Risk of tuberculosis is increased in Behçet’s disease compared to other rheumatological disorders after anti-TNFα treatments: a case series and review of the literature

Background/aimTumor necrosis factor-alfa (TNF-a) antagonists are extensively utilized in the treatment of inflammatory rheumatic diseases and also shown to be effective in Behçet’s disease (BD) patients with major organ involvement. In this study, we aimed to re-evaluate the incidence of tuberculosis (TB) infection after anti-TNFa treatments and to reveal the risk of TB in BD.MethodsData of patients who received anti-TNFa treatment between 2005 and 2018 were assessed retrospectively. Demographic features, TNF-a antagonist type/treatment time, tuberculosis skin test (TST) and QuantiFERON results, isoniazid prophylaxis status, and concomitant corticosteroid (CS) treatments were collected. ResultsA total of 1277 (male/female = 597/680; median age = 49 years) patients were treated with TNF-a antagonist for a median of 33 months (Q1:12, Q3:62). Thirteen (1%) patients developed TB during the follow-up period. Within 13 TB-positive patients, 7 of them had pulmonary, and 7 had extrapulmonary TB. Although, the median time of (month) TNF-a antagonist treatment was higher in TB-positive patients than negative ones, the difference was not statistically significant (48 and 33 months, respectively, p = 0.47). Similarly, TB-positive patients were treated with CSs more than TB-negative patients (80% vs. 60%). Time from the initiation of TNF-a antagonist treatment to the diagnosis of TB had a median of 40 months (Q1-Q3: 22-56). There was a statistically significant increase of TB development in BD patients than non-BD patients after TNF-a antagonists (7.5% vs. 0.8%, respectively, p = 0.007). When we combined our patients with the other series from Turkey, among 12928 patients who received TNF-a antagonists, TB was positive in 12 (3.9%) of 305 BD patients compared to 112 (0.9%) of 12623 non-BD patients (p < 0.00001).ConclusionOur results suggest a higher frequency of TB infections in BD patients with TNF-a antagonists. As biologic agents are increasingly used for major organ involvement in current practice for BD, screening mechanisms should be carefully implemented.     

A T-cell-based enzyme-linked immunospot assay for tuberculosis screening in Chinese patients with rheumatic diseases receiving infliximab therapy

Anti-tumour necrosis factor-α (TNF-α) therapy brought new hopes for treating rheumatic diseases but also increased the risk of infection, including mycobacterium tuberculosis (MTb). Conventional screening tools, such as tuberculin skin test (TST), lack sensitivity or specificity. Recently, T-SPOT.TB has been introduced to detect tuberculosis infection. Reports have proved its superior performance in detecting tuberculosis infection in various patient populations than the TST. To compare the value of a T-cell-based enzyme-linked immunospot assay (ELISPOT) T-SPOT.TB and conventional (TST) in screening and monitoring tuberculosis in patients with rheumatic diseases during infliximab therapy in China. Fifty-eight patients with various rheumatic diseases who received infliximab therapy were enrolled in the trial. Freshly isolated peripheral blood mononuclear cells were stimulated with MTb-specific antigens (ESAT-6 and CFP10), and IFN-γ-producing cells were counted. TST was performed with 1 TU PPD injected intradermally into the volar aspect of forearm. A cutaneous induration with diameter ≥5 mm was considered as positive TST, and an increment ≥5 mm of cutaneous induration was considered as TST conversion. TST and T-SPOT.TB test were carried out at baseline and repeated 12 months after infliximab therapy (if no active TB occurs) or at times when TB occurred. Moreover, all patients were initially evaluated for latent tuberculosis infection (LTBI) with clinical examination and chest radiograph. The McNemar test was used for TST and T-SPOT.TB concordance analysis. Cohen’s kappa coefficient was used to assess strength of the agreement. Among the 58 patients evaluated, 25 (43.1%) had ankylosing spondylitis, 24 (41.4%) had rheumatoid arthritis, 4 (6.9%) had undifferentiated spondyloarthropathy, 3 (5.2%) had psoriatic arthritis and 2 (3.4%) had reactive arthritis. A total of 52 patients (89.7%) had previously received vaccination with Bacille Calmette-Guerin. All of the patients received either single or combination of disease modifying anti-rheumatic drug (DMARDs) therapy, and 16 (27.4%) had previously or presently received glucocorticoid therapy. Before infliximab therapy, 12 patients (20.7%) had positive and 46 (79.3%) had negative TST results, and only 1 (1.7%) had positive T-SPOT.TB. Among 51 patients completing the repeated TST and T-SPOT.TB assay, 7 patients (13.7%) had TST conversion and 4 (7.8%) had positive T-SPOT.TB results. Of 7 patients with TST conversion, 2 patients (28.6%) developed active TB and also had positive T-SPOT.TB results; of 44 patients with no TST conversion, 2 patients (4.5%) had positive T-SPOT.TB and 1 (2.3%) had active TB. If 5 mm was used as the cut-off value of TST, TST and T-SPOT.TB, had an agreement value of 68.6% with a kappa value of 0.166. If 10 mm was used as the cut-off value, the agreement between TST and T-SPOT.TB was 88.2% with a kappa value of 0.338. T-SPOT.TB was more specific than TST in detecting tuberculosis during infliximab therapy in China with high BCG vaccination and high prevalence of TB. It can be used as a reliable tool for TB monitoring during infliximab therapy in Chinese patients with rheumatic diseases. Finally, it is recommended to repeat the TST and T-SPOT.TB periodically during biological treatment.     

Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCG-vaccinated populations

T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.     

Evaluation of the Characteristics of the Enzyme-Linked Immunospot Assay for Diagnosis of Active Tuberculosis in China

The purpose of this study was to evaluate the characteristics of the T-SPOT.TB test for the diagnosis of active tuberculosis (ATB) and to distinguish ATB from other diseases using a receiver operating characteristic (ROC) curve. A total of 535 patients with suspected active tuberculosis were enrolled in the study and divided into ATB and nonactive tuberculosis (NATB) groups, as well as pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) subgroups. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB test for the diagnosis of ATB were 84.95%, 85.12%, 82.94%, 86.93%, 5.71, and 0.18, respectively. The median number of spot-forming cells (SFCs) in the ATB group was higher than that in the NATB group (71 versus 1; P < 0.0001). The sensitivities in the PTB and EPTB subgroups were 92.31% and 81.77%. The areas under the curve (AUC) for the diagnosis of ATB using the T-SPOT.TB, early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) were 0.906, 0.884, and 0.877, respectively. A cutoff of 42.5 SFCs for ATB yielded a positive predictive value of 100%. Our study shows that the T-SPOT.TB test is useful for the diagnosis of ATB. Utilizing an ROC curve to select an appropriate cutoff made it possible to discriminate ATB from NATB.     

Monitoring of Peptide-Specific and Gamma Interferon-Productive T Cells in Patients with Active and Convalescent Tuberculosis Using an Enzyme-Linked Immunosorbent Spot Assay

To establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P > 0.05), but the median number of spot-forming cells/10⁶ cells (SFP value) for positive results was higher by the former than by the latter assay (P < 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P < 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.     

Comparison of two gamma interferon release assays and tuberculin skin testing for tuberculosis screening in a cohort of patients with rheumatic diseases starting anti-tumor necrosis factor therapy

Gamma interferon release assays (IGRAs) are increasingly used for latent Mycobacterium tuberculosis infection (LTBI) screening in patients with rheumatic diseases starting anti-tumor necrosis factor (anti-TNF) therapies. We compared the performances of two IGRAs, an enzyme-linked immunospot release assay (T-SPOT.TB) and an enzyme-linked immunosorbent assay (QuantiFERON-TB Gold In Tube [QFT-GIT]), to that of tuberculin skin testing (TST) for LTBI screening of 157 consecutive rheumatic patients starting anti-TNF therapies. Among 155 patients with valid results, 58 (37%) were positive by TST, 39 (25%) by T-SPOT.TB assay, and 32 (21%) by QFT-GIT assay. IGRAs were associated more strongly with at least one risk factor for tuberculosis (TB) than TST. Risk factors for a positive assay included chest X-ray findings of old TB (TST), advanced age (both IGRAs), origin from a country with a high TB prevalence, and a positive TST (T-SPOT.TB assay). Steroid use was negatively associated with a positive QFT-GIT assay. The agreement rate between IGRAs was 81% (kappa rate = 0.47), which was much higher than that observed between an IGRA and TST. If positivity by either TST or an IGRA was required for LTBI diagnosis, then the rate of LTBI would have been 46 to 47%, while if an IGRA was performed only for TST-positive patients, the respective rate would have been 11 to 17%. In conclusion, IGRAs appear to correlate better with TB risk than TST and should be included in TB screening of patients starting anti-TNF therapies. In view of the high risk of TB in these patients, a combination of one IGRA and TST is probably more appropriate for LTBI diagnosis.     

