Tuberculosis (TB) remains a major global public health problem and is one of the leading causes of morbidity and mortality due to infectious diseases worldwide. According to global estimates, millions of new cases are reported annually, with India contributing a substantial proportion of the global burden¹. While pulmonary tuberculosis is the most common form, extrapulmonary tuberculosis (EPTB) constitutes approximately 15–20% of all cases in immunocompetent individuals and up to 50% in immunocompromised patients². Among the various forms of EPTB, tuberculous lymphadenitis is the most frequent manifestation, particularly affecting cervical lymph nodes³. Lymph node tuberculosis typically presents as painless lymphadenopathy, which may later progress to matting, caseation, or sinus formation. However, the clinical presentation is often nonspecific and overlaps with other conditions such as reactive lymphadenitis, lymphoma, metastatic malignancy, and other granulomatous diseases like sarcoidosis and fungal infections⁴. This overlap creates significant diagnostic challenges, necessitating the use of reliable laboratory methods for accurate diagnosis. Fine needle aspiration cytology (FNAC) has been widely utilized as a first-line diagnostic modality for evaluating lymphadenopathy. It is a minimally invasive, rapid, and cost-effective procedure that provides valuable morphological information. Cytological features suggestive of tuberculous lymphadenitis include epithelioid cell granulomas, Langhans giant cells, and caseous necrosis⁵. Depending on the stage of the disease and host immune response, the cytological patterns may vary from well-formed granulomas to purely necrotic or suppurative lesions⁶. However, despite its high sensitivity, FNAC lacks specificity because similar cytological features can be observed in other granulomatous conditions⁴. Microbiological confirmation of Mycobacterium tuberculosis remains the gold standard for diagnosis. Conventional diagnostic methods such as Ziehl–Neelsen (ZN) staining for acid-fast bacilli (AFB) and mycobacterial culture have inherent limitations. ZN staining has low sensitivity, especially in paucibacillary extrapulmonary samples, while culture methods, though more sensitive, are time-consuming and may take several weeks to yield results⁷. These limitations often lead to delays in diagnosis and initiation of appropriate therapy. The advent of nucleic acid amplification tests (NAATs), particularly the Gene Xpert MTB/RIF assay, has significantly transformed the diagnostic landscape of tuberculosis. This cartridge-based, automated molecular test can detect Mycobacterium tuberculosis complex DNA and rifampicin resistance within a short turnaround time of approximately two hours⁸. Gene Xpert has demonstrated high specificity and good sensitivity even in extrapulmonary samples, making it a valuable diagnostic tool in routine clinical practice⁹. Furthermore, early detection of rifampicin resistance is crucial for the prompt initiation of appropriate anti-tubercular therapy and for preventing the spread of drug-resistant TB. Despite the advantages of both cytopathology and Gene Xpert, discrepancies between the two diagnostic modalities are not uncommon. FNAC may identify granulomatous inflammation in cases where bacillary load is insufficient for molecular detection, resulting in Gene Xpert-negative results¹⁰. Conversely, Gene Xpert may detect mycobacterial DNA in samples that are cytologically inconclusive or lack classical features of tuberculosis. These discordant findings highlight the complementary nature of morphological and molecular diagnostic approaches. In resource-limited settings, where tuberculosis is highly prevalent, reliance on a single diagnostic modality may lead to underdiagnosis or misdiagnosis. Therefore, integrating cytopathology with molecular techniques could enhance diagnostic accuracy, facilitate early treatment initiation, and improve patient outcomes. In this context, the present study aims to evaluate the concordance between FNAC and Gene Xpert MTB/RIF assay in diagnosing lymph node tuberculosis and to assess the utility of a dual diagnostic approach in routine clinical practice.

