In this study, we enrolled 95 patients with presumptive TBLN. Of these, 2 patients were excluded for not meeting the inclusion criteria and 4 patients were excluded for not having sufficient specimens for the study. Of 89 patients included in this study, females accounted for 57.3% (51 of 89). The age of the included patients ranged from 15 to 68 years, with a mean age of 30.9 years (± 11.7). All the patients tested negative for HIV. The majority [96.6% (86 of 89)] of the cases involved lymph node sites in the cervical regions. The specimens were obtained by FNA and direct biopsy for 50 and 39 cases, respectively. The histological examination demonstrated that 82 patients (92.1%) had inflammation features, 2 (2.2%) were considered as sarcoidosis, and 5 cases (5.6%) were malignancy.
Among patients with TBLN, 52 (63.4%) were selected for initial treatment and the remaining 30 (36.6%) were chosen for retreatment. Among these 82 TBLN lymph node specimens, 22 (26.8%) were obtained before anti-TB treatment, 19 (23.2%) from 1 day to 1 month after treatment, 17 (20.7%) from over 1 month to 3 months treatment, 20 (24.4%) from over 3 months treatment, and 4 (4.9%) cases were unknown.
For FFPE samples, granulomas were found in 58.5% (48 of 82) patients with TBLN, whereas AFB were detected in 43.9% (36 of 82) patients. The sensitivity of TB-PCR on FFPE samples (FFPE-PCR) was 69.5% (57 of 82) and was significantly higher than that of ZN staining (χ2 = 10.953, P = 0.001). Not only FFPE-PCR gave positive results in 86.1% (31 of 36) ZN staining positive samples, but also detected additional 56.5% (26 of 46) cases in ZN staining negative samples. When the histological examination was combined with FFPE-PCR, the sensitivity reached up to 82.9% (68 of 82).
The sensitivities of culture and Xpert on fresh samples were 22.0% (18 of 82) and 86.6% (71 of 82), respectively. Apparently, Xpert showed significantly higher sensitivity than culture (χ2 = 69.015, P < 0.0001). Although all the culture positive samples were Xpert positive, Xpert detected almost three times more positive cases compared to culture.
In 2 sarcoidosis and 5 malignant cases, culture, Xpert, ZN staining, and FFPE-PCR tests gave negative results, thereby showing good specificity. In addition, histology also indicated granulomas in 2 sarcoidosis samples, but was ruled out of TB upon combining the clinical information.
These diagnostic methods were also analyzed in patients with TBLN having different anti-TB treatment background. We found that the anti-TB treatment history had a great influence on the sensitivity of culture (Figure 1). In the initially treated group, the sensitivity of culture was 46.2% (24 of 52), whereas in the retreated group, the sensitivity dramatically decreased to 3.3% (1 of 30; χ2 = 16.460, P < 0.0001; Figure 1A). However, histology, ZN staining, Xpert, and FFPE-PCR did not differ significantly in the two groups (P > 0.05; Figure 1A). Furthermore, we compared different diagnostic methods with samples collected before treatment, with anti-TB treatment for 1 day to 1 month, with anti-TB treatment for over 1 month to 3 months, and with anti-TB treatment for over 3 months. The sensitivities of culture were 45.5% (10 of 22), 52.6% (10 of 19), 17.6% (3 of 17), and 0.0% (0 of 20) for the four different time periods, respectively. Yet again, the sensitivity of culture decreased dramatically after 1 month of treatment, and gave no positive results when the treatment lasted over 3 months (Figure 1B). For Xpert, the sensitivity was stable for 3-month treatment with over 86%, but decreased significantly to 70% when the treatment lasted over 3 months (χ2 = 7.798, P = 0.029, Fisher's exact test; Figure 1B). This implies that the length of anti-TB treatment did not affect the sensitivities of histology, ZN staining, and FFPE-PCR (P > 0.05; Figure 1B).
Figure 1. Comparison of sensitivities of different diagnostic methods on lymph node samples with different anti-TB treatment background. (A) Comparison of different diagnostic methods in initial treatment and retreatment groups; (B) Comparison of different diagnostic methods in different anti-TB treatment time groups. *P < 0.05, **P < 0.001, ***P < 0.0001, NSP > 0.05.
