Tuberculosis situation among the pediatric population of Saratov Region (2015-2019)

Subjects and Methods. Incidence and prevalence of tuberculosis among children and adolescents in Saratov Region were retrospectively analyzed for 2015-2019.Results. The incidence of tuberculosis in children reflects general trends in the epidemic situation over the years in Saratov Region. In this region during 10 years of monitoring, tuberculosis incidence among children decreased 4 times by 2019 and it made 2.3 per 100,000 population, and among adolescents, it decreased 1.6 times (16.3 per 100,000 population). The number of children with post-tuberculosis changes detected for the first time increased 3 times in 2019 versus 2015, and the number of adolescents increased 4.5 times not only due to transition to tuberculosis screening in children aged 8-17 years old by skin test with tuberculosis recombinant allergen but also to the examination of children by computed tomography. The incidence in children followed up as Group IVA in 2019 was 15 times higher than this rate in the overall pediatric population. Analysis of the number of children and adolescents who developed tuberculosis and followed up as Groups IV and VI in 2015-2019 showed that the number of children ill with tuberculosis in Group IV was 3 times higher than the number of children in Group VI.

Immunodiagnostic Tests in the Assessment of Specific Sensitization to M. tuberculosis in Children under the Current Epidemical Situation

The objective: to study the degree of specific sensitization according to the results of intradermal immunodiagnostic tests in children with different manifestations of tuberculosis infection under the current epidemic situation.Subjects and Methods. The degree of tuberculin sensitization was studied in 299 children from different age groups (0-14 years old) who were registered for dispensary follow-up in 2018-2019.Based on the results of a tuberculosis recombinant allergen test (TRA), the degree of specific sensitization was studied in children with tuberculin tests conversion (n = 102) and infected with Mycobacterium tuberculosis (MTB) for 2 years or more (n = 165) exposed to tuberculous infection in their families and from healthy environment, and 145 patients under 14 years old with local respiratory tuberculosis.Results. Moderate sensitization to tuberculin predominated in children with converted tuberculin test exposed to tuberculous infection in their families and those from healthy environments. According to the results of TRA test, a high degree of sensitization was observed 4.7 times more frequently in children with tuberculin test conversion exposed to tuberculosis in their families. In children from healthy environments, negative responses to TRA test were registered 2.4 times more frequently. In 73% of children infected with MTB, results of annual Mantoux tests didn’t differ much. Hyperergic reactions to TRA test in infected children exposed to tuberculous infection were 10 times more frequent than in those from healthy environments. A direct correlation was found between the high degree of sensitization to active M. tuberculosis and exposure to a tuberculosis case with a positive result of sputum test (χ2 < 0.001, p < 0.05). In the case of converted tuberculin test and infection with M. tuberculosis for 2 years or more, pronounced and hyperergic reactions to TRA test were registered with equal frequency and did not depend on the timing and duration of infection. A high risk factor and predictor of the disease development is high sensitization of the host to active M. tuberculosis which is found in patients with tuberculosis using TRA test six times more frequently versus Mantoux test (OR 5.951, 95% CI 3.548 and 9.981). The demonstrated results are important for the identification of children facing a high risk to develop active tuberculosis and preventive treatment prescription.

Possibilities of specific immunological tests in patients with TB/HIV coinfection

The objective of the study: to study the possibilities of skin tests with tuberculosis recombinant allergen (TRA) in the diagnosis of tuberculosis in HIV-positive patients in the region with a high prevalence of tuberculosis.Subjects and Methods. Medical files of 85 patients were retrospectively analyzed, all the patients suffered from TB/HIV coinfection and underwent the skin test with TRA.Results. The skin test with TRA was found to be significantly valuable for the diagnosis of tuberculosis in patients with TB/HIV coinfection. The correlation between the intensity of response to the TRA test and CD4+ count was detected (p = 0.011). The lowest values were observed for CD4+ counts below 100 cells/μL. The direct correlation between the intensity of response to TRA and the stage of HIV infection was proved. No effect of the form of tuberculosis, the phase of the tuberculosis disease, or drug sensitivity on the intensity of response to TRA was found. Among tuberculosis patients with bacterial excretion (n = 48), 68.8% of patients responded positively to the TRA test, and in those with no bacterial excretion, the positive reaction was observed in 81.1%.

