Morten Ruhwald

Interferon-γ release assays for the diagnosis of latent Mycobacterium tuberculosis infection: a systematic review and meta-analysis

We conducted a systematic review and meta-analysis to compare the accuracy of the QuantiFERON-TB® Gold In-Tube (QFT-G-IT) and the T-SPOT®.TB assays with the tuberculin skin test (TST) for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). The Medline, Embase and Cochrane databases were explored for relevant articles in November 2009. Specificities, and negative (NPV) and positive (PPV) predictive values of interferon-&ggr; release assays (IGRAs) and the TST, and the exposure gradient influences on test results among bacille Calmette–Guérin (BCG) vaccinees were evaluated. Specificity of IGRAs varied 98–100%. In immunocompetent adults, NPV for progression to tuberculosis within 2 yrs were 97.8% for T-SPOT®.TB and 99.8% for QFT-G-IT. When test performance of an immunodiagnostic test was not restricted to prior positivity of another test, progression rates to tuberculosis among IGRA-positive individuals followed for 19–24 months varied 8–15%, exceeding those reported for the TST (2–3%). In multivariate analyses, the odd ratios for TST positivity following BCG vaccination varied 3–25, whereas IGRA results remained uninfluenced and IGRA positivity was clearly associated with exposure to contagious tuberculosis cases. IGRAs may have a relative advantage over the TST in detecting LTBI and allow the exclusion of M. tuberculosis infection with higher reliability.

Towards tuberculosis elimination: an action framework for low-incidence countries

This paper describes an action framework for countries with low tuberculosis (TB) incidence (<100 TB cases per million population) that are striving for TB elimination. The framework sets out priority interventions required for these countries to progress first towards “pre-elimination” (<10 cases per million) and eventually the elimination of TB as a public health problem (less than one case per million). TB epidemiology in most low-incidence countries is characterised by a low rate of transmission in the general population, occasional outbreaks, a majority of TB cases generated from progression of latent TB infection (LTBI) rather than local transmission, concentration to certain vulnerable and hard-to-reach risk groups, and challenges posed by cross-border migration. Common health system challenges are that political commitment, funding, clinical expertise and general awareness of TB diminishes as TB incidence falls. The framework presents a tailored response to these challenges, grouped into eight priority action areas: 1) ensure political commitment, funding and stewardship for planning and essential services; 2) address the most vulnerable and hard-to-reach groups; 3) address special needs of migrants and cross-border issues; 4) undertake screening for active TB and LTBI in TB contacts and selected high-risk groups, and provide appropriate treatment; 5) optimise the prevention and care of drug-resistant TB; 6) ensure continued surveillance, programme monitoring and evaluation and case-based data management; 7) invest in research and new tools; and 8) support global TB prevention, care and control. The overall approach needs to be multisectorial, focusing on equitable access to high-quality diagnosis and care, and on addressing the social determinants of TB. Because of increasing globalisation and population mobility, the response needs to have both national and global dimensions. Action framework for countries with low tuberculosis incidence: a coherent approach for eliminating tuberculosis http://ow.ly/H03ZZ

Latent Tuberculosis in HIV positive, diagnosed by the M. Tuberculosis Specific Interferon-γ test

BackgroundAlthough tuberculosis (TB) is a minor problem in Denmark, severe and complicated cases occur in HIV positive. Since the new M. tuberculosis specific test for latent TB, the QuantiFERON-TB In-Tube test (QFT-IT) became available the patients in our clinic have been screened for the presence of latent TB using the QFT-IT test. We here report the results from the first patients screened.MethodsOn a routine basis the QFT-IT test was performed and the results from 590 HIV positive individuals consecutively tested are presented here. CD4 cell count and TB risk-factors were recorded from patient files.Main findings27/590(4.6%) of the individuals were QFT-IT test positive, indicating the presence of latent TB infection. Among QFT-IT positive patients, 78% had risk factors such as long-term residency in a TB high endemic area (OR:5.7), known TB exposure (OR:4.9) or previous TB disease (OR:4.9). The prevalence of latent TB in these groups were 13%, 16% and 19% respectively. There was a strong correlation between low CD4 T-cell count and a low mitogen response (P < 0.001;Spearman) and more patients with low CD4 cell count had indeterminate results.ConclusionWe found an overall prevalence of latent TB infection of 4.6% among the HIV positive individuals and a much higher prevalence of latent infection among those with a history of exposure (16%) and long term residency in a high endemic country (13%). The QFT-IT test may indeed be a useful test for HIV positive individuals, but in severely immunocompromised, the test may be impaired by T-cell anergy.

