Background Improvement of the diagnosis of smear-negative pulmonary tuberculosis (PTB) patients and identification of possible immune factors associated with the negative result of sputum will enable early and accurate diagnosis of smear-negative PTB. This study aimed to measure the Mycobacterium load in sputum samples of smear-negative patients and identify cytokines markers associated with smear-negative active pulmonary tuberculosis.
Methods Sputum and heparinised blood samples were collected from 40 smear-negative, 40 smear-positive PTB patients and 21 healthy controls. All sputum samples were analysed by direct ZN stain and conventional PCR to confirm the infection and characterise the bacteria. The load of bacteria in sputum samples was measured using real-time PCR. Blood samples were stimulated with sonicated MTB H37Rv. TH1 (TNF-α, IFN-γ, IL- 1β) and TH2 (IL-10) cytokines were measured using ELISA technique.
Results Eight patients were grade 3+, 23 were grade 2+, 9 were grade 1+ and 40 were negative on smear. 87.5% of smear-negative patients were positive by PCR. Smear-negative PTB patients produced high concentration of IFN-γ compared with smear-positive. IL-10 and TNF-α concentration were significantly lower in smear-negative compared with smear-positive. IL-1β was not significantly different between smear-negatives and smear-positives. Both smear-negative and smear-positive samples produced significantly high IL-10 and TNF-α cytokine compared with the healthy controls, while IFN-γ production was significantly lower in MTB patients. A highly significant correlation between MTB load and cytokines was detected. The mean concentration of IFN-γ was higher in stimulated blood samples of patients with lower bacterial load. In contrast, IL-10 and TNF-α concentration were higher in patients with high bacterial load. The TNF-α and IL-1β were good biomarkers for diagnosis of smear-negatives.
Conclusion Smear-negative PTB produced high TH1 cytokine and low regulatory cytokine compared to smear-positive.
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