Evaluation of Aptamer Fluorescence Microscopy in the Diagnosis of Pulmonary Tuberculosis

Abstract

Sputum smear microscopy for tuberculosis diagnosis has stood the test of time. However, due to its low sensitivity, the positive detection rate for tuberculosis in clinical specimens is not high. To improve the sensitivity of microscopic observation in Mycobacterium tuberculosis (MTB) detection, we developed the MTB-specific aptamer MA1. To further improve the binding reactivity of MA1 with MTB, we constructed a new derivative aptamer with a pocket-stem-loop-structure, MA1-39, and identified it to have high binding reactivity with the MTB reference strain. We developed an aptamer fluorescence microscopy test for MTB based on MA1-39 and evaluated its feasibility for diagnosing pulmonary tuberculosis. Among 56 tested strains, MA1-39 was proven to effectively discriminate MTB from the control strains, including 12 non-tuberculosis mycobacterial (NTM) reference strains, 6 NTM isolates, and 7 other bacteria. Next, this approach was applied to 169 clinical sputum samples from suspected tuberculosis patients and non-tuberculosis controls. Molecular tests together with both clinical and bacteriological identification were used as a protocol to evaluate the efficacy of aptamer detection. Compared with the traditional acid-fast staining light microscope, the aptamer fluorescence microscope showed a higher detection rate for MTB in clinical specimens (48.8% versus 32.6%), and the specificities of the two techniques had almost no significant difference (90.4% versus 94%). In addition, aptamer fluorescence microscopy showed the same positive predictive value (PPV) as staining (84% versus 84.9%), but a higher negative predictive value (NPV; 63% versus 57.3%). In conclusion, the newly established aptamer fluorescence microscopy approach is likely to be a feasible method for microbiological diagnosis of tuberculosis. IMPORTANCE We established an aptamer fluorescence microscopy approach for rapid detection of MTB in clinical sputum samples. The use of aptamer probes was proven to significantly increase the sensitivity of sputum smear microscopy. In resource-limited countries, microscopy is currently a fast, simple, and very common test method in many laboratories, and it will remain the primary means of microbiological diagnosis of tuberculosis in the foreseeable future. Improving detection techniques can further enhance the clinical application value of this ancient diagnostic method. Since aptamer fluorescence microscopy can provide rapid and sensitive results, it may be a feasible and useful method in resource-limited settings.

Keywords: Mycobacterium tuberculosis; aptamer; microscopy; sputum sample; tuberculosis diagnosis.

FIG 1

Mycobacterium tuberculosis (MTB)-specific single-stranded DNA (ssDNA) aptamer MA1 and its derived aptamers. (A) Sequences of MA1 (78-nt) and its derived aptamers MA1-23 (23-nt), MA1-39 (39-nt), and MA1-55 (55-nt). (B) Secondary structures of MA1 and its derived aptamers. (C) Binding reactivity of aptamers with H37Rv determined using an enzyme-linked immunosorbent assay. (D) Binding reactivity of aptamers with H37Rv determined by direct fluorescence microscopy observation (×20). (E) Fluorescence intensity assessed by computer-assisted analysis using Image J. Significance level was set at P < 0.05. BC, blank control; AU, arbitrary units.

FIG 2

The identification of MTB strains by direct MA1-39 fluorescence-microscopy observation (×40). (A) Test in cultured strains. MTB-17 and MTB-23 are two isolates from MTB. Controls 1 and 2 are isolates from non-tuberculosis mycobacteria (NTM) and non-mycobacteria, respectively. (B) Detection in clinical sputum samples. TB-47 is the sputum sample from one TB patient, non-TB-57 is the sputum sample from one non-TB patient.