Improving T-Cell Assays for the Diagnosis of Latent TB Infection: Potential of a Diagnostic Test Based on IP-10

Background There is a need for simple tools such as the M.tuberculosis specific IFN-γ release assays (IGRA) to improve diagnosis of M.tuberculosis-infection in children. The aim of the study was to evaluate the performance of an IP-10 and IL-2 based tests for the diagnosis of M.tuberculosis-infection in recently exposed children from Nigeria. Methodology and Principal Findings Samples were obtained from 59 children at high risk of infection with M.tuberculosis (contacts of adults with smear and culture-positive tuberculosis) and 61 at low risk (contacts of smear-negative/culture-positive tuberculosis or community controls). IP-10 and IL-2 was measured in plasma after stimulation of whole-blood with M.tuberculosis specific antigens and mitogen. Previously developed criteria for positive IP-10 and IL-2 tests were used and the diagnostic performances of the IP-10 and IL-2 tests were compared with the Quantiferon In-Tube (QFT-IT) and the Tuberculin Skin Tests (TST). In response to M.tuberculosis specific antigens, the high-risk children expressed significantly higher levels of IP-10 (1358 pg/ml[IQR 278–2535 pg/ml]) and IL-2 (164 pg/ml[11–590 pg/ml]) than low risk groups 149 pg/ml(25–497 pg/ml), and 0 pg/ml(0–3 pg/ml), respectively. There was excellent agreement (>89%,k>0.80) between IP-10, IL-2 tests and QFT-IT, better than with TST (>74%,k>0.49). The IP-10 and IL-2 responses were strongly associated with M.tuberculosis exposure and with grade of infectiousness of the index cases (p<0.0001). IP-10, IL-2, and TST but not QFT-IT was associated with age of the child in the low risk groups (p<0.02). Conclusions/Significance IP-10 is expressed in high levels and results of the IP-10 test were comparable to the QFT-IT. IL-2 was released in low amounts in response to the antigens and not in response to the mitogen therefore IL-2 seems a less useful marker. We have demonstrated that IP-10 and possibly IL-2 could be alternative or adjunct markers to IFN-γ in the diagnosis infection with M.tuberculosis.

Prednisolone treatment affects the performance of the QuantiFERON gold in-tube test and the tuberculin skin test in patients with autoimmune disorders screened for latent tuberculosis infection†‡

Background: During screening for latent tuberculosis infection (LTBI), before anti‐tumor‐necrosis‐factor‐&agr; treatment, most patients are already receiving immunosuppressive therapy. The objective was to evaluate the performance of the QuantiFERON Gold In‐Tube (QFT‐IT) and the Tuberculin Skin Test (TST). Methods: A prospective multicenter study included 248 patients with ulcerative colitis (39), Crohns disease (54), rheumatoid arthritis (111), and spondylo‐arthropathy (44). Results: QFT‐IT was positive in 7/248 (3%), negative in 229 (92%), and indeterminate in 12 (5%). TST was positive in 54/238 (23%) patients. Chest x‐ray was suspect for tuberculosis in 5/236 (2%), and 35/167 (21%) had ≥1 risk‐factors for infection with Mycobacterium tuberculosis. The main finding was a pronounced negative effect on QFT‐IT and TST performance associated with prednisolone treatment. During prednisolone treatment interferon gamma (IFN‐&ggr;) response to mitogen stimulation was impaired (median IFN‐&ggr; response 4.9 IU/mL; interquartile range [IQR] 0.8 to ≥10.0) compared to patients 1) not receiving corticosteroids (median ≥10.0; IQR 5.0 to ≥10.0; P = 0.0015) or 2) receiving long‐acting corticosteroids (median >10.0; IQR 9.7 to >10.0; P = 0.0058). Prednisolone treatment was strongly associated with negative TST, adjusted odds ratio (AOR) 0.22 (0.1–0.8; P = 0.018), and with an increased risk of indeterminate QFT‐IT results AOR 16.1 (4.1–63.2; P < 0.001), whereas no negative effect was found for long‐acting corticosteroids. Doses of ≥10 mg prednisolone were associated with a 27% risk of indeterminate results. Single use of azathioprine, methotrexate, or 5‐aminosalicylate (5‐ASA) did not affect the test results. Conclusions: Oral prednisolone severely suppressed QFT‐IT and TST performance, whereas the long‐acting corticosteroids methotrexate, azathioprine, and 5‐ASA did not have a similar detrimental effect. Patients should be screened for LTBI with QFT‐IT or TST prior to initiation of prednisolone therapy and negative QFT‐IT or TST results interpreted with caution in patients treated with any corticosteroid until further data are available. (Inflamm Bowel Dis 2011;)

IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as biomarkers for infection with M. tuberculosis in a whole blood based T-cell assay

