two groups 209 patients without contact with patients wih TB and 93 patients with contact with patients with TB. We have included in the table 2. Now we have modified the table 2 the previous columns now are files, and the previous files now are columns. 5. Similarly those with previous TB should lead Table 1 in terms of the number of positive test. We have taken on board the valuable suggestion of the reviewer and analyzed separately patients with previous or current TB in a new column in Table 1. 6. Table 1 should be amended to include the criteria for giving preventive treatment - the categories patients with (or without) past or current TB or treatment for LTBI are too broad. We have changed this remaining category in Table 1 and Table 2 for Patients with positive TST. We also have changed this remaining category in the test. 7. Table 4 should be simplified to give a clear message and include only data, which show significant variation. According to the reviewer s recommendation, we have simplified the table 4 by including only significant variables. Discretionary Revision 1. There is increasing interest in the grey zone for the different tests. The data could be used to show how many are in these zones, perhaps using 5-15 mm for the tuberculin skin test. This could be combined with the number of indeterminate results and the CD4 cell count to complement the literature on these aspects. The reviewer raises again an interesting point, and we are grateful for his contribution to improve the paper. Unfortunately, the quantification of the induration with the tuberculin test was not recorded in all cases in our study, and therefore we cannot provide this information in the manuscript. Reviewer: Mengistu Legesse MAJOR COMPULSORY REVISIONS

- The authors found a much higher LTBI prevalence when using T-SPOT.TB compared to QFG or TST. Essentially, they conclude that the discrepancy must be due to a higher T-SPOT.TB sensitivity. Although this may well be true and previous studies support this finding, the authors should address the importance/reliability of USP Microscope compared to ELISPOT plate reader in measuring/counting SFU in the method part We thank the reviewer for his valuable suggestion. Accordingly, we have incorporated a new paragraph in the Discussion section: It should be stated, however, that we did not use the most reliable method for counting SFU, the automated ELISPOT plate reader, and therefore misclassification of some of our results can not be ruled out. - A higher rate of indeterminate results was found with the T-SPOT.TB than QFG. On the other hand, the authors discussed that T-SPOT.TB may be less affected by advanced immunosuppression than QFG test (which is not clear). Thus, the possible source of indeterminate results in T.SPOT.TB needs to be assessed /discussed Following the suggestion of the reviewer, we have included the main reasons explaining the number of indeterminate results with the T.SPOT.TB (page 12, second paragraph). We also included a new reference. It number is 22. We also change de number of the rest of the references. - More positive results were common in patients with past, current history of TB or LTBI by the three tests. What does this imply (re-infection, re-activation or persistent immune response)? How the authors define LTBI? What was the importance (objective) of including those individuals with past history of M. tuberculosis infection? These issues should be addressed more thoroughly in the introduction and discussion parts. We are grateful for the constructive comments of the reviewer. The past TB was referred by the patient or collected from clinical records. Current TB diagnosis was based on the results of clinical and radiologic examination and/or isolation of M. tuberculosis from sputum or other specimen. LTBI was defined by a previous positive TST performed in our clinic or in another clinic. The patients with past, current history of TB or LTBI had a higher frequency of positive tests presumably because of a persistent immune response. Those patients were included in the study as positive controls, to assess the yield and reliability of the tests in the subset of patients who retained persistent immune response. We have included this information in the Methods and Discussion sections (page 5, last paragraph and page 13, first paragraph). - The presentation and interpretation of results of factors associated with LTBI are not clear both in the text and in table 4. According to the reviewer recommendation we have simplified the table 4 by including only significant variables.

