Evaluation of OMNIgene®•SPUTUM reagent for mycobacterial culture.

SETTING: National Mycobacterium Reference Laboratory, Borstel, Germany. OBJECTIVE: To evaluate the effectiveness of OMNIgene®•SPUTUM (OM-S) reagent in comparison with a method using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) with regard to mycobacterial recovery and contamination of broth and solid cultures. DESIGN: Sputum samples from patients with tuberculosis and other respiratory diseases underwent decontamination with NALC-NaOH-based (MycoDDR™) or OM-S reagent. The decontamination procedure was assigned by block randomisation. Samples were inoculated on Löwenstein-Jensen, Stonebrink and MGIT™ (Mycobacterial Growth Indicator Tubes). Mycobacterial recovery from samples spiked with Mycobacterium tuberculosis following decontamination was determined. RESULTS: Eighty-five samples were randomised to NALC-NaOH and 84 to OM-S reagent. Mycobacterial recovery was significantly lower for samples processed with OM-S reagent compared with the NALC-NaOH method across all media types. Culture contamination was lower with NALC-NaOH reagent on solid media (9.4-12.9% vs. 28.6-29.8%). Growth was not observed in MGIT among samples spiked with 10 600-16 800 colony-forming units of M. tuberculosis following decontamination with OM-S reagent. CONCLUSION: Low mycobacterial recovery, especially in MGIT, observed in the present study suggests that OM-S reagent might not be compatible with the MGIT system. More extensive field evaluations of the OM-S reagent are warranted to demonstrate a significant benefit over currently used methods

Effect of HIV on the frequency and number of Mycobacterium tuberculosis-specific CD4+ T cells in blood and the airways in latent tuberculosis infection

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). There is extensive depletion of Mycobacterium tuberculosis (M.tuberculosis)-specific CD4+ T cells in blood in early HIV infection, but little is known about responses in the lungs at this stage. Given that mucosal organs are a principal target for HIV-mediated CD4 destruction, we investigated M.tuberculosis-specific responses in bronchoalveolar lavage (BAL), in persons with latent TB infection and untreated HIV-1 co-infection with preserved CD4 counts. M.tuberculosis-specific CD4+ cytokine responses (IFN-, TNF- and IL-2) were discordant in frequency and function between BAL and blood. Responses in BAL were 15-fold lower in HIV-infected compared to uninfected persons (p=0.048), whilst blood responses were 2-fold lower (p=0.006). However, an increase in T cells in the airways in HIV-infected persons resulted in the overall number of M.tuberculosis-specific CD4+ cells in BAL being similar. Our study highlights the important insights gained from studying TB immunity at the site of disease during HIV infection

Treatment responses in multidrug-resistant tuberculosis in Germany.

BACKGROUND: Excellent treatment outcomes have recently been reported for patients with multi/extensively drug-resistant tuberculosis (M/XDR-TB) in settings where optimal resources for individualised therapy are available. OBJECTIVE: To ascertain whether differences remain in treatment responses between patients with M/XDR-TB and those with non-M/XDR-TB. METHOD: Patients with TB were prospectively enrolled between March 2013 and March 2016 at five hospitals in Germany. Treatment was conducted following current guidelines and individualised on the basis of drug susceptibility testing. Two-month and 6-month sputum smear and sputum culture conversion rates were assessed. A clinical and radiological score were used to assess response to anti-tuberculosis treatment. RESULTS: Non-M/XDR-TB (n = 29) and M/XDR-TB (n = 46) patients showed similar rates of microbiological conversion: 2-month smear conversion rate, 90% vs. 78%; culture conversion rate, 67% vs. 61%; time to smear conversion, 19 days (IQR 10-32) vs. 31 days (IQR 14-56) (P = 0.066); time to culture conversion, 39 days (IQR 17-67) vs. 39 days (IQR 6-85) (P = 0.191). Both clinical and radiological scores decreased after the introduction of anti-tuberculosis treatment. CONCLUSION: There were no significant differences in scores between the two groups until 6 months of treatment. Under optimal clinical conditions, with the availability of novel diagnostics and a wide range of therapeutic options for individualised treatment, patients with M/XDR-TB achieved 6-month culture conversion rates that were compatible with those in patients with non-M/XDR-TB

Bronchoalveolar Lavage Enzyme-Linked Immunospot for Diagnosis of Smear-Negative Tuberculosis in HIV-Infected Patients

Peripheral blood interferon-gamma release assays (IGRAs) have sub-optimal sensitivity and specificity for diagnosis of active pulmonary tuberculosis (TB). However, assessment of local immune responses has been reported to improve the accuracy of TB diagnosis.We enrolled HIV-infected adults with cough ≥2 weeks' duration admitted to Mulago Hospital in Kampala, Uganda and referred for bronchoscopy following two negative sputum acid-fast bacillus smears. We performed an ELISPOT-based IGRA (T-SPOT.TB®, Oxford Immunotec, Oxford, UK) using peripheral blood and bronchoalveolar lavage (BAL) fluid mononuclear cells, and determined the accuracy of IGRAs using mycobacterial culture results as a reference standard.94 HIV-infected patients with paired peripheral blood and BAL IGRA results were included. The study population was young (median age 34 years [IQR 28-40 years]) and had advanced HIV/AIDS (median CD4+ T-lymphocyte count 60 cells/µl [IQR 22-200 cells/µl]). The proportion of indeterminate IGRA results was higher in BAL fluid than in peripheral blood specimens (34% vs. 14%, difference 20%, 95% CI 7-33%, p = 0.002). BAL IGRA had moderate sensitivity (73%, 95% CI 50-89%) but poor specificity (48%, 95% CI 32-64%) for TB diagnosis. Sensitivity was similar (75%, 95% CI 57-89%) and specificity was higher (78%, 95% CI 63-88%) when IGRA was performed on peripheral blood.BAL IGRA performed poorly for the diagnosis of smear-negative TB in a high HIV/TB burden setting. Further studies are needed to examine reasons for the large proportion of indeterminate results and low specificity of BAL IGRA for active TB in high HIV/TB burden settings

