Author's response to reviews Title: Role of Interferon-gamma Release Assays in the Diagnosis of Pulmonary Tuberculosis in Patients with Advanced HIV infection Authors: Adithya Cattamanchi (acattamanchi@medsfgh.ucsf.edu) Isaac Ssewenyana (sewyisaac@yahoo.co.uk) J. Lucian Davis (lucian.davis@ucsf.edu) Laurence Huang (lhuang@php.ucsf.edu) William Worodria (worodria@yahoo.com) Saskia den Boon (sdenboon@muucsf.org) Samuel Yoo (yoouga@yahoo.com) Alfred Andama (andamalf@yahoo.com) Philip C Hopewell (phopewell@medsfgh.ucsf.edu) Huyen Cao (Huyen.Cao@cdph.ca.gov) Version: 2 Date: 4 January 2010 Author's response to reviews: see over
Dear Editor, We appreciate your continued consideration of our manuscript entitled Role of Interferon-gamma Release Assays in the Diagnosis of Pulmonary Tuberculosis in Patients with Advanced HIV infection (MS: 2012368177307881). The reviewers comments have helped us to improve the manuscript. Below, we provide detailed responses to each of the reviewers comments and indicate changes made to the manuscript based on the comments. Sincerely, Adithya Cattamanchi, MD Assistant Professor of Medicine, UCSF REVIEWER 1 Major compulsory concerns: 1) Although sputum culture of MTB is considered highly sensitive (80-93%) and highly specific (98%) the lack of clinical follow-up of patients with negative cultures does compromise the diagnostic certainty. This may be particularly important for advanced HIV infection, where paucibacillary disease and extrapulmonary TB occur more frequently. The absence of mycobacterial speciation also compromises the specificity of the cultures, as culture of nontuberculous mycobacteria, which occur more frequently in HIV-infected people in some geographical areas, would not have been detected in this study. The authors should address these issues in the discussion. We agree with the reviewer that mycobacterial culture is not a perfect reference standard. However, culture is the best available and most commonly used reference standard in TB diagnostic studies. Follow-up data is useful in patients with negative cultures who improve without anti-tb treatment these patients can definitely be considered to have an alternate diagnosis (ie, not TB). But there is less diagnostic certainty in patients who improve with empiric anti-tb treatment. These patients could have culture-negative TB or another disease process that resolves independent of anti-tb medicines (e.g., viral pneumonia) or that may respond to treatment with anti-tb medicines (e.g., bacterial pneumonia). We also agree that isolation of NTM species is a potential concern in HIVinfected patients. However, NTM species are not known to be common in East Africa and are more likely to be isolated with liquid culture media (culture was performed on solid media in our study). To address the reviewer s concern
regarding culture as a gold standard, we added the following to Paragraph 5 of the Discussion: Second, mycobacterial culture on Lowenstein-Jensen media is an imperfect reference standard. A more sensitive culture technique (such as liquid culture) may have decreased the number of false positive IGRA results and increased the number of true positive results, resulting in higher sensitivity and specificity.[26] In addition, we did not perform speciation tests to exclude the possibility of isolating non-tuberculous mycobacteria. 2) Why were individuals without smear results excluded? Was the decision to perform the T SPOT test dependent on smear status (in view of the fact that the reference standard was culture)? It is not stated how many people had negative smears and were not referred for bronchoscopy (it is only stated that people with negative smears had a bronchoscopy IF referred by their clinicians). Please clarify. We excluded patients without smear results because one of our principal objectives was to delineate the performance of T-SPOT.TB in smear-negative patients. There were only 4 patients who were excluded based on lack of smear results. Inclusion of these patients did not materially change our estimates of diagnostic accuracy. With respect to bronchoscopy, we had access to BAL fluid culture results as our study was nested within a larger study looking at causes of pneumonia in HIVinfected patients. Only 30% of the smear-negative patients enrolled in our study underwent bronchoscopy. However, among those who underwent bronchoscopy, BAL results had a small impact on outcome classification only 3 patients were identified to have culture-positive TB by BAL fluid results alone. Should the editor prefer, we can report results based on sputum culture alone. Minor essential concerns 3) Why was blood collected in citrate dextrose? As far as I am aware the manufacturers recommend sodium citrate, sodium heparin or lithium heparin tubes. Could this be a factor in the high rate of indeterminate results? The citrate dextrose tubes were part of a parallel protocol in the same patient cohort that included assessment of intracellular cytokines and sequencing (which preclude the use of heparin). In our preliminary testing, the use of citrate dextrose tubes yielded comparable results to sodium heparin tubes for the TSPOT assays (Dr. Cao, personal communication). Therefore, we do not feel this was a factor in the high rate of indeterminate results.