The Diagnostic Importance of Recombinant Allergen IgE Testing in Patients with Hymenoptera Venom Allergy: Comparison of Two Methods

Adults with systemic anaphylactic reactions (SAR) to insect sting show often multiple-positivity of serum-specific IgE (sIgE) to Hymenoptera venoms. Unnecessary long-lasting venom-specific immunotherapies (VIT) in false-positive patients increase the risk of recurrent SAR. This report aims to analyze the diagnostic importance of recombinant allergen IgE testing in patients with SAR to Hymenoptera sting.In 82 patients we measured sIgE to honeybee venom (HBV), wasp venom (WV) and hornet venom (HV) extracts, recombinant phospholipase A2 from HBV (sIgE-rApi m1), recombinant antigen 5 from WV (sIgE-rVes v5), and cross-reactive carbohydrate determinants-CCD-bromelain by ImmunoCAP. We analyzed the correlation of ImmunoCAP and Immunoblot for HBV and WV extracts, rApi m1, and rVes v5 in 39/82 patients. According to the history of the culprit insect, we compared sensitivity and specificity between the two methods.The severity of the SAR does not depend on the sIgE level to venom extracts and recombinant allergens. Fifty-one percent of the patients had a multiple-positivity to HBV/WV or HBV/WV/HV extracts. Severe SAR and CCD-sIgE were more frequent in multiple-positive than single-positive patients. CCD-sIgE were more frequent in HBV allergic patients than WV and HV allergic patients. There was a significant correlation between levels of sIgE to venom extracts and recombinant allergens measured by ImmunoCAP and Immunoblot. ImmunoCAP has higher sensitivity and specificity than Immunoblot for diagnosis of SAR to Hymenoptera venoms.IgE testing to recombinant CCD-free allergens is necessary for the adequate selection of long-lasting VIT, especially in patients with multiple sensitivities to venom extracts.

Microarray-Based Allergy Diagnosis: Quo Vadis?

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

A Challenge for Allergologist: Application of Allergy Diagnostic Methods in Mast Cell Disorders

Primary and secondary mast cell activation syndromes (MCAS) can occur in patients with mastocytosis. During the past few years our knowledge about the pathogenesis and disease-triggering mechanisms in MCAS and mastocytosis have increased substantially. Whereas mastocytosis is characterized by an accumulation of neoplastic (clonal) mast cells (MC) in various organ systems, MCAS is defined by a massive and systemic activation of these cells. Mast cells are crucial effector cells in allergic diseases, thus their elevated number and activation can cause severe anaphylactic reactions and MCAS in patients with mastocytosis. However, these cells may also degranulate spontaneously or degranulate in response to non-allergic triggers leading to clinical symptoms. In mastocytosis patients, such symptoms may lead to the diagnosis of a primary MCAS. The diagnosis of a concomitant allergy in mastocytosis patients is challenging. In these patients, a mixed form (primary and secondary) of MCAS may be diagnosed. These patients may also suffer from life-threatening anaphylactic reactions when exposed to allergens. In these cases, the possibility of severe side effects of in vivo provocations can sometimes also limit diagnostic evaluations. In the current article, we discuss the diagnosis and management of patients suffering from mastocytosis and concomitant MCAS, with special emphasis on novel diagnostic tests and management, including allergen microarrays, recombinant allergen analysis, basophil activation tests, optimal prophylaxis, and specific therapies.

Preventive Tuberculosis Services Reduces the Risk of Local Forms of Tuberculosis Development in Children on Immunosuppressive Therapy: Retrospective Cohort Study

Background. Long-term immunosuppressive therapy in children (including genetically engineered biologic drugs, GEBD) is associated with a high risk of local tuberculosis (TB) development. Objective. The aim of the study was to examine efficacy of tuberculosis services in children with high risk of developing tuberculosis associated with immunosuppressive therapy.Methods. The study included children at the age from 0 to 17 years on immunosuppressive therapy due to autoimmune disease and who were referred to phthisiatrician consultation. The incidence of TB was estimated one year after in groups receiving preventive TB services (isoniazid and pyrazinamide for 3–6 months) due to the high risk of TB development (contact with TB patients and/or controversial or positive test results with tubercular recombinant allergen) or not receiving such therapy (no indications for preventive treatment, parents’ refusal). The source of any data was medical documentation.Results. Preventive tuberculosis service was performed in 167 (60%) out of 279 children included in the study, 112 children did not receive such treatment (5 cases — parents’ refusal, 107 cases — lack of indications for preventive treatment). TB was detected in 1 (0.6%) child after one year in the preventive treatment group, and in 14 (12.5%) children (p < 0.001) in the group without preventive treatment. Thoracic lymph nodes tuberculosis was diagnosed in 4 (27%) patients among all who has developed TB, tuberculous primary complex — in 3 (20%) patients, focal tuberculosis in 7 (46%) patients, disseminated tuberculosis in 1 (7%) patient.Conclusion. Preventive tuberculosis service reduces the risk of tuberculosis in children on administration of immunosuppressive drugs, including GEBD.

CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

Background: Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. Objective: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. Methods: Recombinant forms of Lol_p_1 and Lol_p_5 proteins from ryegrass pollen (RGP) and Api_m_1 from honeybee venom (BV) were produced, biotinylated and tetramerized with streptavidin-fluorophore conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. Results: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api_m_1 and Lol_p_1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single tube assay enabled rapid detection of sensitization to both Lol_p_1 and Lol_p_5 in RGP-allergic patients and discriminated between controls, BV-allergic and RGP-allergic patients. Conclusion: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.