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.

Machine Learning Algorithms Evaluate Immune Response to Novel Mycobacterium tuberculosis Antigens for Diagnosis of Tuberculosis

RationaleTuberculosis diagnosis in children remains challenging. Microbiological confirmation of tuberculosis disease is often lacking, and standard immunodiagnostic including the tuberculin skin test and interferon-γ release assay for tuberculosis infection has limited sensitivity. Recent research suggests that inclusion of novel Mycobacterium tuberculosis antigens has the potential to improve standard immunodiagnostic tests for tuberculosis.ObjectiveTo identify optimal antigen–cytokine combinations using novel Mycobacterium tuberculosis antigens and cytokine read-outs by machine learning algorithms to improve immunodiagnostic assays for tuberculosis.MethodsA total of 80 children undergoing investigation of tuberculosis were included (15 confirmed tuberculosis disease, five unconfirmed tuberculosis disease, 28 tuberculosis infection and 32 unlikely tuberculosis). Whole blood was stimulated with 10 novel Mycobacterium tuberculosis antigens and a fusion protein of early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP) 10. Cytokines were measured using xMAP multiplex assays. Machine learning algorithms defined a discriminative classifier with performance measured using area under the receiver operating characteristics.Measurements and main resultsWe found the following four antigen–cytokine pairs had a higher weight in the discriminative classifier compared to the standard ESAT-6/CFP-10-induced interferon-γ: Rv2346/47c- and Rv3614/15c-induced interferon-gamma inducible protein-10; Rv2031c-induced granulocyte-macrophage colony-stimulating factor and ESAT-6/CFP-10-induced tumor necrosis factor-α. A combination of the 10 best antigen–cytokine pairs resulted in area under the curve of 0.92 ± 0.04.ConclusionWe exploited the use of machine learning algorithms as a key tool to evaluate large immunological datasets. This identified several antigen–cytokine pairs with the potential to improve immunodiagnostic tests for tuberculosis in children.

Mycobacterium microti: Not Just a Coincidental Pathogen for Cats

Public interest in animal tuberculosis is mainly focused on prevention and eradication of bovine tuberculosis in cattle and wildlife. In cattle, immunodiagnostic tests such as the tuberculin skin test or the interferon gamma (IFN-γ) assay have been established and are commercially available. Feline tuberculosis is rather unknown, and the available diagnostic tools are limited. However, infections with Mycobacterium tuberculosis complex members need to be considered an aetiological differential diagnosis in cats with granulomatous lymphadenopathy or skin nodules and, due to the zoonotic potential, a time-efficient and accurate diagnostic approach is required. The present study describes 11 independent cases of Mycobacterium microti infection in domestic cats in Switzerland. For three cases, clinical presentation, diagnostic imaging, bacteriological results, immunodiagnostic testing, and pathological features are reported. An adapted feline IFN-γ release assay was successfully applied in two cases and appears to be a promising tool for the ante mortem diagnosis of tuberculosis in cats. Direct contact with M. microti reservoir hosts was suspected to be the origin of infection in all three cases. However, there was no evidence of M. microti infection in 346 trapped wild mice from a presumptive endemic region. Therefore, the source and modalities of infection in cats in Switzerland remain to be further elucidated.

Bedaquiline in the treatment regimen of multidrugresistant tuberculosis (clinical observation)

Current trials are underway to design chemotherapy regimens that include bedaquiline to treat children with rifampicin and isoniazid resistant mycobacterium tuberculosis. This clinical observation is interesting from the viewpoint of experience of the potential effective and safe use of the new anti-tuberculosis drug bedaquiline (Sirturo) in children under 12 years of age, as the publications of clinical trials of the drug use in children is limited. The female patient was diagnosed with primary tuberculosis complex complicated by infusional pleurisy based on the epidemiological anamnesis (family contact with tuberculosis patients), immunodiagnostic tests – a positive ATR-test result (Diaskin-test), pathological changes in the lung tissue and intrathoracic lymph nodes found on the chest CT scans. The diagnosis was confirmed by detection of MBT in pleural effusion by PCR and liquid culture method (BACTEC MGIT-960), which are resistant to isoniazid and rifampicin (multidrug resistance, MDR). The girl was enrolled in a clinical trial and treated according to chemotherapy regimen IV including a new anti-tuberculosis drug, which she received for 6 months. There was a positive clinical and X-ray dynamics of the tuberculosis process with minimal residual changes throughout the entire course of chemotherapy followed by confirmed clinical cure of TB (after 24-month follow-up). Based on the example of this clinical observation, it can be concluded that bedaquiline is safe and showed good clinical efficacy in the chemotherapy regimen to treat MDR MBT in children under 12 years of age.

Renal echinococcosis; the parasite, host immune response, diagnosis and management

Renal echinococcosis is a rare parasite-caused disease of humans and animals; it makes up about 4% of confirmed cases of cystic echinococcosis. It is a zoonotic disease that occurs in the intermediate hosts harboring the larval stage, the hydatid cyst, of Echinococcus spp. The renal involvement is often asymptomatic or with unspecific signs. Its diagnosis is mostly based on imaging technique. Immunodiagnostic tests are not applicable. Furthermore, because the disease is not common, our knowledge about its different aspects is scarce. In this review, the parasite, host immune response, diagnosis, and management of renal echinococcosis are described.

Designing a Recombinant Multi-Epitope Antigen of Echinococ-cus granulosus to Diagnose Human Cystic Echinococcosis

Background: Cystic echinococcosis can cause severe disease and probable death in humans. Epitopes of its antigens play a key role in the sensitivity and specificity of immunodiagnostic tests. Methods: Epitope prediction software programs predict the most antigenic linear B-cell epitopes of AgB (8 kD), Ag5, and Ag95. Six such epitopes were predicted and connected by “Gly-Ser” linker and synthesized. The purity of the concentrated recombinant multi-epitope protein was assessed by 15% SDS-PAGE. Overall, 186 serum samples were collected from the Loghman Hakim Hospital and different laboratories, Tehran, Iran, from July 2016 to February 2017. Patients infected with hepatic hydatid cysts, patients infected by other parasites and viruses, and healthy individuals were used to detect the anti-CE IgG using recombinant multi-epitope protein. Results: Forty-one samples out of 43 cases of hydatidosis were diagnosed correctly as positive, and two were negative. In addition, six negative cases of healthy individual group were diagnosed as positive and negative with rMEP-ELISA and the commercial kit, respectively. Therefore, these six samples were considered as false positive using our method. In addition, a diagnostic sensitivity of 95.3% (95% CI, 84.19% to 99.43%) and a specificity of 95.0% (95% CI, 89.43% to 98.14%) were obtained using optimum cutoff value (0.20). The sensitivity and specificity of the commercial kit was 100%. Conclusion: Our findings showed high diagnostic accuracy of the ELISA test using the developed recombinant protein, which encourages the use of this recombinant multi-epitope protein for rapid serological diagnosis of hydatidosis.