Rifapentine access in Europe: growing concerns over key tuberculosis treatment component

[No abstract available]Support statement: C. Lange is supported by the German Center of Infection Research (DZIF). All other authors have no funding to declare for this study. Funding information for this article has been deposited with the Crossref Funder Registry

Supplementary Material for: Bifunctional T-Cell-Derived Cytokines for the Diagnosis of Tuberculosis and Treatment Monitoring

<b><i>Background:</i></b> Diagnosis and treatment monitoring of patients with tuberculosis remain challenging. <b><i>Objective:</i></b> We have evaluated whether <i>Mycobacterium</i>-specific interferon (IFN)-γ and interleukin (IL)-2 bifunctional cytokine immune response assays improve the diagnosis of and correlate to treatment response in pulmonary tuberculosis. <b><i>Methods:</i></b> Early secretory antigenic target (ESAT)6/culture filtrate protein 10 (CFP10), microsomal triglyceride transfer protein 65 (MTP65) and the purified protein derivative (PPD) tuberculin-specific immune profiles were investigated in peripheral blood mononuclear cells from 19 patients with culture-confirmed tuberculosis and 23 healthy community controls (HCCs; 82.6% with latent <i>M. tuberculosis</i> infection) using a novel fluorescence-based dual-colour enzyme-linked immunospot (EliSpot) technology (FluoroSpot). <b><i>Results:</i></b> The frequency of ESAT6/CFP10-induced IFN-γ+IL-2- producing cells was elevated (p < 0.001), whereas the percentages of specific IFN-γ-IL-2+ (p = 0.002) and IFN-γ+IL-2+ double producing cells (p = 0.037) were diminished in tuberculosis patients in comparison to HCCs. A 3-host marker model using a combination of those IFN-γ and IL-2 single-cell responses showed 93.8% sensitivity and 77.8% specificity for tuberculosis. During tuberculosis treatment, the PPD-induced immune responses shifted from an IFN-γ+IL-2- dominated profile towards a balance of IFN-γ-IL-2+ and IFN-γ+IL-2+ double producing cells (all p ≤ 0.05). <b><i>Conclusions:</i></b> The addition of antigen-specific IL-2 production to IFN-γ responses by EliSpot in IFN-γ release assays increases diagnostic sensitivity for active tuberculosis

Bronchoalveolar Lavage Enzyme-linked Immunospot for a Rapid Diagnosis of Tuberculosis: A Tuberculosis Network European Trialsgroup Study

Rationale: The rapid diagnosis of pulmonary tuberculosis (TB) is difficult when acid fast bacilli (AFB) cannot be detected in sputum smears

Bronchoalveolar lavage enzyme-linked immunospot for a rapid diagnosis of tuberculosis: a Tuberculosis Network European Trialsgroup study.

The rapid diagnosis of pulmonary tuberculosis (TB) is difficult when acid fast bacilli (AFB) cannot be detected in sputum smears.Following a proof of principle study, we examined in routine clinical practice whether individuals with sputum AFB smear-negative TB can be discriminated from those with latent TB infection by local immunodiagnosis with a Mycobacterium tuberculosis-specific enzyme-linked immunospot (ELISpot) assay.Subjects suspected of having active TB who were unable to produce sputum or with AFB-negative sputum smears were prospectively enrolled at Tuberculosis Network European Trialsgroup centers in Europe. ELISpot with early-secretory-antigenic-target-6 and culture-filtrate-protein-10 peptides was performed on peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage mononuclear cells (BALMCs). M. tuberculosis-specific nucleic acid amplification (NAAT) was performed on bronchoalveolar lavage fluid.Seventy-one of 347 (20.4\%) patients had active TB. Out of 276 patients who had an alternative diagnosis, 127 (46.0\%) were considered to be latently infected with M. tuberculosis by a positive PBMC ELISpot result. The sensitivity and specificity of BALMC ELISpot for the diagnosis of active pulmonary TB were 91 and 80\%, respectively. The BALMC ELISpot (diagnostic odds ratio [OR], 40.4) was superior to PBMC ELISpot (OR, 10.0), tuberculin skin test (OR, 7.8), and M. tuberculosis specific NAAT (OR, 12.4) to diagnose sputum AFB smear-negative TB. In contrast to PBMC ELISpot and tuberculin skin test, the BALMC ELISpot was not influenced by previous history of TB.Bronchoalveolar lavage ELISpot is an important advancement to rapidly distinguish sputum AFB smear-negative TB from latent TB infection in routine clinical practice