Study Design and Setting: This prospective observational study was conducted in the Departments of Microbiology and Pathology at a tertiary care teaching hospital over a period of 12 months. The study aimed to evaluate the diagnostic concordance between fine needle aspiration cytology (FNAC) and Gene Xpert MTB/RIF assay in patients presenting with lymphadenopathy clinically suspected of tuberculous etiology. Study Population: A total of 120 consecutive patients presenting to outpatient and inpatient departments with lymph node enlargement were enrolled in the study. Patients of all age groups and both genders were included. A detailed clinical history including duration of swelling, associated symptoms such as fever, weight loss, night sweats, and past history of tuberculosis was recorded. Physical examination findings such as size, site, consistency, matting, and presence of sinus formation were documented. Inclusion Criteria • Patients with lymphadenopathy persisting for more than two weeks • Clinical suspicion of tuberculous lymphadenitis • Patients consenting to undergo FNAC and molecular testing Exclusion Criteria • Patients already receiving anti-tubercular therapy • Known cases of malignancy involving lymph nodes • Patients with bleeding disorders contraindicating FNAC • Inadequate or insufficient aspirated material Sample Size and Sampling Technique A total sample size of 120 was determined based on feasibility and patient load during the study period. Consecutive sampling technique was used to avoid selection bias. Procedure for Sample Collection Fine needle aspiration was performed under aseptic conditions using a 22–23 gauge needle attached to a 10 mL disposable syringe. The most prominent or accessible lymph node was selected for aspiration. In cases of multiple lymph nodes, the largest node or one showing signs of fluctuation was chosen. The aspirated material was divided into two portions: 1. Cytopathological examination 2. Gene Xpert MTB/RIF assay In cases where material was scanty, priority was given to preparing adequate cytology smears, and residual material was used for molecular testing. Cytopathological Examination Smear Preparation and Staining Aspirated material was smeared onto clean glass slides. Air-dried smears were stained with May-Grünwald Giemsa (MGG), while alcohol-fixed smears were stained with Hematoxylin and Eosin (H&E). Additional smears were stained using Ziehl–Neelsen (ZN) staining for detection of acid-fast bacilli (AFB). Cytological Evaluation Smears were examined independently by experienced cytopathologists. Cytomorphological features were analyzed, including: • Presence of epithelioid cell granulomas • Caseous necrosis • Langhans giant cells • Background inflammatory infiltrate Based on cytological findings, cases were categorized into the following groups: 1. Granulomatous lymphadenitis with necrosis 2. Granulomatous lymphadenitis without necrosis 3. Suppurative lymphadenitis 4. Reactive lymphadenitis Cases showing granulomas with or without necrosis, as well as suppurative lesions with strong clinical suspicion, were considered suggestive of tuberculosis. Ziehl–Neelsen Staining: ZN staining was performed on selected smears to detect AFB. Slides were examined under oil immersion (1000× magnification). AFB positivity was graded according to standard guidelines. However, due to the known low sensitivity in extrapulmonary samples, ZN staining was considered supplementary. Gene Xpert MTB/RIF Assay: Sample Processing A portion of the aspirated material was transferred into a sterile container and processed according to the manufacturer’s protocol for Gene Xpert MTB/RIF assay. The sample was mixed with the provided sample reagent in a 2:1 ratio and incubated for 15 minutes at room temperature with intermittent shaking to ensure proper liquefaction and inactivation. Test Procedure The processed sample was transferred into the Gene Xpert cartridge, which was then loaded into the Gene Xpert instrument. The system performs automated sample processing, nucleic acid amplification, and real-time detection of Mycobacterium tuberculosis complex DNA. Interpretation of Results Results were categorized as: • MTB detected (with semi-quantitative grading: very low, low, medium, high) • MTB not detected • Rifampicin resistance detected • Rifampicin resistance not detected • Invalid/error results (retested) Quality Control Measures • All staining procedures were performed using standardized protocols. • Internal quality control of Gene Xpert was ensured through built-in controls in each cartridge. • Periodic calibration of the Gene Xpert instrument was maintained. • Duplicate slide examination was performed in doubtful cases to reduce observer bias. Data Collection and Management All clinical, cytological, and molecular data were recorded in a predesigned proforma. Data included demographic details, clinical features, cytological diagnosis, ZN staining results, and Gene Xpert findings. Statistical Analysis Data were entered into Microsoft Excel and analyzed using SPSS software (version XX). Sensitivity, Specificity, PPV and NPV was calculated. Gene Xpert was considered as the reference standard for microbiological confirmation. Concordance between FNAC and Gene Xpert was assessed using Cohen’s kappa coefficient (κ) and interpreted as: • <0.20: Poor agreement • 0.21–0.40: Fair agreement • 0.41–0.60: Moderate agreement • 0.61–0.80: Substantial agreement • 0.80: Almost perfect agreement A p-value of <0.05 was considered statistically significant. Ethical Considerations The study was conducted after obtaining approval from the Institutional Ethics Committee (Ref No: GVPIHCMT/IEC/20250909/07). Written informed consent was obtained from all participants prior to inclusion in the study. Patient confidentiality was strictly maintained throughout the study. Outcome Measures The primary outcome was to assess the concordance between FNAC and Gene Xpert in diagnosing lymph node tuberculosis. Secondary outcomes included evaluation of diagnostic accuracy of each method and detection of rifampicin resistance.