Characterization of Mycobacterium tuberculosis isolated from cancer patients with suspected tuberculosis infection in Egypt: identification,prevalence, risk factors and resistance pattern

Data are sparse on Mycobacterium tuberculosis infection among patients with cancer in Egypt. We sought to detect the presence of tuberculosis (TB) disease among patients with malignant conditions and suspected TB and to study the main risk factors. Also, we compared different diagnostic procedures and detected the antimicrobial susceptibility of M. tuberculosis isolates against rifampin and isoniazid. One hundred patients were included in this study, all of them had malignant conditions and were suspected by the clinicians of having TB. Identification of M. tuberculosis in different specimens was performed by smear microscopy, followed by Lowenstein–Jensen medium and Mycobacterium growth indicator tube (MGIT) cultures and artus^® real-time PCR. In addition, an indirect MGIT anti-TB susceptibility test was carried out against rifampin and isoniazid. A total of 76% of studied cases were found to be TB positive. The frequencies of TB-positive cases in the bronchogenic, haematological and solid tumour malignancy groups were 21%, 25% and 30%, respectively. Significant differences between pulmonary and extrapulmonary TB in different malignancy groups were recorded. Real-time PCR showed the highest overall diagnostic efficiency. Multidrug-resistance of M. tuberculosis to both rifampin and isoniazid was detected in 28.6% of examined isolates. Infection in cancer patients with TB was significantly more often recorded among elderly patients and those suffering from poverty. Pulmonary TB is more common than extrapulmonary TB in patients with malignancy. Real-time PCR is the most accurate and rapid method for TB diagnosis. MGIT-rifampin resistance may be used as a reliable marker for detection of multidrug-resistant TB. Diagnosis and instituting treatment course for active or latent TB infection are crucial before starting anticancer therapy.     

Frequency,Risk Factors,and Outcome of Active Tuberculosis following Allogeneic Hematopoietic Stem Cell Transplantation

We aimed to investigate the frequency, risk factors, and outcome of active tuberculosis (TB) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This retrospective, nested, case-control study reviewed data from 6236 patients who received allo-HSCT from January 2008 to December 2018 at a single center; thirty-three patients (0.5%) with active TB and 99 controls without active TB after allo-HSCT were identified. We performed propensity score matching by randomly selecting 3 controls for each identified active TB patient according to the time of transplantation and follow-up period. History of pretransplant active TB previously treated and inactive at time of transplantation (P< .001) was an independent risk factor. No significant differences in overall survival (P= .342), nonrelapse mortality (P= .497), or incidence of relapse (P= .807) were found. Thirty (90.9%) patients were treated with 4-drug (isoniazid, rifampicin/three rifapentine, pyrazinamide, and ethambutol) or 3-drug combination first-line therapy, with a response rate of 76.7%. Twenty-six (78.8%) patients were treated with first-line and second-line combined therapy, and the response rate was 76.9%. Five (15.2%) patients developed hepatotoxicity. In conclusion, history of pretransplant active TB previously treated and inactive at time of transplantation was an independent risk factor of active TB after allo-HSCT. No significant differences in prognosis between the TB and control groups were found. More studies are needed to help develop standardized therapeutic strategies for patients with post-transplant TB.     

Diagnosis of Active Tuberculosis in China Using an In-House Gamma Interferon Enzyme-Linked Immunospot Assay

