Lithuania is a country with a high incidence of tuberculosis (TB), despite a high coverage with bacille Calmette-Guerin (BCG) vaccination. Until now the only method used to detect latent TB infection was the tuberculin skin test (TST). However, TST may have a cross reactivity to the BCG vaccine and to environmental mycobacteria. The aim of this study was to conduct assessments of the diagnostic accuracy of the T-cell based test (T SPOT TB) for TB in children who had previously been BCG vaccinated and compare these with the results of the TST. Between January 2005 and February 2007, children with bacteriologically confirmed TB, children having contacts with a case of infectious pulmonary TB and children without any known risk for TB were tested with both the TST and T SPOT TB. The TST and T SPOT TB tests were positive for all patients in the „culture-confirmed TB“ group. Whereas, in the „high risk for TB“ group, the TST was positive for 60%, but the T SPOT TB test, only for 17.8%. Meanwhile the results for the „low risk for TB“ group were 65.4% and 9.6%, respectively. A correlation between the TST and T SPOT TB was obtained in the "culture-confirmed TB" group where the TST ≥15 mm (r = 0.35, p < 0.001). The T-cell based method is more objective than the TST for identifying latent TB infection in children who had been previously BCG vaccinated. This method could be useful in countries like Lithuania where there is a high incidence of TB despite a high coverage with BCG vaccination. It may also help to avoid unnecessary chemoprophylaxis when TST reactions are false-positive.

https://link.springer.com/content/pdf/10.1186%2F1471-2466-9-41.pdf

T-cell-based diagnosis of tuberculosis infection in children in Lithuania: a country of high incidence despite a high coverage with bacille Calmette-Guerin vaccination

In this study, an electrochemical immunosensor was introduced for the detection of tuberculosis (TB) via utilization of a modified electrode containing a quantum dot (CdSe/ZnS QD) and functionalized silica nanoparticles (SiNPs) on screen-printed carbon electrode (SPCE) CdSe/ZnS QD/SiNPs/SPCE, by employing indirect enzyme-linked immunosorbent assay (ELISA). Here, the fabricated electrode was linked to the biocatalytic action of enzyme catalase through antigen-antibody binding for the detection of the antigen (CFP10-ESAT6) by means of producing a differential pulse voltammetry (DPV) current. The characterization and cyclic voltammetry (CV) of the modified electrode showed good electrochemical behavior and enhanced high electron transfer between the electrode and analyte. Moreover, the active surface area was 4.14-fold higher than the bare SPCE. The developed method showed high selectivity towards CFP10-ESAT6 compared with the other TB proteins. The detection of CFP10-ESAT6 also showed a linear response towards different concentrations of CFP10-ESAT6 with R2 = 0.9937, yielding a limit of detection (LOD) of as low as 1.5 × 10-10 g/mL for a linear range of 40 to 100 ng/mL of CFP10-ESAT6 concentration. The proposed method showed good reproducibility of target analyte with a relative standard deviation of 1.45%.

https://www.mdpi.com/1996-1944/13/1/149/pdf

Surface Enhanced CdSe/ZnS QD/SiNP Electrochemical Immunosensor for the Detection of Mycobacterium Tuberculosis by Combination of CFP10-ESAT6 for Better Diagnostic Specificity.

Tuberculosis is a major public health problem worldwide. Indeed, a third of the world population is infected with Mycobacterium tuberculosis and more than 8 million new cases of tuberculosis each year. Pulmonary tuberculosis is the most common location. Its diagnosis is difficult and often established with a delay causing a spread of infection. The diagnosis of tuberculosis infection is mainly based on immunological tests represented by the tuberculin skin test and detection of gamma interferon, while the diagnosis of pulmonary tuberculosis is suspected on epidemiological context, lasting general and respiratory symptoms, contrasting usually with normal lung examination, and a chest radiography showing suggestive lesions. The radioclinical feature may be atypical in patients with extreme ages and in case of immunodeficiency. Confirmation of tuberculosis is bacteriological. Conventional bacteriological methods remain the reference. Innovative tests using the technique of molecular biology have improved the diagnosis of tuberculosis in terms of sensitivity and especially speed. However, those techniques are of limited use.

