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. 2021 Mar 15:11:634915.
doi: 10.3389/fcimb.2021.634915. eCollection 2021.

Aptamer Detection of Mycobaterium tuberculosis Mannose-Capped Lipoarabinomannan in Lesion Tissues for Tuberculosis Diagnosis

Yuanyuan Zhou  1   2   3 , Huan Xiong  1   2   3 , Rong Chen  4   5 , Lixia Wan  4   5 , Ying Kong  1   2   3 , Jianwei Rao  1   2   3 , Yan Xie  1   2   3 , Chaolin Huang  4   5 , Xiao-Lian Zhang  1   2   3
Affiliations

Aptamer Detection of Mycobaterium tuberculosis Mannose-Capped Lipoarabinomannan in Lesion Tissues for Tuberculosis Diagnosis

Yuanyuan Zhou et al. Front Cell Infect Microbiol. .

Abstract

Tuberculosis (TB) is the leading infectious cause of mortality worldwide. However, the diagnosis of TB, especially extrapulmonary TB (EPTB) diagnosis from lesion tissues, remains a challenge. Nucleic acid aptamers are analogous to antibodies and have advantages of easier modification, high specificity, and affinity. Mannose-capped lipoarabinomannan (ManLAM) is a unique surface lipoglycan component or constantly released from mycobacterium tuberculosis (M.tb) cell wall, which makes it a perfect candidate biomarker for TB diagnosis. Our present study aims to establish M.tb ManLAM aptamer-based immunohistochemistry (IHC) method for TB diagnosis. We performed TB diagnosis using 263 formalin-fixed paraffin-embedded tissue samples including 213 TB samples (pulmonary TB (PTB) and EPTB), and 8 samples from latent TB infection (LTBI) high risk subjects, and 42 samples from other non-TB patients with ManLAM aptamer-based IHC and routine laboratory TB diagnostic methods parallelly. The sensitivity and specificity of the ManLAM aptamer-based IHC were 86.38% and 92.86%, with much higher sensitivity than those of mycobacterial culture (9.66%) and acid-fast staining (AFS) (43.01%) and comparability to Interferon-gamma Release Assay (IGRA) (84.38%) and GeneXpert (79.31%). High agreement between ManLAM based-IHC and IGRA or GeneXpert for TB diagnosis were observed. Furthermore, ManLAM aptamer-based IHC combination with other routine TB laboratory diagnostic methods significantly increased the sensitivity up to 88.64%-97.92%. As our knowledge, this is the first report about aptamer-based IHC for disease diagnosis. Thus, ManLAM aptamer-based IHC has potentials for TB diagnosis, including PTB, and EPTB, and assists the diagnosis of LTBI with high effectiveness, feasibility, and easy production.

Keywords: ManLAM; aptamer; diagnosis; lesion tissue; tuberculosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Much higher immunohistochemistry (IHC) scores in tuberculosis (TB) patients than those in non-TB patients. (A) Comparison of IHC scores between non-TB and TB patients, cutoff value: 0.0580 (dotted line). Data are presented as median ± IQR (inter-quantile range). (B) ROC curve for IHC score for TB patients vs. non-TB patients. (C) Comparison of IHC scores between non-TB and LTBI/PTB/EPTB patients. (D) ROC curve for IHC score for latent TB infection (LTBI) patients vs. non-TB patients, PTB patients vs. non-TB patients, and EPTB patients vs. non-TB patients. (E) The sensitivities of ManLAM based-IHC, Culture, AFS, IGRA, GeneXpert, or their combination for TB patients. The sensitivities were statistically evaluated with statistical proportion test (R prop.test). *P < 0.05, ****P < 0.0001, (Mann-Whitney U test, vs. non- TB) in (A, C).
Figure 2
Figure 2
Representative images and comparison between acid-fast staining and ManLAM-based immunohistochemistry (IHC) on lesion tissues. Representative images of (A) lesion sections of intestinal tuberculosis (TB) patient, (B, C) tissues of lymph node TB patients, and (D) lesion sections of body lumbar TB. TB, tuberculosis. Black arrows indicate positive signals (AFS by red staining, or aptamer-based IHC by brown granular staining). Samples with a score less than the cutoff value (0.0580) were designated as “-”, (≥0.0580 and <0.5010) as “+”, (≥0.5010 and <0.8780) as “++”, >0.8780 as “+++”. The numbers (0.5010 and 0.8780) were determined by calculating the 50% and 75% percentile of the Scores.
Figure 3
Figure 3
Representative images and comparison between acid-fast staining (AFS) and ManLAM aptamer-based IHC in lesion tissues with negative AFS. Representative images of (A) tissues of pulmonary tuberculosis (TB) patient, (B) lesion sections of lymph node TB patient, (C) lesion sections of LTBI patient, (D) lesion sections of non-TB patient, (E, F) lesion sections of two NTM patients. TB, tuberculosis. NTM, nontuberculous mycobacteria. Arrows indicate positive signals (aptamer-based immunohistochemistry (IHC) by brown granular staining; AFS by red staining). Samples with a score less than the cutoff value (0.0580) were designated as “-”, (≥0.0580 and <0.5010) as “+”, (≥0.5010 and <0.8780) as “++”, >0.8780 as “+++”. The numbers (0.5010 and 0.8780) were determined by calculating the 50% and 75% percentile of the Score.
Figure 4
Figure 4
Comparison of aptamer- and antibodies-based immunohistochemistry (IHC)/immunofluorescence for tuberculosis (TB) diagnosis in TB mouse model. Images of M.tb H37Rv infected mouse lung sections (A) and uninfected mouse lung sections (B) detected by acid-fast staining (AFS), aptamer-based IHC, anti-ManLAM, anti-Ag85B, or anti-Rv2645 pAbs. Arrows indicate positive signals (aptamer-based IHC by brown granular staining; AFS by red staining). (C). Images of infected mouse lung sections detected by AF488 labeled ManLAM aptamer-based immunofluorescence, anti-Rv1579, anti-Rv2645 pAbs developed with AF488-anti-IgG and Rhodamine B (RB). RB is a kind of M.tb specific fluorescence dye, used as a positive control. Samples with a score less than the cutoff value (0.0580) were designated as “-”, (≥0.0580 and <0.5010) as “+”, (≥0.5010 and <0.8780) as “++”, >0.8780 as “+++”. The numbers (0.5010 and 0.8780) were determined by calculating the 50% and 75% percentile of the Score.
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