BackgroundIFN-γ responses to M. tuberculosis antigens are used as in-vitro diagnostic tests for tuberculosis infection. The tests are highly specific but sensitivity may be impaired due to immuno-suppression. The objective of this small exploratory study was to compare three novel biomarkers for in-vitro diagnosis of tuberculosis – MCP-1, MCP-3 and IL-1RA – with the current established biomarker IFN-γ and the newly described IP-10 and MCP-2.MethodsWhole blood from 8 patents with active tuberculosis and from 7 healthy controls was stimulated with M. tuberculosis specific antigens and mitogen in the Quantiferon In Tube test tubes. Levels of biomarkers were measured using Luminex and ELISA (IFN-γ).ResultsWe found all five new biomarkers were expressed in significantly higher concentrations compared to IFN-γ. IP-10 and MCP-3 levels in the un-stimulated samples were higher in patients compared with controls.ConclusionAll biomarkers had diagnostic potential as they could differentiate between the patients and the controls. IP-10 and MCP-2 seemed most promising as they were expressed in high levels with antigen stimulation and were low in the un-stimulated samples. Further studies are needed to explore the potential of these highly expressed novel biomarkers individually and in combination.

A multicentre evaluation of the accuracy and performance of IP-10 for the diagnosis of infection with M. tuberculosis

IP-10 has potential as a diagnostic marker for infection with Mycobacterium tuberculosis, with comparable accuracy to QuantiFERON-TB Gold In-Tube test (QFT-IT). The aims were to assess the sensitivity and specificity of IP-10, and to evaluate the impact of co-morbidity on IP-10 and QFT-IT. 168 cases with active TB, 101 healthy controls and 175 non-TB patients were included. IP-10 and IFN-γ were measured in plasma of QFT-IT stimulated whole blood and analyzed using previously determined algorithms. A subgroup of 48 patients and 70 healthy controls was tested in parallel with T-SPOT.TB IP-10 and QFT-IT had comparable accuracy. Sensitivity was 81% and 84% with a specificity of 97% and 100%, respectively. Combining IP-10 and QFT-IT improved sensitivity to 87% (p < 0.0005), with a specificity of 97%. T-SPOT.TB was more sensitive than QFT-IT, but not IP-10. Among non-TB patients IP-10 had a higher rate of positive responders (35% vs 27%, p < 0.02) and for both tests a positive response was associated with relevant risk factors. IFN-γ but not IP-10 responses to mitogen stimulation were reduced in patients with TB and non-TB infection. This study confirms and validates previous findings and adds substance to IP-10 as a novel diagnostic marker for infection with M. tuberculosis. IP-10 appeared less influenced by infections other than TB; further studies are needed to test the clinical impact of these findings.

IP-10 release assays in the diagnosis of tuberculosis infection: current status and future directions

The current state-of-the-art tests for infection with Mycobacterium tuberculosis – the IFN-γ release assays – rely on accurate measurement of the cytokine IFN-γ. Many other potential biomarkers are expressed in concert with IFN-γ, and IP-10 in particular has shown promising results. IP-10 is produced in large amounts, allowing for the development of new and simplified test platforms, such as lateral flow. In this review, we summarize the results of 22 clinical studies exploring the use of IP-10 as an alternative marker to IFN-γ. The studies report that diagnostic accuracy of IP-10 is on par with IFN-γ, but also that IP-10 may be more robust in young children and in HIV-infected individuals with low CD4 cell counts. We conclude the review by presenting limitations of the published works and outline recent developments and future directions.

Point-of-care diagnosis of tuberculosis: past, present and future

Diagnosis represents only one aspect of tuberculosis (TB) control but is perhaps one of the most challenging. The drawbacks of current tools highlight several unmet needs in TB diagnosis, that is, necessity for accuracy, rapidity of diagnosis, affordability, simplicity and the ability to generate same‐day results at point‐of‐care (POC). When a return visit is required to access test results, time to treatment is prolonged, and default rates are significant. However, a good diagnostic tool is also critically dependent on obtaining an adequate biological sample. Here, we review the accuracy and potential impact of established and newer potential POC diagnostic tests for TB, including smear microscopy, the Xpert MTB/RIF assay (Cepheid) and the Determine TB lipoarabinomannan antigen test (Alere). Novel experimental approaches and detection technologies for POC diagnosis of active TB, including nucleic acid amplification tests, detection of volatile organic compounds or metabolites, mass spectroscopy, microfluidics, surface‐enhanced Raman spectroscopy, electrochemical approaches, and aptamers among others, are discussed. We also discuss future applications, including the potential POC diagnosis of drug‐resistant TB and presumed latent TB infection. Challenges to the development and roll‐out of POC tests for TB are also reviewed.

A novel liposomal adjuvant system, CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.