Supplementary Data for the journal article "Cervical Lymph Node TB: Diagnostic yield and patient profile"

<b>BACKGROUND:</b> Cervical lymph node tuberculosis (CL-TB) is the most prevalent form of extrapulmonary TB, yet it remains underdiagnosed in endemic settings due to nonspecific symptoms and inconsistent diagnostic pathways. We aimed to identify socio-demographic and clinical predictors of CL-TB in patients attending a tertiary hospital in Bangladesh and evaluate the diagnostic yield of available tests. <b>METHODS:</b> <b>Study design:</b>This cross-sectional study was nested within the "Diagnostic Algorithm for Peripheral Lymph Node Tuberculosis Using Portable Station/Mobile-TB-Lab" study, a multi-country diagnostic evaluation spanning Germany, Uganda, and Bangladesh. In Bangladesh, the study site was Bangladesh Medical University (BMU). The study population was selected from the patients with lymphadenitis (LN swelling) arriving at the BMU Internal Medicine Outpatient Department (OPD), presenting with one or more enlarged, palpable cervical LNs, and either chronic low-grade fever (&gt;2 weeks), or a history of contact with TB patients, unexplained weight loss, or cough. Patients with known diagnoses of cancer or infections other than tuberculosis were excluded from the study. We screened 3,619 patients from January 2024 to August 2024 and enrolled 104 patients (Figure 1). <b>Study procedure:</b>Following informed consent, we collected demographic and clinical data from each patient using a semi-structured questionnaire. The study physician performed a routine physical examination, maintaining the privacy of the patients. Fine Needle Aspiration (FNA) from the affected LNs was then performed, adhering to standard aseptic techniques for microscopic examination for AFB and cytological evaluation. The remaining samples were utilized for AFB culture, Real-Time Multiplex PCR (RT-PCR (Multiplex)), and GeneXpert (Cepheid, USA). We described the methodology for Culture, Real-Time Multiplex PCR, and GeneXpert in the supplementary file.Following the dispatch of diagnostic findings, we informed patients and assisted them in registering at the DOT center to ensure TB treatment. The diagnostic findings were used to stratify participants into CL-TB and non-CL-TB groups (52 in each group) based on the following operational definitions.CL-TB Cases: Any participant regarded by the clinicians as a presumptive case of tuberculous lymphadenitis (TBLN) and is positive through either AFB microscopy and/or cytopathology and/or culture and/or RT-PCR (Multiplex) and/or GeneXpert. Non-CL-TB: Participants with presumptive CL-TB with negative diagnostic test results following AFB microscopy, cytopathology, culture, RT-PCR (Multiplex), and GeneXpert (Figure 1).As we considered any positive test indicative of TB in line with clinical practice and used a composite reference standard, diagnostic yield was reported instead of test accuracy. We chose this approach due to the limitations of culture as a gold standard for LNTB, including its suboptimal sensitivity and a reduced incubation period of 8 weeks in our study.<b>Sample size:</b>We calculated the sample size considering the incidence of CL-TB in Bangladesh for the multi-country study. Assuming a CL-TB incidence of 3% among presumed participants presenting at a tertiary-level hospital derived from a working paper, and a confidence interval (CI) of 95% with a precision rate of 3.5%, the sample size was estimated at ninety-one participants. We considered an attrition rate of 10%, bringing the final sample size to 101. However, we successfully enrolled 104 participants after screening 3,619 patients. <b>Statistical analysis:</b>We used Stata 15 (by StataCorp LLC) for the statistical analysis of the data. Descriptive analysis determined the frequency and percentage of each variable. Chi-square tests were performed to assess the relationship between the independent and outcome variables. We used binary and multivariate logistic regression to estimate the association between outcome and independent variables following adjustments for statistically significant factors derived from the univariate analyses. Given the modest sample size (N = 104), we considered a p-value of &lt;0.10 and a 95% CI to indicate statistical significance during analysis, as this threshold has been recommended in small-sample studies to reduce the risk of Type II error and to help identify emerging patterns for further validation. <b>Ethical approval:</b>Since this was a multi-country study (PR-23031), the Ethical Review Committee (ERC) of icddr,b, Leipzig University, Germany, and Makerere University, Uganda, approved it. We executed this study following the Declaration of Helsinki.<br><b>RESULTS: </b>Among 104 participants, 52 were confirmed to have CL-TB by either microscopy, cytopathology, GeneXpert, PCR, or culture. Younger age (18–30 years) and purulent aspirate appearance were independently associated with CL-TB. Cytopathology showed the highest diagnostic yield (82.7%), followed by GeneXpert (71.2%) and PCR (67.3%), while microscopy and culture detected &lt;7% of CL-TB cases. <b>CONCLUSION: </b>Combinations of cytopathology with GeneXpert or PCR yielded near-perfect CL-TB detection. This was the first study in Bangladesh to utilize LN aspirates directly for PCR alongside cytopathology, GeneXpert, microscopy, and culture, demonstrating a feasible diagnostic approach in high-burden settings like Bangladesh and offering improved detection and reduced delay.