A total of 120 patients with clinically suspected tuberculous lymphadenitis were included in the study. All samples were subjected to cytopathological examination and Gene Xpert MTB/RIF assay. 1. Demographic Profile Out of the 120 patients, 70 (58.3%) were females and 50 (41.7%) were males, with a female predominance (M:F ratio = 1:1.4). The age of patients ranged from 5 to 68 years, with a mean age of 32.4 ± 12.6 years. The majority of patients belonged to the 21–40 years age group, accounting for 54 cases (45%), followed by 41–60 years (30 cases, 25%), <20 years (24 cases, 20%), and >60 years (12 cases, 10%). This indicates that tuberculous lymphadenitis predominantly affects the young and middle-aged population. 2. Clinical Characteristics The most common site of lymphadenopathy was cervical lymph nodes, observed in 84 cases (70%), followed by axillary nodes in 18 cases (15%), inguinal nodes in 10 cases (8.3%), and other sites (supraclavicular, submandibular) in 8 cases (6.7%). The duration of lymph node swelling ranged from 2 weeks to 6 months. Constitutional symptoms such as fever (60%), weight loss (48%), and night sweats (35%) were frequently reported. Matting of lymph nodes was observed in 40 cases (33%), while sinus formation was seen in 10 cases (8.3%). 3. Cytopathological Findings Based on FNAC, cytological patterns were categorized as follows: • Granulomatous lymphadenitis with necrosis: 60 cases (50%) • Granulomatous lymphadenitis without necrosis: 25 cases (20.8%) • Suppurative lymphadenitis: 15 cases (12.5%) • Reactive lymphadenitis: 20 cases (16.7%) Thus, 85 out of 120 cases (70.8%) were considered cytologically suggestive of tuberculosis (granulomatous ± necrosis and suppurative lesions with clinical suspicion). Ziehl–Neelsen staining for AFB was positive in 30 cases (25%), predominantly in smears showing necrosis and suppuration. AFB positivity was highest in granulomatous lymphadenitis with necrosis (20 cases), followed by suppurative lesions (8 cases), and was rarely seen in granulomatous lesions without necrosis (2 cases). 4. Gene Xpert MTB/RIF Results Gene Xpert MTB/RIF assay detected Mycobacterium tuberculosis in 78 out of 120 cases (65%), while 42 cases (35%) were negative. Among the positive cases, semi-quantitative grading showed: • Very low: 20 cases (25.6%) • Low: 30 cases (38.5%) • Medium: 18 cases (23.1%) • High: 10 cases (12.8%) Rifampicin resistance was detected in 6 cases (5% of total cases and 7.7% of Gene Xpert positives), while the remaining 72 cases were rifampicin-sensitive. 5. Correlation Between Cytopathology and Gene Xpert A detailed comparison between FNAC and Gene Xpert results is shown below: Cytology Category Total Cases Gene Xpert Positive Gene Xpert Negative Granulomatous with necrosis 60 50 10 Granulomatous without necrosis 25 12 13 Suppurative 15 10 5 Reactive 20 6 14 Gene Xpert positivity was highest in granulomatous lymphadenitis with necrosis (83.3%), followed by suppurative lesions (66.7%). Lower positivity was observed in granulomatous lesions without necrosis (48%), indicating a possible lower bacillary load. Interestingly, 6 cases categorized as reactive lymphadenitis on cytology were Gene Xpert positive, suggesting early or subclinical infection. 6. Concordance Analysis The concordance between FNAC and Gene Xpert was assessed as follows: • Both FNAC and Gene Xpert positive: 72 cases • FNAC positive, Gene Xpert negative: 13 cases • FNAC negative, Gene Xpert positive: 6 cases • Both negative: 29 cases The overall concordance rate was 85% (102/120 cases). Cohen’s kappa coefficient (κ) was calculated to be 0.70, indicating substantial agreement between the two diagnostic modalities. 7. Diagnostic Accuracy Considering Gene Xpert as the reference standard: • Sensitivity of FNAC: 92.3% • Specificity of FNAC: 69% • Positive Predictive Value (PPV): 84.7% • Negative Predictive Value (NPV): 82.9% Alternatively, when FNAC was considered as the screening tool: • Sensitivity of Gene Xpert: 84.7% • Specificity of Gene Xpert: 69% These findings suggest that FNAC is highly sensitive in detecting tuberculous lymphadenitis, while Gene Xpert provides higher specificity and microbiological confirmation. 8. Discordant Cases Analysis A total of 19 discordant cases (15.8%) were observed: FNAC Positive, Gene Xpert Negative (13 cases) • Majority were granulomatous lymphadenitis without necrosis • Possible reasons include low bacillary load and sampling variability Gene Xpert Positive, FNAC Negative (6 cases) • Cytology reported as reactive lymphadenitis Indicates early infection or inadequate sampling for cytological interpretation

