Granulomatous inflammation represents a patterned host response to persistent antigenic stimulation and is encountered across a broad spectrum of infectious and non-infectious conditions [1]. In lymph nodes, granulomatous lymphadenitis remains a diagnostically important entity because it can reflect treatable infections, systemic inflammatory disorders, or less common immune-mediated and toxic exposures [2,3]. From a practical pathology standpoint, granuloma type (necrotizing versus non-necrotizing), the presence of suppuration, and the accompanying background reaction can narrow the differential diagnosis, but these morphologic cues are not fully specific [1-3].

In countries with a high tuberculosis burden, tuberculous lymphadenitis constitutes a major proportion of extrapulmonary tuberculosis, frequently presenting as painless cervical lymphadenopathy [4-6]. Histopathology typically demonstrates epithelioid granulomas with or without caseous necrosis and multinucleated giant cells;

however, necrosis is not universal and the appearance can be modified by host immunity, duration of illness, and partial treatment [5,6]. Moreover, several fungal infections can mimic tuberculous granulomas, and necrotizing granulomas can occur in non-tuberculous settings, complicating etiologic attribution when ancillary tests are unavailable [2,3].

Conventional microbiological confirmation using Ziehl–Neelsen staining has limited sensitivity in lymph node tissue because the disease is often paucibacillary [4,6]. Molecular assays based on automated nucleic acid amplification, including cartridge-based platforms (CBNAAT/Xpert MTB/RIF), have improved diagnostic yield and allow rapid detection of rifampicin resistance, even when smear microscopy is negative [7-9,11,12]. Recent evidence supports using molecular testing alongside histopathology to enhance diagnostic confidence and reduce indeterminate categorization in granulomatous lymphadenitis [9,10].

Fungal lymphadenitis is less frequent than tuberculous lymphadenitis in routine biopsy practice, yet it carries substantial clinical implications because management requires organism-directed antifungal therapy. Histochemical stains such as periodic acid–Schiff and Gomori methenamine silver remain central for demonstrating fungal elements in tissue, particularly when culture is unavailable or delayed [13,14]. Given the overlapping morphology between mycobacterial and fungal granulomas, understanding test concordance in routine diagnostic workflows is essential for improving reporting accuracy and guiding clinicians toward appropriate therapy.

In routine tertiary-care practice, excision biopsy is often performed after an initial cytology workup or when nodes persist despite empiric therapy. At this point, clinicians require an actionable etiologic report, yet laboratories frequently face constraints in performing culture for mycobacteria and fungi. Consequently, histopathology combined with a limited set of rapid ancillary tests becomes the pragmatic pathway for decision-making. Quantifying the yield and agreement of these tests within a defined biopsy cohort provides a basis for standardizing reporting language and for selecting cases in which additional testing is most informative.

Objectives: To describe the demographic, clinical, and histopathological features of granulomatous lymphadenitis in lymph node biopsies and to assess concordance between histopathological interpretation and mycobacterial/fungal detection methods (Ziehl–Neelsen stain, CBNAAT, PAS, and GMS).

Study design and setting: This observational study was conducted in the Department of Pathology, Government Medical College, Karimnagar, Telangana, India, over 8 months (May 2025 to December 2025). All lymph node biopsy specimens received during the study period were processed as per departmental protocols.

Study population: Consecutive lymph node biopsies that demonstrated granulomatous inflammation on routine hematoxylin and eosin (H&E) examination were included until a sample size of 100 was reached. Clinical details (age, sex, symptoms, and nodal site) were obtained from requisition forms and case records where available.

Inclusion criteria: (i) Excision biopsy or core biopsy of lymph node tissue with adequate material for histopathological evaluation, and (ii) microscopic evidence of epithelioid granulomas with or without necrosis.

Exclusion criteria: (i) Poorly preserved tissue or inadequate biopsy material, (ii) biopsies showing primary or metastatic malignancy without granulomatous inflammation, and (iii) repeat biopsies from the same patient during the study period.

