Patients and specimen collection
One hundred twenty-nine patients with suspicion of osteoarticular TB in the Jiangmen Central Hospital orthopedics department from 2016 to 2020 were included in this study. The enrolled patients suffered from severe bone damage and had to undergo inpatient surgery. Of them, 39 cases were excluded based on diagnostic criteria for skeletal TB, including clinical presentation, pathological examination, radiographic images, and the efficacy of antimycobacterial therapy (6–24 months). The remaining 90 subjects were confirmed to have skeletal TB. After surgery, FFPE tissues were prepared according to standard procedures and serial sections for each case were used for histopathological analysis and DNA isolation. In addition, all samples were subjected to pathological examination, qPCR, and AFS. The study was carried out by the Declaration of Helsinki and approved by the Ethics Committee of the Jiangmen Central Hospital. Informed consent was obtained from all participants.
DNA extraction
Five slices (6 μm) per case were pooled from paraffin blocks of skeletal tissues for DNA extraction. Genomic DNA was isolated manually using the TIANGEN FFPE DNA Kit (#DP330, TIANGEN Biotech; Beijing, China) according to the manufacturer’s protocol. In brief, the sections were vortexed vigorously in 1 mL xylene for 1 min and then centrifuged at 12,000 rpm for 2 min. After removing xylene, 1 mL absolute ethanol was added and vortexed for 20 s, followed by centrifugation at 12,000 rpm for 2 min. Next, the ethanol was pipetted off, and the Eppendorf tube was left at room temperature to evaporate residual ethanol. Then, 1 mL TE (pH = 9.9) was added and rotated at 95 °C (1000 rpm, 10 min), ended by centrifugation at 12,000 rpm for 2 min. After carefully removing the supernatant, the pellet was collected. The resulting DNA was stored at − 20 °C until use.
Quantitative PCR (qPCR)
Aliquots from the extracted DNA were submitted to mycobacteria qPCR. We used a commercial kit (Guangdong Bright-Innovation BioMed Co., Ltd., Shunde, China) to target three mycobacterial DNA sequences, IS6110, HSP65, and ITS, respectively, for differential detection of M. tuberculosis, M. abscessus complex (MABC) and M. avium complex (MAC). In fact, DNA sequences of various subspecies of MABC including M. abscessus, M. massiliense and M. bolletii or MAC including M. intracellulare and M. avium, are highly homologous. In addition, the treatment regimen for patients affected by the different subspecies of MABC or MAC is consistent. Therefore, we designed primers and probes to target MABC and MAC, rather than their subspecies. The qPCR assay consisted of a DNA template, forward primer, reverse primer, probe, and PCR mix buffer in a 25 µL. The cycling conditions shared for three targets were as follows: 1 cycle at 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s, one cycle at 72 °C for 30 s, and collection of Taqman probe fluorescence signal at 60 °C for 30 s according to the manufacturer’s instructions. Analyses of M. tuberculosis, MABC and MAC were separately conducted in three tubes for each DNA sample. A blank control, negative control, and a positive sample were tested in parallel in each qPCR run. The housekeeping gene β-actin (ACTB) was used as an internal reference to control for the absence/presence of inhibitors and the quality of the samples after extraction. The cycle threshold (Ct) values were measured at a fixed fluorescence threshold. Samples with Ct > 40 were considered negative. Repeated experiments were conducted for cases with qPCR-negative but AFS-positive. All analyses were performed on a SLAN®96P Real-Time PCR System (Shanghai Hongshi Medical Technology, China).
Acid-fast bacillus staining (AFS)
Qualified pathologists used a modified Wade-Fite staining technique for AFS. Briefly, tissue sections (6 μm) were oven-baked at 65 °C for 2 h and deparaffinized in a mixture of gasoline and turpentine for two changes, 30 min each. Then, sections were rinsed with running water for 5 min and dropped into a basic Fuchsin solution at room temperature for 10 min, followed by washing and differentiation with 20% sulfuric acid for 3 min. As a result, the sections should be pale pink or colorless under microscopy. Finally, sections were counter stained with 0.1% methylene blue, dehydrated in graded alcohols, dipped in xylene to clear, and mounted in neutral gums. Acid-fast bacilli, including leprosy bacilli and tuberculosis bacilli, were colored bright red, nuclei, and background were stained blue.
Statistical analysis
Statistical analyses were calculated using the SPSS 26.0 software. The Mann–Whitney U test was used for continuous variables between two groups. All tests were two-sided, and p < 0.05 was considered statistically significant. To assess the sensitivity and specificity of qPCR along with AFS and further compare their ability to diagnose skeletal TB, we constructed receiver operating characteristic (ROC) curves and calculated the area under the curve (AUC) using deLong's test of the R Programming Language. The percentage of mycobacteria DNA relative to human cell DNA was calculated using the following formula: 2(Ct ACTB − Ct Target) × 100 [10].