Tuberculous lymphadenitis remains the most common form of extrapulmonary tuberculosis, particularly in high-burden countries like India. The present study evaluated the diagnostic concordance between fine needle aspiration cytology (FNAC) and Gene Xpert MTB/RIF assay in 120 clinically suspected cases, highlighting the complementary role of cytomorphology and molecular diagnostics in routine clinical practice. In this study, a female predominance (58.3%) was observed, with the majority of patients belonging to the 21–40 years age group. This demographic pattern is consistent with previous studies, which have reported higher prevalence among young adults and females, possibly due to greater healthcare-seeking behavior and immunological factors⁸. Cervical lymph nodes were the most commonly involved site (70%), aligning with established literature that identifies cervical lymphadenopathy as the hallmark presentation of tuberculous lymphadenitis³. FNAC demonstrated a high sensitivity (92.3%) in the present study, reaffirming its role as a reliable initial diagnostic tool. The most frequent cytological pattern observed was granulomatous lymphadenitis with necrosis (50%), which is considered highly suggestive of tuberculosis⁵. Similar findings have been reported by Pandit et al., who emphasized the diagnostic value of necrotizing granulomas in FNAC smears⁸. However, granulomatous inflammation without necrosis and suppurative patterns were also observed, reflecting the spectrum of host immune responses and disease stages⁶. These variations highlight the inherent limitation of FNAC in differentiating tuberculosis from other granulomatous conditions such as sarcoidosis and fungal infections⁴. Ziehl–Neelsen (ZN) staining showed a relatively low positivity rate (25%), which is consistent with its known limited sensitivity in extrapulmonary and paucibacillary specimens⁷. This reinforces the need for more sensitive diagnostic modalities, particularly in lymph node tuberculosis where bacillary load is often low. The Gene Xpert MTB/RIF assay detected Mycobacterium tuberculosis in 65% of cases, demonstrating high specificity and rapid turnaround time. The assay also identified rifampicin resistance in 5% of cases, which is clinically significant as it enables early detection of drug-resistant tuberculosis and timely initiation of appropriate therapy⁹. Comparable studies have reported Gene Xpert sensitivity ranging from 60% to 85% in extrapulmonary samples, supporting its utility in lymph node tuberculosis⁹. The concordance rate between FNAC and Gene Xpert in this study was 85%, with a Cohen’s kappa value of 0.70, indicating substantial agreement. This finding is in agreement with previous studies that have reported concordance rates ranging from 75% to 90%¹⁰. Despite this high level of agreement, discordant results were observed in 15.8% of cases, underscoring the limitations of each diagnostic modality when used in isolation. FNAC-positive but Gene Xpert-negative cases (13 cases) were primarily granulomatous lymphadenitis without necrosis. This may be attributed to low bacillary load, uneven distribution of organisms, or inadequate sample volume for molecular testing¹⁰. In such cases, cytomorphological evidence plays a crucial role in guiding clinical decision-making, especially in endemic regions where empirical therapy is often initiated based on cytological findings. Conversely, Gene Xpert-positive but FNAC-negative cases (6 cases) were reported as reactive lymphadenitis on cytology. These findings may represent early stages of infection where characteristic granulomatous features have not yet developed, or may be due to sampling error during FNAC. Similar observations have been documented in previous studies, highlighting the superior sensitivity of molecular techniques in detecting mycobacterial DNA even in the absence of classical cytological features⁹. A notable observation in the present study was the strong correlation between necrosis on cytology and Gene Xpert positivity. Cases with granulomatous lymphadenitis with necrosis showed the highest