Histopathological evaluation: Formalin-fixed, paraffin-embedded tissue sections (3–4 µm) were stained with H&E for primary assessment. Granulomas were categorized as necrotizing (caseous necrosis present) or non-necrotizing. Two pathologists independently reviewed the H&E slides; discrepant impressions were resolved by joint review to minimize interpretive variability. When multiple nodes or blocks were available, the block showing maximal necrosis or granuloma density was prioritized for ancillary testing. The presence of multinucleated giant cells (Langhans-type or foreign-body type), suppuration, and associated lymphoid hyperplasia was documented using predefined reporting variables aligned with published histopathology frameworks [1-3].

Mycobacterial and fungal detection: Ziehl–Neelsen staining was performed on representative sections for acid-fast bacilli detection. CBNAAT (Xpert MTB/RIF platform) was performed on biopsy tissue as per manufacturer-based workflows used for clinical diagnosis, targeting rapid detection of Mycobacterium tuberculosis complex and rifampicin resistance [11,12]. Fungal evaluation used periodic acid–Schiff and Gomori methenamine silver stains on selected sections, particularly when suppuration, necrosis with limited epithelioid response, or yeast/hyphal-like structures were suspected on H&E [13,14].

Operational definitions and concordance: Histopathology was considered ‘suggestive of tuberculosis’ when epithelioid granulomas were accompanied by caseous necrosis and/or strong supportive morphology. Microbiological confirmation of tuberculosis was defined as positivity on Ziehl–Neelsen stain and/or CBNAAT. Fungal lymphadenitis was defined by demonstration of fungal elements on PAS and/or GMS in an appropriate tissue context. Concordance was assessed by comparing histopathological categorization with microbiological results.

Data analysis and ethics: Data were summarized as mean ± standard deviation for age and as frequencies and percentages for categorical variables. The study used de-identified laboratory data and was conducted after approval by the institutional ethics committee, with confidentiality maintained throughout.

A total of 100 consecutive lymph node biopsy specimens showing granulomatous inflammation were evaluated. The clinicodemographic profile, histomorphology, and ancillary test results are summarized in Tables 1–4.

Among the 100 participants, 56 were male and 44 were female (male-to-female ratio 1.27:1). The mean age was 34.6 ± 13.8 years (range: 8–72 years), and the largest proportion belonged to the 21–40 year age group (48%). Painless lymphadenopathy was the predominant presentation (72%), followed by fever (38%), weight loss (31%), and night sweats (18%). Cervical lymph node involvement was most common (68%), with axillary (17%), inguinal (11%), and generalized lymphadenopathy (4%) less frequent (Table 1).

Table 1. Demographic and Clinical Characteristics of Study Participants (N = 100)

*Multiple symptoms may coexist.

On histopathology, epithelioid cell granulomas were present in all biopsies. Caseous necrosis was identified in 62% and non-caseating granulomas in 38%. Multinucleated giant cells were noted in 74% of cases, with Langhans-type morphology more frequent than foreign-body type. Associated lymphoid hyperplasia was observed in 41% (Table 2).

Table 2. Histopathological Features of Granulomatous Lymphadenitis (N = 100)

Mycobacterial detection by Ziehl–Neelsen stain demonstrated acid-fast bacilli in 34% of biopsies, whereas CBNAAT was positive in 46%, indicating a higher diagnostic yield for molecular testing (Table 3). CBNAAT positivity was higher in necrotizing granulomas (58%) than in non-caseating granulomas (26%). Fungal elements were demonstrated in 8% of biopsies on PAS and GMS stains; these cases predominantly showed non-caseating granulomas with prominent suppurative inflammation. None of the fungal-positive biopsies showed AFB on Ziehl–Neelsen stain or CBNAAT positivity, supporting a distinct infectious etiology (Tables 3 and 4).

Table 3. Mycobacterial and Fungal Detection by Special Stains and Molecular Methods (N = 100)

On concordance analysis, histopathology was interpreted as suggestive of tuberculosis in 70 cases. Of these, 43 showed microbiological confirmation by AFB staining and/or CBNAAT (61.4%), while 27 lacked microbiological confirmation. Overall, 22 biopsies remained etiologically indeterminate after negative mycobacterial and fungal detection, underscoring the limitations of single-modality diagnosis in granulomatous lymphadenitis (Table 4).