MINOR ESSENTIAL REVISIONS 1. Abstract - 1.1. Background: tells us the objective of the study rather than the rationale of the study We have taken on board the reviewer s suggestion, and have added two new sentences in the Background: Diagnosis and treatment of latent tuberculosis infection (LTBI) is the most effective strategy to control tuberculosis (TB) among patients with HIV infection. The tuberculin skin test (TST) was the only available method to identify LTBI. The aim of the present work was to.. - 1.2. Methods: indicate the period of the study We have included the period of the study in Methods: Prospective study from January 2009 to October 2010 carried out in consecutive patients cared for in a single institution in Spain. IGRAS and tuberculin skin test (TST) were performed simultaneously. - 1.3. Results: the statistical difference between the positive results found using TST and QFG (12.3% VS 7.5%, P= 0.05) is not significant. even the difference between TST and T-SPOT.TB (12.3% VS 18.5%, P=0.04) is not strong. We have taken on board the reviewer s suggestion and have changed Compared with TST, more positive results were observed with T-SPOT.TB (12.3% vs. 18.5%; p=0.04) and less with QFG (12.3% vs. 7.5%; p=0.05) for TST, GFG and T-SPOT.TB were positive in 12.5%, 7.5% and 18.5% cases, respectively - 1.4. Among patients without past history of M. tuberculosis infection (n= 302), TST results were not available in 25 (8.3%), while 20 (6.6%) were positive by TST. I think the percentage (6.6%) should be re-calculated for those patients whose TST results are available (N= 277). This correction needs to be made through all the manuscript. We have recalculated the percentage of patients with positive TST, and corrected the old value through all the manuscript. - 1.5. Mention some significantly associated factors like CD4 counts with the diagnostic positivity of LTBI Following the recommendation of the reviewer, we have added the factors associated with the diagnostic positivity of LTBI (Abstract, last sentence of the Results section): Patients with a CD4 cell count greater than 500 cells/µl and prior stay in prison were more likely to have a diagnosis of LTBI by TST and/or QFG and/or T.SPOT.TB (adjusted odds ratio [aor]: 3.76; 95% CI, 1.4 9.89; and aor: 3.3; 95% CI, 1.3 8.3, respectively). 2. Background 2.1. Page 3, second paragraph, second line: Mycobacterium tuberculosis, needs to be abbreviated as M. tuberculosis in bracket.

We have abbreviated Mycobacterium tuberculosis accordingly 2.2. Page 4, first paragraph, line seven: In this investigation, patients with a broad spectrum of HIV disease were studied the meaning of patients with a broad spectrum of HIV disease is not clear. We have modified the sentence according to the recommendations of the reviewer: patients with a broad spectrum of HIV disease for. patients with different stages of HIV disease. 3. Methods 3.1 Page 4, second paragraph, was a treatment given when the IGRAs/TST results found positive? To explain the management of patients with LTBI, the following paragraph has been included: All patients with a positive tuberculin test who had not been previously treated received chemoprophylaxis with isoniazide for 6-9 months. Patients with a positive IGRA plus a positive tuberculin also received chemoprophylaxis. Patients with a positive IGRA but negative tuberculin test were individually managed by their doctors in charge, who decided to start chemoprophylaxis or not according to other accompanying factors. 3.2. Page 5, first paragraph, line 5, it is not clear how was the skin induration measured? We have taken on board the reviewer s suggestion, and we have rewritten the sentence: Induration was measured at 48-72 hours after the inoculation. The size of the induration equal or higher than 5 mm was considered positive. 3.3. Page 5, second paragraph, it is unclear how blood samples were collected for QFG assay? Was it collected directly into each tube? According to the reviewer s recommendation we have clarified and re-written the sentence about the collection of the samples for the QFG assay For QFG, whole blood was collected from each patient and inoculated in three heparinized tubes of 1 ml each: one containing TB antigens (ESAT-6, CFP-10, and TB7.7), a positive control tube containing phytohemagglutinin, and a null control 3.4. Page 5, the same paragraph, how was the level of IFN- determined? was the interpretation made using software developed by the company? We have taken on board the reviewer s suggestion, and added the following sentence: Optical densities were interpreted by using specific software provided by the manufacturer 3.5. Page 6, first paragraph, last line, references are needed for the reliability of USP Microscope

