The clinical utility of antigen-specific interferon (IFN)-c release assays (IGRAs) using pleural mononuclear cells, for the diagnosis of tuberculosis (TB), requires clarification.We compared the diagnostic utility of unstimulated pleural IFN-c levels with several pleural antigen-specific T-cell IGRAs (early secretory antigenic target-6 and culture filtrate protein-10 (T-SPOT.1TB, QuantiFERON1-TB Gold In-tube), purified protein derivative (PPD) and heparinbinding haemagglutinin (HBHA)) in 78 South African TB suspects. Test results were compared against a clinical score and a reference standard.Out of 74 evaluable subjects 48, seven and 19 had definite, probable and no TB, respectively. 11 (15%) out of 74 pleural samples (nine (19%) out of 48 of the definite TB cases) had total cell counts that were inadequate for T-cell processing. In the remaining 63 samples, the sensitivity, specificity, positive predictive value and negative predictive value of different diagnostic methods were as follows. Maximal bioclinical score: 54, 89, 92 and 43%, respectively; T-SPOT.1TB: 86, 60, 84 and 64%, respectively; QuantiFERON1-TB Gold In-tube: 57, 80, 87 and 44%, respectively; HBHA-specific IGRA: 59, 31, 64 and 27%, respectively; PPD-specific IGRA: 81, 40, 76 and 46%, respectively; and pleural fluid unstimulated IFN-c: 97, 100, 100 and 94%, respectively. Unstimulated IFN-c was the most accurate test for distinguishing TB from non-TB effusions in a high-burden setting. The antigen-specific T-cell IGRAs were limited by suboptimal accuracy and the inability to isolate sufficient mononuclear cells to perform the assay.

Background: The diagnosis of smear-negative pulmonary tuberculosis (TB) is problematic. There are limited data on the profile of alveolar TB antigen-specific T cells, and their utility for the rapid immunodiagnosis of pulmonary TB is unclear. Methods: Antigen-specific interferon c (IFNc) responses to the RD-1 antigens ESAT-6 and CFP-10 (T-SPOT.TB and QuantiFERON-TB-Gold-In-Tube), heparin-binding haemagglutinin and purified protein derivative were evaluated, using alveolar lavage cells, in 91 consecutively recruited South African patients suspected of having TB. Results: Of 85 evaluable patients (29% HIV+), 24, 11, 48 and 2 had definite TB, probable TB, non-TB and an uncertain diagnosis, respectively. Between 34% (T-SPOT.TB) and 41% (QuantiFERON-TB-Gold-In-Tube) of all test results were inconclusive. Failure of the positive control was significantly higher with the QuantiFERON-TBGold-In-Tube than with T-SPOT.TB (85% vs 46% of inconclusive results; p = 0.001). Using staphylococcal enterotoxin B, compared with phytohaemagglutinin, substantially reduced failure of the positive control (25% to 3%; p = 0.02). In evaluable samples, when the definite and non-TB groups were used for outcome analysis, the percentage sensitivity, specificity, positive predictive value and negative predictive value for T-SPOT.TB (>20 spots/million alveolar mononuclear cells) and QuantiFERON-TB-Gold-In-Tube (0.35 IU/ml) were 89, 94, 89 and 94% (n = 55) and 55, 86, 77 and 69% (n = 46), respectively. Rapid diagnosis of TB was achieved more frequently with T-SPOT.TB than with smear microscopy (14/24 (58%) vs. 7/24 (29%) of definite TB cases; p = 0.02). Heparin-binding haemagluttinin and purified protein derivative alveolar lymphocyte IFNc responses had poor performance outcomes. Conclusion: Provided evaluable results are obtained, the RD-1, but not the heparin-binding haemagglutinin or purified protein derivative, alveolar lymphocyte IFNc ELISPOT response is a useful rapid immunodiagnostic test for TB. However, test utility in high-burden settings may be limited by the high proportion of inconclusive results.

Mutations in the m.13094T>C MT-ND5 gene have been previously described in three cases of Leigh Syndrome (LS). In this retrospective, international cohort study we identified 20 clinically affected individuals (13 families) and four asymptomatic carriers. Ten patients were deceased at the time of analysis (median age of death was 10 years (range: 5·4 months−37 years, IQR = 17·9 years). Nine patients manifested with LS, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and one with Leber hereditary optic neuropathy. The remaining nine patients presented with either overlapping syndromes or isolated neurological symptoms. Mitochondrial respiratory chain activity analysis was normal in five out of ten muscle biopsies. We confirmed maternal inheritance in six families, and demonstrated marked variability in tissue segregation, and phenotypic expression at relatively low blood mutant loads. Neuropathological studies of two patients manifesting with LS/MELAS showed prominent capillary proliferation, microvacuolation and severe neuronal cell loss in the brainstem and cerebellum, with conspicuous absence of basal ganglia involvement. These findings suggest that whole mtDNA genome sequencing should be considered in patients with suspected mitochondrial disease presenting with complex neurological manifestations, which would identify over 300 known pathogenic variants including the m.13094T>C.

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