Table 4. Concordance Between Histopathology and Microbiological Detection

Percentages for confirmation within the histology-suggestive subgroup are calculated using 70 as the denominator.

The present study characterizes granulomatous lymphadenitis in a biopsy cohort from a tertiary-care pathology service in Telangana and highlights the practical value of combining morphology with rapid detection methods. The demographic profile showed a young-adult predominance with mild male preponderance, and cervical lymphadenopathy formed the major clinical pattern. This aligns with the recognized epidemiology of tuberculous lymphadenitis as a common form of extrapulmonary tuberculosis in endemic regions and with the tendency for patients to present with painless cervical nodes [4-6].

Histopathologically, necrotizing granulomas constituted nearly two-thirds of biopsies, and multinucleated giant cells were frequent. Although caseous necrosis remains a strong morphologic clue for tuberculosis, granuloma morphology alone is not specific and can overlap with other infectious and non-infectious causes, particularly when clinical context is limited [1-3]. The current dataset also underscores this limitation: a substantial proportion of biopsies interpreted as suggestive of tuberculosis lacked microbiological confirmation. Discordance can arise from low bacillary burden, uneven organism distribution within tissue, prior anti-tubercular therapy, or alternative etiologies that produce similar patterns [4,5,9].

The molecular detection yield (CBNAAT positivity 46%) exceeded Ziehl–Neelsen positivity (34%), supporting the known advantage of nucleic acid amplification in paucibacillary extrapulmonary disease [4,7-9]. Automated cartridge-based assays were developed to provide rapid, standardized detection of Mycobacterium tuberculosis complex and rifampicin resistance and have expanded their role beyond pulmonary samples [11,12]. In the present series, CBNAAT positivity was higher in necrotizing granulomas than in non-necrotizing patterns, consistent with the expectation of higher bacillary load in necrotizing lesions. Importantly, CBNAAT also detected mycobacterial DNA in a subset of non-caseating granulomas, emphasizing that early or partially treated tuberculosis can lack classic necrosis and that molecular testing can strengthen interpretation in such settings [5,9].

Fungal lymphadenitis accounted for 8% of biopsies, predominantly associated with suppurative, non-caseating granulomas. This finding reinforces the need to consider fungal infections within the granulomatous differential diagnosis, particularly when tissue shows suppuration or when mycobacterial tests are negative. Histochemical stains remain essential for direct tissue demonstration of fungal organisms and can provide a timely diagnostic direction in routine laboratories [13,14].

Taken together, these observations support a tiered diagnostic approach for granulomatous lymphadenitis: detailed morphologic classification, targeted Ziehl–Neelsen staining, and early deployment of CBNAAT in suspected cases, with PAS/GMS applied when morphology suggests fungal disease or when mycobacterial testing is negative despite granulomatous inflammation. Such an integrated workflow can reduce diagnostic ambiguity and improve clinicopathological communication in settings where culture is not routinely available.

This single-center study evaluated only biopsy-confirmed granulomatous lymphadenitis and therefore does not represent all patients with lymphadenopathy in the community. Mycobacterial and fungal cultures were not routinely performed, limiting species-level confirmation and antimicrobial susceptibility correlation. Clinical follow-up and radiologic correlation were incomplete for some cases, and comprehensive workup for non-tuberculous mycobacteria and non-infectious granulomatous disorders was not available in all patients.

In this tertiary-care biopsy cohort, granulomatous lymphadenitis predominantly affected young adults and most often involved cervical lymph nodes. Necrotizing granulomas were common, yet reliance on morphology alone left a meaningful fraction of cases without microbiological corroboration. CBNAAT demonstrated a higher diagnostic yield than Ziehl–Neelsen staining and supported etiologic classification even in non-caseating granulomas, highlighting its value in paucibacillary disease. Fungal elements were identified in a small but clinically important subset, largely associated with suppurative non-caseating patterns, underscoring the role of PAS and GMS in selected cases. An integrated diagnostic strategy that links morphologic patterns with targeted mycobacterial and fungal detection improves reporting confidence and can guide timely, organism-specific therapy.

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