Gamma interferon (IFN-γ) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-γ enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-γ responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-γ ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure (r = −0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-γ ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.Tuberculosis (TB) is a leading cause of morbidity and mortality throughout the world, with 95% of cases and 97% of all deaths occurring in high-prevalence countries, such as China, where the prevalence of active TB is as high as 367/100,000 population (10). For the effective and efficient control of TB in these countries, rapid diagnosis and treatment for active-TB patients are the mainstays of the TB control program. However, the current widely used tests, including acid-fast staining of sputum, mycobacterial culture, and antibody test, are not satisfactory for this purpose (4).Recently, commercial immunodiagnostic tests for TB infection have been introduced. These tests are based on the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) and include a whole-blood gamma interferon (IFN-γ) enzyme-linked immunosorbent assay (QuantiFERON-TB Gold in-tube [QFT-IT]; Cellestis Ltd, Victoria, Australia) and an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxfordshire, United Kingdom). Both tests have shown promising results in the detection of latent TB infection (LTBI) (1, 11). In addition, some clinical data also suggest the potential to use these IFN-γ assays for the differential diagnoses of active tuberculosis (3, 8, 9). However, the sensitivities and specificities of these assays varied among the different populations studied, due mostly to the different HLA genetic backgrounds, the prevalence of TB infection, and the coverage of Mycobacterium bovis BCG vaccination (11).In contrast to the wide use of IFN-γ assays for the diagnosis of M. tuberculosis infection in Europe and America, the utilization of IFN-γ assays in China is scarce, and no data are available to evaluate the diagnostic value of the QFT-IT assay mainly because of the high cost of these kits. Since China ranks second on the list of 22 countries with the highest tuberculosis burden in the world (11), the aim of this study was to evaluate the usefulness of IFN-γ assays in the diagnosis of active tuberculosis in the Chinese population in mainland China. Thus, we developed and evaluated an in-house IFN-γ ELISPOT assay by using recombinant ESAT-6 protein and peptide pools derived from ESAT-6 and CFP-10 for diagnosis of active TB. We also compared the performance of our ELISPOT assay with that of the commercial QFT-IT assay and analyzed the influence of clinical manifestation on the accuracy of the ELISPOT assay.     

Western Blotting Is an Efficient Tool for Differential Diagnosis of Paracoccidioidomycosis and Pulmonary Tuberculosis

Sputum and sera from 134 patients screened for tuberculosis (TB) were analyzed to investigate TB and paracoccidioidomycosis (PCM). Of these patients, 11 (8.2%) were confirmed to have TB, but six (4.5%) were positive only for PCM. All patients with PCM presented anti-43-kDa-component antibodies in Western blotting (WB) assays, while in the TB-positive patients these antibodies did not appear. This preliminary study suggests WB as a potential tool for differential laboratory diagnosis between TB and PCM.     

Diminished Adherence and/or Ingestion of Virulent Mycobacterium tuberculosis by Monocyte-Derived Macrophages from Patients with Tuberculosis

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [³H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and ³H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.     

Immunoglobulin A (IgA) and IgG Immune Responses against P-90 Antigen for Diagnosis of Pulmonary Tuberculosis and Screening for Mycobacterium tuberculosis Infection

The purpose of the present study was to evaluate the usefulness of detection of serum immunoglobulin A (IgA) and IgG antibodies directed against the mycobacterial P-90 antigen for the diagnosis of active pulmonary tuberculosis (PTB) among symptomatic individuals and for the detection of Mycobacterium tuberculosis infections among close contacts of PTB patients. Two commercially available enzyme immunoassay (EIA) kits (IgA EIA-TB [EIA-IgA] and IgG EIA-TB [EIA-IgG]; Kreatech Diagnostics) were evaluated in a blinded fashion by using stored serum samples from 268 individuals, including 69 patients with PTB, 41 patients with diseases other than tuberculosis (TB), 12 subjects with healed PTB, 39 close contacts of PTB patients, and 107 healthy volunteers. For the EIA-IgA, the sensitivity was 74% and the specificity was 68% when a cutoff determined by a receiver operator characteristic curve was used. For the EIA-IgG, the sensitivity was 69% and the specificity was 64%. The EIA-IgA was positive for 54% of healthy close contacts of PTB patients but only 8% of healthy controls without contact with a PTB patient or a prior personal history of TB (P < 0.001). The relatively low sensitivities and specificities of these serologic tests make them poor tools for the diagnosis of PTB among patients with suspected PTB. However, the relatively high prevalence of positive EIA-IgA results among healthy close contacts of PTB patients warrants further evaluation of this test with close contacts and other populations at risk for recent M. tuberculosis exposure and development of disease.     

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.     

Use of Antibodies in Lymphocyte Secretions for Detection of Subclinical Tuberculosis Infection in Asymptomatic Contacts

We have previously demonstrated that Mycobacterium bovis BCG-specific immunoglobulin G antibodies in lymphocyte secretions (ALS) can be employed as a marker for active tuberculosis (TB). We aimed to determine whether the ALS method allows detection of subclinical TB infection in asymptomatic individuals. A prospective study of family contacts (FCs) of patients with active TB and healthy controls was performed. Thirteen of 42 FCs had high ALS responses, including 6 FCs who subsequently developed active TB. No correlation was observed between the tuberculin skin test and the ALS responses in the FCs (r = 0.1, P = 0.23). Among patients with active TB, BCG-specific ALS responses steadily declined from the time of diagnosis through 6 months following antimycobacterial chemotherapy (P = 0.001). The ALS assay enabled detection of infection in exposed symptom-free contacts, who are at greater risk for developing active TB. The method may also allow discrimination between effective treatment of active infection and suboptimal response to therapy.     

Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of Mycobacterium bovis BCG in Serodiagnosis of Tuberculosis

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of ≥1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.     

Acceptability of Sputum Specimens for Diagnosing Pulmonary Tuberculosis

The evaluation of the quality of a sputum specimen prior to bacterial culture has been an accepted practice. However, optimal sputum criteria for pulmonary tuberculosis (TB) are not well established. We investigated indicators for sputum acceptability in tuberculosis cultures and acid-fast bacilli (AFB) smear. A post-hoc analysis of a randomized trial with 228 sputum specimens from 77 patients was conducted. In the trial, pulmonary TB suspects were requested for collecting three sputum specimens. We performed both TB study (AFB smear and M. tuberculosis culture) and Gram staining in each specimen. By using generalized estimating equations, the association between sputum characteristics and positive TB testings were analyzed. Although acceptable specimens for bacterial pneumonia showed higher TB-culture positive rates than unacceptable specimens (adjusted odds ratio [aOR]=1.66; 95% confidence interval [CI]=1.11-2.49), a specimen with ≥25 white blood cells/low-power field was the better predictor for positive M. tuberculosis cultures (aOR=2.30; 95% CI=1.48-3.58) and acid-fast bacilli smears (aOR=1.85; 95% CI=1.05-3.25). Sputum leukocytosis could be an indicator of sputum acceptability for diagnosing pulmonary tuberculosis.

Graphical Abstract

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Efficacy of isoniazid salvage therapy for latent tuberculosis infection in patients with immune-mediated inflammatory disorders – A retrospective cohort study in Taiwan

Background

Active tuberculosis (TB) in patients with latent tuberculosis infection (LTBI) was associated with use of biological agents for immune-mediated inflammatory disorders (IMIDs). For decreasing active TB, isoniazid prophylaxis therapy was administered before biologic therapy among IMID patients with LTBI. However, for patients who had been received biologics for a long time with unknown status of LTBI or exposure history of active TB, the prevalence of LTBI and efficacy of isoniazid therapy were unclear.

Glycolipids of Mycobacterium tuberculosis Strain H37Rv Are Potential Serological Markers for Diagnosis of Active Tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Prospective Evaluation of a Whole-Blood Test Using Mycobacterium tuberculosis-Specific Antigens ESAT-6 and CFP-10 for Diagnosis of Active Tuberculosis

A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.     

Description of Pediatric Tuberculosis Evaluated in a Referral Center in Istanbul Turkey

Purpose

Diagnosis of tuberculosis (TB) in children is more challenging than in adults. This study aimed to describe demographical, clinical and laboratory findings of children diagnosed with tuberculosis in Turkey, including the issues of contact tracing, culture positivity and forms of the disease.

Materials and Methods

Clinical and laboratory data of 51 children with a mean age of 8.0±4.6 years who were diagnosed with TB were retrospectively reviewed. Main diagnostic tools included tuberculin skin test, chest X-ray, sputum/gastric aspirate culture with sensitivity testing, and direct microscopy for acid-fast bacilli on available samples. Clinical characteristics and outcomes of the patients were examined.

Results

Thirty-six (70.6%) children were diagnosed with intra-thoracic and 15 (29.4%) with extra-thoracic tuberculosis. Twenty-eight of the patients had a positive Bacillus Calmette-Guérin vaccine scar (28/51, 54.9%) and 23/51 (45.1%) had a positive tuberculin skin test. An adult TB contact was identified in 27 (52.9%) of the cases. On direct microscopy, acid-fast bacilli were found in nine (17.6%) patients and positive culture for Mycobacterium tuberculosis was found in 19 (37.3%). Drug resistance to isoniazid was detected in four (7.8%). One patient with nephrotic syndrome and miliary tuberculosis died during follow-up. All other patients responded well to the treatment.

Conclusion

Focusing on active contact tracing among all household contacts of tuberculous cases may be helpful in early identification and controlling childhood disease, even in regions with low disease prevalence. Adopting a suspicious and proactive approach in this particular age group is warranted.