In this study, 1 patient with TBLN reported enlargement of the lymph node after 40 days of treatment. To clarify whether it resulted from failure of the treatment or was just a paradoxical reaction, biopsy was performed. After biopsy, both Xpert and FFPE-PCR showed positive results. To elaborate, quantification of the M. tuberculosis amount by FFPE-PCR revealed a huge amount of bacilli, 8.3 × 105 cfu/mL (1660 cfu/per section), in the sample. Detection of Ag85B antigen from M. tuberculosis by immunohistochemistry showed abundant M. tuberculosis bacilli in the tissue (Figure 2B and C) However, culture gave negative results and ZN staining only showed a few AFB in the sample (Figure 2A). Both molecular test and immunohistochemistry only detect nucleic acid or protein but not the intact live bacilli. However, culture only detects live bacilli whereas ZN staining only detects intact bacilli. These results suggested that most bacilli detected in this case were cell wall-deficient and might be in a nonactive state or not alive. Rifampicin susceptibility test by FFPE-HRM also showed no mutation in rpoBRRDR region, suggesting rifampicin sensitive case. From these results, we concluded that anti-TB treatment was effective and the enlargement of lymph node was just a paradoxical reaction.
Figure 2. Ziehl-Neelsen staining and M. tuberculosis Ag85B immunohistochemistry staining on lymph node tissues from 2 patients with TBLN having different anti-TB treatment background. (A to C) from a patient with TBLN after 40 days of anti-TB treatment; (D to F) from a patient with TBLN without anti-TB treatment. (A and C) ZN staining of AFB (× 100 oil immersion); (B, C, E, and F) immunohistochemistry staining to detect Ag85B expression (× 40 magnification).
All these results indicated that Xpert was the most sensitive test, and FFPE-PCR, histology, and ZN staining descended from higher sensitivity to lower sensitivity. Of all, culture was the most insensitive method and its sensitivity was dramatically influenced by the anti-TB treatment history.
In this study, rifampicin susceptibility was tested by culture, Xpert, and HRM on FFPE samples (FFPE-HRM), all of which gave successful DST results for 22, 69, and 30 samples, respectively (Table 1). Apparently, Xpert showed significantly higher success rate than that of culture (χ2 = 54.535, P < 0.0001) and FFPE-HRM (χ2 = 38.764, P < 0.0001). The success rates of culture and FFPE-HRM were not significantly different (P > 0.05), and these two methods showed rather complementary results. Only eight samples were successful by both culture and FFPE-HRM; however, 14 samples were successful by culture but failed FFPE-HRM, and 22 samples were successful by FFPE-HRM but failed culture (Table 1).
Counts Xpert Successful Xpert Failed Culture successful Culture failed Culture successful Culture failed FFPE-HRM successful 8 (1 rifampicin resistant) 22 (0 rifampicin resistant) 0 0 FFPE-HRM failed 14 (1 rifampicin resistant) 25 (1 rifampicin resistant) 0 13
Table 1. Performance of Xpert MTB/RIF, mycobacterial culture, and high-resolution melting curve tests on determining rifampicin susceptibility
Evidently, the success rate of culture was significantly affected by anti-TB treatment. In the initially treated group, the success rate of culture was 40.4% (21 of 52), whereas in the retreated group, the success rate dramatically decreased to 3.3% (1 of 30; χ2 = 13.304, P < 0.001; Figure 3A). The success rates were 36.4% (8 of 22), 47.4% (9 of 19), 17.6% (3 of 17), and 0.0% (0 of 20) in the four different time periods of receiving anti-TB treatment, respectively. Once again, the success rate of culture decreased dramatically after 1 month of treatment and gave no positive results when the treatment lasted over 3 months (χ2 = 14.948, P = 0.001; Figure 3B). However, the anti-TB treatment history did not affect the success rate of Xpert or FFPE-HRM (P > 0.05; Figure 3).
Figure 3. Comparison of performance of different rifampicin susceptibility tests on lymph node samples with different anti-TB treatment background. (A) Comparison of different rifampicin susceptibility tests in initial treatment and retreatment groups; (B) Comparison of different rifampicin susceptibility tests in different anti-TB treatment time groups. *P < 0.01, **P < 0.001, NSP > 0.05.
The Xpert method detected all three rifampicin-resistant samples, two of which were confirmed by culture, and one was confirmed by both culture and FFPE-HRM (Table 1). The rifampicin-resistant rate detected by culture, Xpert, and FFPE-HRM was 9.1% (2 of 22), 4.3% (3 of 69), and 3.3% (1 of 30). All the DST results of culture or FFPE-HRM were in perfect match with Xpert, in addition, the DST result from eight samples that was detected by both culture and FFPE-HRM was in perfect accordance.
These result suggested that the anti-TB treatment background had significant influence on the success rate of culture, but not on molecular DSTs. Moreover, genotypic DSTs were in good accordance with phenotypic DST. The Xpert method was superior to culture and FFPE-HRM in detecting rifampicin resistance.