Discretionary revisions 4) It is stated that 16 ml of blood were collected from each individual. Why was it not possible to isolate 250 000 PBMCs in this case? It should rather be stated how much blood was collected for this particular assay. We clarified in the Methods that 16 ml of blood was collected for the T-SPOT.TB assays. At the time of enrollment, a study officer collected approximately 16 ml of blood for the T-SPOT.TB assay in anticoagulant-citrate-dextrose tubes. However, we too were surprised that we could not isolate sufficient PBMCs from all patients. We suspect that this was related both to the profound HIV-related immunosuppression and critical illness of our hospitalized population. However, there were too few patients with insufficient PBMCs (N=24) to adequately investigate these possibilities. 5) The authors state that further studies should focus on new TB antigens that will enable differentiation between latent and active disease. In the same way new host markers, other than IFN-g, may also need to be identified that will enable this distinction between infection phenotypes. We agree with the reviewer and modified the last sentence of the Conclusion: Further research and innovation is needed to identify M. tuberculosis antigens and host markers that are more sensitive and specific for active tuberculosis.
REVIEWER 2 1. The IGRA was not useful in smear negative TB but it would be interesting to see how chest x-ray findings modified the results presented. Were CXR findings accessible? Unfortunately, CXR findings were not recorded in the study database and are not accessible. 2. Abstract conclusion: please specify the population i.e. hospitalized population with a high prevalence of HIV. We revised the conclusion as suggested by the reviewer: An ELISPOT-based IGRA detected a high prevalence of latent tuberculosis infection in a hospitalized population of tuberculosis suspects with advanced HIV/AIDS but had limited utility for diagnosis of active tuberculosis in a high prevalence setting. 3. Figure 1 should show further details including the number of indeterminate IGRAs, those that could not be done due to low cell numbers, the number positive in each category etc otherwise the reader struggles. We revised Figure 1 as suggested by the reviewer. 4. Did the indeterminate results correlate with the CD4 counts? We had previously mentioned there was no correlation between indeterminate results and CD4 counts in our study population. But we agree it is more informative to provide the actual data. Therefore, we added the following sentence to paragraph 3 of the Results: There was no difference in the proportion of indeterminate test results among patients with a CD4+ T-lymphocyte count 50 cells/µl (33/109, 30%), 51-200 cells/µl (15/60, 25%), and > 200 cells/µl (6/43, 14%) (p=0.11). 5. Discussion: para 4..post-test should read pre-test? The reviewer is correct. We fixed the error: A diagnostic test should substantially change the pre-test probability of disease and influence treatment decisions in order to have clinical utility.
6. Discussion para 3: refs- 8, 10, 17, and 18.these refs pertain to low burden settings see Thorax, 2009 (PMID 19592392) for outcomes pertaining to a high burden setting it would be more relevant here. We appreciate the reviewer s suggestion. We included the suggested reference and modified the sentence as follows: The specificity of T-SPOT.TB for active pulmonary TB has ranged from 45-95%, with studies in both low burden[8, 10, 17, 18] and high burden[22] countries reporting values below 65%. 7. Table 2 should reflect the PPV and NPV too. We did not include PPV and NPV, as these test characteristics are highly dependent on prevalence of disease in the study population. We believe the sensitivity and specificity estimates and likelihood ratios best reflect performance characteristics relevant to other settings and therefore present these in Tables 2 and 3.