Alfred Lardizabal

Prospective Comparison of the Tuberculin Skin Test and 2 Whole-Blood Interferon-γ Release Assays in Persons with Suspected Tuberculosis

BACKGROUND Interferon-gamma release assays (IGRAs) are attractive alternatives to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, the inability to definitively confirm the presence of most M. tuberculosis infections hampers assessment of IGRA accuracy. Although IGRAs are primarily indicated for the detection of latent tuberculosis infection, we sought to determine the sensitivity of the TST and 2 whole-blood IGRAs (QuantiFERON-TB assay [QFT] and QuantiFERON-TB Gold assay [QFT-G]) in situations in which infection is confirmed by recovery of M. tuberculosis by culture. METHODS We conducted a prospective, multicenter, cross-sectional comparison study in which 148 persons suspected to have tuberculosis were tested simultaneously with the TST, QFT, and QFT-G. RESULTS M. tuberculosis was cultured from samples from 69 (47%) of 148 persons suspected to have tuberculosis; the TST induration was > or = 5 mm for 51 (73.9%) of the 69 subjects (95% confidence interval [CI], 62.5%-82.8%). The QFT indicated tuberculosis infection for 48 (69.6%) of the 69 subjects (95% CI, 57.9%-79.2%) and was indeterminate for 7 (10.1%). The QFT-G yielded positive results for 46 (66.7%) of the 69 subjects (95% CI, 54.9%-76.7%) and indeterminate results for 9 subjects (13.0%). If subjects with indeterminate QFT-G results were excluded, 46 (76.7%) of 60 subjects (95% CI, 64.6%-85.6%) had positive TST results, and the same number of subjects had positive QFT-G results. HIV infection was associated with false-negative TST results but not with false-negative QFT-G results. CONCLUSIONS The TST, QFT, and QFT-G have similar sensitivity in persons with culture-confirmed infection. As with the TST, negative QFT and QFT-G results should not be used to exclude the diagnosis of tuberculosis in persons with suggestive signs or symptoms.

A Summary of Meeting Proceedings on Addressing Variability around the Cut Point in Serial Interferon-γ Release Assay Testing

On June 13, 2012, a group of key stakeholders, leaders, and national experts on tuberculosis (TB), occupational health, and laboratory science met in Atlanta, Georgia, to focus national discussion on the higher than expected positive results occurring among low-risk, unexposed healthcare workers undergoing serial testing with interferon-γ release assays (IGRAs). The objectives of the meeting were to present the latest clinical and operational research findings on the topic, to discuss evaluation and treatment algorithms that are emerging in the absence of national guidance, and to develop a consensus on the action steps needed to assist programs and physicians in the interpretation of serial testing IGRA results. This report summarizes its proceedings.

Profiling T Cell Activation Using Single-Molecule Fluorescence In Situ Hybridization and Flow Cytometry

Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity–based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single–molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.

Update: prevention and treatment of tuberculosis.

Tuberculosis remains one of the leading infectious disease killers globally. A significant reservoir of infected individuals and disease activity remains, particularly in the countries of the world which have the least economic resources to treat latent and active disease. This has set the stage, over the decades, for the development of multi-drug resistant isolates now easily spread around the world with increased trans-national migration.Tuberculosis treatment and control requires a global approach with international organizations such as the World Health Organization (WHO), and the International Union Against Tuberculosis and Lung Disease (IUATLD) overseeing National Tuberculosis Control Programs and seeking financial support from governmental as well as non-governmental sources. Directly Observed Therapy Short Course (DOTS) and DOTS Plus remain the cornerstones in the treatment of drug sensitive and drug resistant diseases respectively.Research activity should focus in developing early diagnostic tools and drugs which would be able to effectively treat tuberculosis cases for shorter periods of time in the new millennium.

Diagnosis of Latent Tuberculosis Infection

For 2015, tuberculosis (TB) incidence in the United States has plateaued at 3.0 per 100,000. This remains the lowest case rate since recording started. On the global level, although the TB epidemic is larger than previously estimated, TB deaths and incidence rate continue to fall. For both low and high incidence countries, accelerating the decline in TB incidence towards elimination goals requires that more emphasis be placed on strengthening systems for detection and treatment of latent TB infection (LTBI) in addition to improving TB care globally. Here, we review the tuberculin skin test and gamma interferon release assays currently available for the detection of LTBI.

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1–coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM.

Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status.

RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. Moreover, NK cells contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry. Multi-scale, single-cell analyses may satisfy the need to determine disease stage and therapy response for tuberculosis and other infectious pathologies.

Drug-resistant tuberculosis: how are we doing?