positivity (83.3%), suggesting that necrotic material contains a higher bacillary load. This finding is consistent with earlier reports that emphasize the importance of targeting necrotic areas during aspiration to improve diagnostic yield¹⁰. Suppurative lesions also demonstrated moderate Gene Xpert positivity, indicating that tuberculosis should be considered in the differential diagnosis of suppurative lymphadenitis, particularly in endemic settings. The diagnostic accuracy of FNAC and Gene Xpert highlights their complementary roles. FNAC, with its high sensitivity, serves as an excellent screening tool, enabling rapid identification of suspected cases. On the other hand, Gene Xpert provides microbiological confirmation and detects rifampicin resistance, thereby enhancing specificity and guiding appropriate therapy⁶. The integration of both modalities reduces the likelihood of false-negative and false-positive diagnoses, ultimately improving patient outcomes. From a public health perspective, early and accurate diagnosis of lymph node tuberculosis is essential to reduce disease transmission and prevent complications. The World Health Organization recommends the use of molecular diagnostic tests such as Gene Xpert as the initial test in suspected cases of extrapulmonary tuberculosis⁶. However, in resource-limited settings, reliance solely on molecular methods may not be feasible due to cost constraints and infrastructure requirements. In such scenarios, FNAC remains a valuable and accessible diagnostic tool. The findings of this study support the implementation of a dual diagnostic approach combining cytopathology and Gene Xpert. This approach is particularly beneficial in high-burden settings, where rapid diagnosis and early initiation of treatment are critical. Additionally, the detection of rifampicin resistance by Gene Xpert has significant implications for tuberculosis control programs, as it facilitates early identification of multidrug-resistant TB cases. However, the study has certain limitations. The absence of culture as a gold standard may affect the assessment of diagnostic accuracy. Additionally, the sample size, although adequate, may not fully represent the broader population. Future studies incorporating culture and histopathological correlation, along with larger sample sizes, would provide more comprehensive insights. In conclusion, the present study demonstrates that FNAC and Gene Xpert are complementary diagnostic modalities in lymph node tuberculosis. While FNAC provides rapid and sensitive detection of granulomatous inflammation, Gene Xpert offers specific confirmation and drug resistance profiling. The combined use of these techniques significantly enhances diagnostic accuracy and should be adopted as a standard approach in the evaluation of suspected tuberculous lymphadenitis.

The present study highlights the diagnostic utility of a dual approach combining fine needle aspiration cytology (FNAC) and Gene Xpert MTB/RIF assay in the evaluation of lymph node tuberculosis. FNAC proved to be a highly sensitive, rapid, and cost-effective initial screening tool, capable of identifying characteristic cytomorphological features of tuberculous lymphadenitis across different stages of disease. However, its specificity remains limited due to overlap with other granulomatous conditions. Gene Xpert, on the other hand, demonstrated high specificity and the added advantage of rapid molecular confirmation of Mycobacterium tuberculosis along with detection of rifampicin resistance. The substantial concordance observed between the two modalities reinforces their complementary roles in clinical practice. The presence of discordant cases emphasizes that reliance on a single diagnostic method may lead to underdiagnosis or misdiagnosis. Therefore, integrating cytopathology with molecular diagnostics significantly enhances overall diagnostic accuracy, reduces delays in treatment initiation, and improves patient outcomes. In high-burden and resource-limited settings, adopting this dual diagnostic strategy can strengthen tuberculosis control programs by enabling early detection, appropriate therapy, and identification of drug-resistant cases. Hence, routine use of both FNAC and Gene Xpert is strongly recommended in suspected lymph node tuberculosis.

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