We have modified and added the reference of the software of USB Microscope according your recommendations. The new sentence says: The SFU were counted with an Veho - VMS- 001 USB Microscope with the Microcapture software [16] Reference: [16] User instructions. Microcapture Software. Available in: http://videopen.narod.ru/usb-ustroistva/usb-mikroskop-200x-8-led/microcapture.pdf We have changed the references because we have added a new reference 16. 3.6. Was the SFU count performed by one person? We have added: SFU were counted by one observer in case of doubt by two observers 4. Results 4.1. Page 8, first paragraph, was there any unresponsive (anergy) result by the TST? (as anergy is common in HIV-infected individuals) The reviewer raises an interesting question. Unfortunately, no additional tests were performed to quantify how many of the negative results of the TST were indeed patients with no LTBI or anergic patients. We have included a sentence at the end of the Discussion section addressing this: The rate of anergic patients was also unknown because no additional skin tests to the TST were performed 4.2. page 7, last paragraph, last line, needs to be corrected as Table 1 shows... those patients with and with no... We thank the reviewer for making us aware of this mistake. We have corrected the sentence: Table 1 shows the differences between those patients with and with no past or current TB or positive TST. 4.3. Page 8, second paragraph, Compared with TST, more positive results were observed with T-SPOT.TB according to manufacturer s criteria (12.3% versus 18.5%; p=0.04) and less with QFG (12.3% versus 7.5%; p=0.05). The number of indeterminate or invalid results was lower for QFG than for T-SPOT.TB (2.7% versus 7.2%; p=0.002) We have taken on board the reviewer s suggestion and have changed Compared with TST, more positive results were observed with T-SPOT.TB (12.3% vs. 18.5%; p=0.04) and less with QFG (12.3% vs. 7.5%;p=0.05) for TST, GFG and T-SPOT.TB were positive in 12.5%, 7.5% and 18.5% cases, respectively. 4.4. As it is described in data analysis section the p-values are generated from Pearson chi-square. However, the appropriate test statistic is McNemar s chi-square. Authors should reanalyzed this part of the data appropriately and also indicate the method used here in the data analysis section.

We have re-analyzed data specified by the reviewer by using McNemar s test, and indicated it in Methods section 4.5. Page 8, last paragraph, the percentage (6.6%) of TST positive individuals should be corrected (or describe, how it was calculated ). According to the recommendations of the reviewer, we have changed the denominator to 277. The new percentage obtained of positive TST individuals of 7.2% has been changed all through the manuscript. 4.6. Page 9, Is the OR mentioned in the text an adjusted or crude? What is associated with what in Table 4? I can not see the logistic regression results (OR or 95%CI). In table 4, only univariate analyses have been included. Associations in the table represent the factors linked with positive results for each test. ORs adjusted for all significant variables of univariate analyses have only been shown in the text. For clarification purposes, we have incorporated this information in the footnote of Table 4. We have added in the statistical analysis section that the OR are adjusted OR (aor). 4.7 Under each table, it is described that Date are no (%) of patients, what is a date? We are sorry for the mistake. We have changed Date are no (%) for Data are no (%) of patients. 4.8 Table 3, column 2, row 4, is the range from 0.339-609? Please see also row 6, range from 0.315-583? We thank the reviewer, it has been also a mistake, We have modified in column 2, row 4 the range (it should say: 0.339-0.609 ), and row in 6 ( 0.315-0.583 ). 4.9 is BCG defined as Bacille Calmette-Guérin or Bacille de Calmette et Guérin? Bacille de Calmette et Guérin is in French and Bacille Calmette-Guérin in English. We have taken on board the reviewer s suggestion and have changed from French language to English 5.1. Discussion 5.1 Page 11, first paragraph, line 8, in studies carried out... the sensitivity was 94% for T-SPOT.TB (Ref 22), and 67% for QFG (Ref 23) it is not appropriate to compare the sensitivities of the two tests in two independent studies. The results would be varied, if both tests would have been used in the same study According to the reviewer recommendation we have modified the original sentence and we have transformed it into two sentences. In studies carried out in countries with low prevalence of TB, sensitivity was 94% for T-SPOT.TB [22]. In the study of Sauzullo et al the sensitivity of QFG was 67%, not higher than that of TST in head-to-head comparison [25].