IN 1972, Annik Rouillon pointed out that ‘to default is the natural reaction of normal, sensible people. The person who continues to swallow drugs or have injections with complete regularity in the absence of encouragement and help from others is the abnormal one.’1 Six years later, Grzybowski and Enarson compared poor treatment with no treatment and found, presciently and paradoxically, that poor treatment is worse than no treatment.2 The 5-year survival rate in their no treatment group, while perhaps infl uenced by different times, different nutrition, different support, however, can still, however, serve as a surrogate control group to compare to current treatment regimens in gauging effi cacy. That comparison with treatment for multidrug-resistant tuberculosis (MDR-TB) is not good news! In this issue of the Journal, Chiang et al. compare the Grzybowski no treatment data2 with average current outcomes of MDR-TB treatment in World Health Organization (WHO) Global Tuberculosis Control Reports, and fi nd the overall proportion of treatment success was 48%—recognizing, of course, that some excellent MDR treatment results do exist.3 But in many countries these results are still alarmingly close to the no treatment patients reported by Grzybowski. Chiang et al. put the blame for these dismal results squarely on the length of the WHO-recommended regimen (total duration at least 20 months),3 and point out that in some studies 20–50% of MDR patients are severely challenged by adverse drug effects, countering Rouillon’s 1972 admonition about treatment support being key.1 The solution, according to Chiang et al., is to shorten the regimen, as has been done with the ‘Bangladesh Regimen’, in appropriate patients to a total duration of 9 months. Results have been excellent (87.9% and 86.1% treatment success), and reasonable even in patients with quinolone resistance.3 These regimens are promising enough in appropriate patients that a new study pitting the short regimen against the WHO-endorsed longer course regimen is under way, with results due in 2016 (Stream Study). Chiang et al. argue, however, that we cannot wait for this confi rmation, and that we need to urgently adopt a short regimen in appropriate patients under proper conditions. We strongly agree with them. Furthermore, with a simple algorithm utilizing Xpert® MTB/RIF and the short MDR regimen, the WHO has endorsed its use in projects that adhere to approval by a national ethics board; delivery of treatment under operational research conditions in accordance with international standards; and monitoring of the program by an independent monitoring board.3 The TB community has often been slow to react to developments, citing ‘the need for more data’, but events sometimes drive reaction. The short regimen requires a critical component, the fl uoroquinolone. But, as opposed to a time when the TB community exclusively had its own drugs, with other disciplines afraid to use them, we now share several drug classes, and therefore drug resistance, as in quinolones, which is increasing in several countries. Chiang et al. essentially point out that the train has left the station with an effective, short regimen for MDR-TB. It is past time to get it into appropriate patients, and, hopefully, make MDR-TB management more effective.

Tuberculosis State Is Associated with Expression of Toll-Like Receptor 2 in Sputum Macrophages

Mycobacterium tuberculosis is an intracellular pathogen that parasitizes the host macrophage. While approximately two billion people are infected worldwide, only 5 to 10% become diseased with pulmonary tuberculosis, at least in the absence of comorbidities. Tuberculosis control requires development of noninvasive methods probing the host immune status to help distinguish latent infection from active tuberculosis. With such methods, high-risk individuals could be targeted for treatment before disease manifestation. Previous investigations have been based on examination of peripheral blood cells or, more rarely, lung macrophages obtained with invasive procedures, such as bronchoalveolar lavages. Here we show that differences exist in the expression of a surface protein (Toll-like receptor 2) between macrophages recovered from the sputum of individuals in different diagnostic groups: i.e., infection free, latent tuberculosis infection, and active pulmonary tuberculosis. Thus, phenotypic analysis of local macrophages obtained with noninvasive procedures can help distinguish among tuberculosis infection stages. ABSTRACT During tuberculosis, macrophages are critical for both pathogen survival and host immune activation. Since expression of particular cell surface markers reflects cell function, we used flow cytometry to measure the abundance of surface markers associated with polarity, lipid uptake, or pattern recognition on macrophages found in induced sputum. Nine macrophage surface markers were examined from three groups of donors: infection-free, latent tuberculosis infection, and active pulmonary tuberculosis. Using a trend test, we found that expression of Toll-like receptor 2 was greater from absence of infection to latent infection and from latent infection to active tuberculosis. The results point to the possibility that innate immune cell phenotypes be used to distinguish among tuberculosis infection stages. Moreover, this study shows that readily accessible sputum macrophages have potential for tuberculosis diagnosis and prognosis. IMPORTANCE Mycobacterium tuberculosis is an intracellular pathogen that parasitizes the host macrophage. While approximately two billion people are infected worldwide, only 5 to 10% become diseased with pulmonary tuberculosis, at least in the absence of comorbidities. Tuberculosis control requires development of noninvasive methods probing the host immune status to help distinguish latent infection from active tuberculosis. With such methods, high-risk individuals could be targeted for treatment before disease manifestation. Previous investigations have been based on examination of peripheral blood cells or, more rarely, lung macrophages obtained with invasive procedures, such as bronchoalveolar lavages. Here we show that differences exist in the expression of a surface protein (Toll-like receptor 2) between macrophages recovered from the sputum of individuals in different diagnostic groups: i.e., infection free, latent tuberculosis infection, and active pulmonary tuberculosis. Thus, phenotypic analysis of local macrophages obtained with noninvasive procedures can help distinguish among tuberculosis infection stages.