Interferon Gamma Release Assays for the Diagnosis of Latent TB Infection in HIV-Infected Individuals in a Low TB Burden Country

<div><h3>Background</h3><p>Interferon gamma release assays (IGRAs) are used to diagnose latent tuberculosis infection. Two IGRAs are commercially available: the Quantiferon TB Gold In Tube (QFT-IT) and the T-SPOT.TB. There is debate as to which test to use in HIV+ individuals. Previous publications from high TB burden countries have raised concerns that the sensitivity of the QFT-IT assay, but not the T-SPOT.TB, may be impaired in HIV+ individuals with low CD4+ T-cell counts. We sought to compare the tests in a low TB burden setting.</p> <h3>Methodology/Principal Findings</h3><p>T-SPOT.TB, QFT-IT, and tuberculin skin tests (TST) were performed in HIV infected individuals. Results were related to patient characteristics. McNemar’s test, multivariate regression and correlation analysis were carried out using SPSS (SPSS Inc). 256 HIV infected patients were enrolled in the study. The median CD4+ T-cell count was 338 cells/µL (range 1–1328). 37 (14%) patients had a CD4+ T-cell count of <100 cells/µL. 46/256 (18% ) of QFT-IT results and 28/256 (11%) of T-SPOT.TB results were positive. 6 (2%) of QFT-IT and 18 (7%) of T-SPOT.TB results were indeterminate. An additional 9 (4%) of T-SPOT.TB results were unavailable as tests were not performed due to insufficient cells or clotting of the sample. We found a statistically significant association between lower CD4+ T-cell count and negative QFT-IT results (OR 1.055, p = 0.03), and indeterminate/unavailable T-SPOT.TB results (OR 1.079, p = 0.02).</p> <h3>Conclusions/Significance</h3><p>In low TB prevalence settings, the QFT-IT yields more positive and fewer indeterminate results than T-SPOT.TB. Negative results on the QFT-IT and indeterminate/unavailable results on the T-SPOT.TB were more common in individuals with low CD4+ T-cell counts.</p> </div

Association between positive T-SPOT.TB test results and patient variables.

<p>Association between positive T-SPOT.TB test results and patient variables.</p

Association between positive QFT test results and patient variables.

<p>Association between positive QFT test results and patient variables.</p

Factors associated with discordant IGRA results.

<p>Multivariate model also included age and gender as independent predictor variables.</p

Baseline characteristics of study population (n = 256).

<p>Baseline characteristics of study population (n = 256).</p

Proteomic Profiling of Serological Responses to <i>Aspergillus fumigatus</i> Antigens in Patients with Invasive Aspergillosis

<i>Aspergillus fumigatus</i> is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the <i>A. fumigatus</i> proteins that trigger the production of <i>Aspergillus</i>-specific IgG antibodies during the course of IA. To characterize the serological response to <i>A. fumigatus</i> protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from <i>A. fumigatus</i>. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after <i>in vitro</i> stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA

Patient demographics.

<p>^a Healthcare-associated infections were defined as (i) index positive blood culture collected ≥48hrs after hospital admission, and no signs or symptoms of the infection noted at time of admission; OR (ii) index positive blood culture collected <48hrs after hospital admission if any of the following criteria are met: received intravenous therapy in an ambulatory setting in the 30 days before onset of BSI, attended a hospital clinic or haemodialysis in the 30 days before onset of BSI, hospitalised in an acute care hospital for ≥ 2 days in the 90 days prior to onset of BSI, resident of nursing home or long-term care facility.</p><p>^b<i>Staphylococcus aureus</i> bacteraemia was defined as uncomplicated if all of the following criteria were met: exclusion of endocarditis; no evidence of metastatic infection; absence of implanted prostheses; follow-up blood cultures at 2–4 days culture-negative for <i>S</i>. <i>aureus</i>; defervescence within 72 h of initiating effective therapy. Percentages shown are of entire <i>S</i>. <i>aureus</i> BSI population.</p><p>^† Three patients had chronic diabetic foot ulcers as a source of their <i>S</i>. <i>aureus</i> BSI, and in all cases the contiguous underlying bone was also found to be infected.</p><p>MRSA = methicillin-resistant <i>Staphylococcus aureus</i>. NA = not applicable. BSI = bloodstream infection.</p><p>Data are displayed as median (interquartile range) and number (percentage). <i>P</i> values are calculated by Mann-Whitney and Fisher’s exact test respectively.</p

Prior exposure to <i>S</i>. <i>aureus</i> increases IFNγ secretion by CD4⁺ and CD8⁺ T cells during subsequent infection.

<p>Groups of mice were exposed to <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via an i.p. injection on d 0, 7 and 14. Prior exposed mice were then re-challenged with an i.p. injection of <i>S</i>. <i>aureus</i> (5x10⁸ CFU) on d 35 alongside a control group of naïve mice. At indicated time points post-challenge the peritoneal cavity was lavaged with PBS to assess IFNγ secretion by ELISA (A). n = 15 per group. At 3 h post challenge peritoneal cells were isolated to assess the proportions of IFNγ-producing CD4⁺ and CD8⁺ T cells using flow cytometry (B). Results expressed as mean ± SEM and representative FACS plots. n = 5 per group. *p<0.05, **p<0.005, ***p<0.001.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection is associated with increased IFNγ production.

<p>Serum from <i>S</i>. <i>aureus</i> and <i>E</i>.<i>coli</i> bloodstream infection patients was collected on day 7 ± 2 post-initial bacteraemia and assessed for IFNγ (A) and IL-17A (B) by ELISA. Results expressed as individual patient values with median indicated by bar, n = 11–24 per group. *p<0.05.</p

Transfer of <i>S</i>. <i>aureus</i> antigen-specific peritoneal Th1 cells protects against subsequent <i>S</i>. <i>aureus</i> infection via enhanced macrophage responses.

<p>Groups of mice received transfers of 5x10⁶<i>S</i>. <i>aureus</i> specific Th1 cells originating from the peritoneal cavity of previously exposed mice via i.p. injection. Another group of mice received a transfer of 5x10⁶ naive splenic CD3⁺ cells as a control. At 3 h post transfer both groups of mice were challenged with <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via i.p. injection. At 72 h post-bacterial challenge the bacterial burden was assessed in the peritoneal cavity, kidneys and spleen (A). Results expressed as log₁₀ CFU/ml with mean indicated by bars. At indicated time points post-bacterial challenge, the peritoneal cavity was lavaged with PBS to assess CXCL1 and CCL5 secretion by ELISA (B,C). Results expressed as mean ± SEM. At indicated time points post-challenge, the absolute numbers of macrophages (F4/80⁺Ly6G^-) were assessed in the peritoneal cavity by flow cytometry (D). MHCII expression by infiltrating macrophages was determined 24 h post infection (E). Absolute numbers of neutrophils (Ly6G⁺CD11b⁺) in the peritoneal cavity were assessed at the indicated time points post-challenge (F). Results expressed as mean ± SEM. n = 5–8 mice per group. Data pooled from 3 independent experiments. *p<0.05, **p<0.005.</p