Primary infection and pulmonary tuberculosis

Resume L’intradermoreaction cutanee a la tuberculine, couramment utilisee depuis un siecle pour le diagnostic d’infection tuberculeuse, presente de nombreux inconvenients. De nouveaux tests diagnostiques ont ete recemment introduits. Ils mesurent soit la production d’interferon-γ dans le sang total, soit le nombre de lymphocytes T producteurs d’interferon-γ apres stimulation in vitro par des proteines specifiques de M. tuberculosis, absentes du BCG et de la plupart des mycobacteries atypiques. Le gain en specificite permet de reduire les resultats faux positifs chez les sujets vaccines, evitant ainsi le cout de chimioprophylaxies inutiles et potentiellement toxiques. Le gain en sensibilite, identifiant les infections tuberculeuses latentes parmi les sujets ayant une IDR faussement negative, permet d’accroitre les performances diagnostiques dans les populations les plus a risques de progresser vers la tuberculose maladie, a savoir les patients immunodeprimes. L’evaluation de ces tests doit desormais se focaliser sur certains points qui restent a preciser : leur sensibilite chez l’enfant et le sujet immunodeprime, leurs valeurs predictives positive et negative et l’interpretation de leur variation eventuelle au cours du temps, que les patients soient traites ou non.

Apport des tests de quantification de la libération d’interféron gamma par les lymphocytes T sensibilisés pour le diagnostic des infections tuberculeuses

The immune response against mycobacterium tuberculosis (MTB) is cell mediated. T-cells become sensitized when they encounter MTB antigens and subsequently activated effector T-cells produce a number of cytokines including interferon- gamma (INF-gamma) to fight the infecting organisms. Demonstration of either production of INF-gamma or presence of effector T-cells sensitized to MTB specific antigens in vitro can be diagnostic for TB infection. Aim of this study was to determine the efficacy of commercially available T-SPOT.TB kit which is used for the in vitro diagnosis of TB infection and to determine if this test has any cross reactivity in leprosy patients. Blood sample was taken from 30 sputum AFB positive, 30 sputum AFB negative healthy controls and 10 cases of paucibacillary leprosy patients. The blood samples were processed to separate peripheral blood mononuclear cells. The final cell suspensions were cultured along with MTB specific antigens namely- Early Secretory Antigenic Target (ESAT-6) and Culture Filtrate Protein (CFP 10) along with negative and positive controls. The production of INF-gamma was demonstrated by enzyme linked immunospot (ELISPOT) assay technique. All AFB positive samples produced INF-gamma after exposure to MTB specific antigens. 4 (16.6%) of healthy controls were also found reactive for INF-gamma. The sensitivity and "specificity" for active disease of the ELISPOT (T-SPOT.TB) in respect to AFB microscopy was 100% and 85.7% respectively. Assessment of CMI against tuberculosis, by demonstrating effector T-cell sensitized to MTB antigens can be use to aid the diagnosis of tuberculosis. T-SPOT.TB has no cross reactivity with leprosy patients.

In-vitro assessment of cell-mediated immunity by demonstrating effector-t cells for diagnosis of tuberculosis in Nepalese subjects.

Tuberculosis is one of the biggest global health problems. One-third of the world’s population (2 billion) is latently infected with tuberculosis. The tuberculin skin test is commonly used to diagnose tuberculosis infection. This test has poor specificity and sensitivity, cross-reactivity with bacille Calmette-Guerin vaccination and many environmental mycobacteria, and poor sensitivity (only 75–90% in active tuberculosis). Mycobacterium tuberculosis activates a strong T cell-mediated immune response. That is why, a better marker for tuberculosis infection could be the presence of mycobacteria specific interferon-y-secreting T cells. These cells can be identified in blood or any other sample, which contains T cells. The test specificity is 99.9% (in low-risk control groups), and the sensitivity is 97.2% (in subjects with culture-confirmed active disease). New in vitro diagnostic test of tuberculosis, based on tuberculosis-induced immunological mechanisms, seems to be more specific and useful as previous methods.

/pdf/research-for-practice-a-new-in-vitro-test-for-identification-53168f0l6g.pdf

Research for practice: a new in vitro test for identification of tuberculosis infection.

