Diagnosis of tuberculous lymphadenopathy: the role of droplet digital PCR

Background: Tuberculous lymphadenopathy (TB-LAP) is the most common form of extrapulmonary TB and remains diagnostically challenging due to its paucibacillary nature. We evaluated the diagnostic performance of droplet digital PCR (ddPCR) for detecting Mycobacterium tuberculosis (Mtb) DNA in formalin-fixed paraffin-embedded (FFPE) lymph node specimens. Methods: We retrospectively analysed 102 lymph node FFPE specimens with microbiologically confirmed or pathology-suspected TB-LAP. We performed ddPCR targeting the IS6110 gene and correlated it with pathological findings and mycobacterial culture results. Diagnostic performance was assessed using receiver operating characteristic analysis. Results: Pathological findings included granulomatous inflammation (GI) (n = 67), caseating GI (CGI) (n = 22), CGI with acid-fast bacilli (AFB) (n = 7), GI with AFB (n = 1), and atypical findings (n = 5). There was a graded association between ddPCR and pathological severity (P for trend = 0.002), with the highest levels in CGI plus AFB and the lowest in non-TB-LAP cases. Mtb DNA concentration (copies/μL) was significantly higher in AFB-positive specimens (mean (x̄) = 22.9, standard deviation (SD) = 61.5) compared with AFB-negative specimens (x̄ = 0.6, SD = 0.8) (P = 0.044). Among 52 specimens sent for mycobacterial culture, 9 (17%) were culture-positive, and these specimens had significantly higher ddPCR values (P = 0.025). Receiver operating characteristic analysis yielded an area under the curve of 0.715 (95% confidence interval = 0.536-0.894) for AFB positivity. Using a cutoff value of >1.25 copies/μL resulted in 50% sensitivity and 86% specificity. Lowering the cutoff to >0.33 copies/μL increased sensitivity to 88%, but reduced specificity to 51%. Conclusions: We found that ddPCR quantitatively detects Mtb DNA in lymph node FFPE specimens and correlates with pathological severity and culture positivity. Although the overall diagnostic accuracy was moderate, ddPCR demonstrated distinct sensitivity and specificity profiles at different cutoff values and may serve as a useful adjunctive diagnostic tool when interpreted in conjunction with clinical and histopathological findings in TB-LAP.