Background and objective: Lithuania belongs to the group of countries with a high-incidence of tuberculosis (TB). Some scientific studies show that the interferon-gamma release assay is more accurate and correlates more highly with TB exposure as compared to the tuberculin skin test (TST). This study aimed at comparing the efficacy between the T SPOT TB and TST for diagnosing TB among Lithuanian adults. Materials and methods: Individuals with diagnosed TB, healthcare workers with known risk for TB and individuals without any known risk for TB underwent clinical examinations, interviews about their history of TB exposure and chest radiography. Then the TST and the T SPOT TB were performed on patients. Results: A positive T SPOT TB was more common in the group with diagnosed TB compared to healthcare workers and the low risk for TB groups (97.5%, 36.4%, and 0%, respectively, P 0.05). Agreement between TST and T SPOT TB was poor (kappa 0.14, P > 0.05). T SPOT TB had higher specificity and sensitivity compared to TST (area under the ROC 0.9 ± 0.04, P 0.05). Conclusions: The T SPOT TB showed greater accuracy in diagnosing TB than TST did. Positive T SPOT TB result but not the TST was more common in patients with diagnosed TB.

/pdf/use-of-interferon-gamma-release-assay-and-tuberculin-skin-3pzqvfir27.pdf

Use of interferon-gamma release assay and tuberculin skin test in diagnosing tuberculosis in Lithuanian adults: A comparative analysis

Tuberkuliozė (TB) yra viena seniausių ir grėsmingiausių, placiai pasaulyje paplitusių lėtinių infekcinių ligų, kurios plitimo priežastys dažnai yra socialinės. Darbo tikslas. Įvertinti socialinius ir gyvensenos veiksnius, galincius turėti įtakos vaikų užsikrėtimui ir sergamumui TB. Tyrimo medžiaga ir metodai. Anketinės apklausos būdu istirti vaikai, sergantys TB (n=23), užsikrėte TB (n=13) ir sveiki (n=31). Jaunesnių nei 14 metų vaikų tėvai atsakė į klausimus apie vaiko gyvenimo sąlygas (atskirą kambarį, ventiliaciją bute), vaiko maitinimosi reguliarumą, artimą kontaktą su serganciu bakteriologiskai patvirtinta TB, tėvų issilavinimą, seimos sudėtį, žalingus vaiko ir tėvų įprocius. Rezultatai. Tyrime dalyvavo 31 berniukas ir 36 mergaitės, amžiaus vidurkis – 14,3 ± 2,1 metų. Įvertinus vaiko maitinimosi skaicių per dieną, nustatyta, kad 21,7 proc. TB susirgusių vaikų maitinosi reciau nei tris kartus per dieną, nors reiksmingo skirtumo tarp tirtų grupių nenustatyta (p=0,075). Vertinant vaiko rūkymą, nustatyta, kad užsikrėtusiųjų TB (7,7 proc.) ir sveikų tiriamųjų (3,2 proc.) grupėse buvo statistiskai reiksmingai mažiau rūkancių vaikų nei serganciųjų TB grupėje (30,4 proc.). 30,4 proc. susirgusiųjų TB ir 7,7 proc. užsikrėtusių TB vaikų gyveno namuose be ventiliacijos. Vertinant tėvų rūkymą, alkoholinių gėrimų vartojimą, seimos statusą, reiksmingo skirtumo tarp tirtų grupių nenustatyta. Net 47,8 proc. vaikų, susirgusių bakteriologiskai patvirtinta TB, turėjo artimą kontaktą su asmeniu, serganciu TB. Vertinant tėvų issilavinimą, pastebėta, jog serganciųjų TB grupėje žymiai daugiau buvo tėvų su nebaigtu viduriniu ir viduriniu issilavinimu (56,5 proc.). Isvados. Sio tyrimo duomenimis, vaikams susirgti ar užsikrėsti TB ypac didele įtaką turi sie veiksniai: artimas kontaktas su serganciuoju TB, vaiko rūkymas, ventiliacijos gyvenamojoje patalpoje nebuvimas. Reiksminiai žodžiai: tuberkuliozė, socialiniai veiksniai, žalingi įprociai, gyvensena.

Edita Hansted

Papers

Research for practice: a new in vitro test for identification of tuberculosis infection.

Use of interferon-gamma release assay and tuberculin skin test in diagnosing tuberculosis in Lithuanian adults: A comparative analysis

Socialiniai ir gyvensenos veiksniai, galintys turėti įtakos vaikų užsikrėtimui ir sergamumui