Assessing the Combined Diagnostic Value of Serum ACE Levels, Mantoux Test and Lymphopenia in Uveitis Patients from a Tertiary Eye Care Center in Southern India

Introduction

Uveitis encompasses a heterogeneous group of intraocular inflammatory disorders involving the uveal tract and adjacent ocular structures, and remains a significant cause of visual morbidity worldwide.^1,2 It accounts for approximately 3–10% of blindness in developed countries and up to 25% in developing regions such as India, where infectious etiologies predominate.^1,2 The etiological spectrum of uveitis is broad, including infectious, autoimmune, and idiopathic causes, often presenting with overlapping clinical features.^3,4 Establishing an accurate diagnosis is critical, as management strategies vary substantially depending on the underlying etiology and may be vision- or life-saving.^4,5 Among the various causes of uveitis, granulomatous conditions such as ocular tuberculosis and ocular sarcoidosis pose a particular diagnostic challenge.^6–8 Both entities share similar clinical presentations, including chronic inflammation, posterior segment involvement, and systemic associations.^6,8 However, their management differs fundamentally. Tuberculosis requires prolonged antimicrobial therapy, whereas sarcoidosis is primarily treated with corticosteroids and immunosuppressive agents.^7,9 Misdiagnosis can therefore lead to inappropriate treatment, worsening of disease, and systemic complications.^6,7 In regions with a high burden of tuberculosis, differentiating between these two conditions becomes even more critical yet challenging.^9,10 In clinical practice, a combination of clinical findings and supportive laboratory investigations is often employed to arrive at a diagnosis.⁴ Serum angiotensin-converting enzyme (ACE) has been widely studied as a biomarker for sarcoidosis, reflecting granuloma burden due to macrophage activation.^8,11 Similarly, the Mantoux test serves as an indirect indicator of Mycobacterium tuberculosis exposure,^12,13 while lymphopenia has been associated with systemic immune dysregulation in sarcoidosis.¹⁴ However, the diagnostic accuracy of these tests varies considerably, and their interpretation is often confounded by overlapping disease processes¹⁵ and regional epidemiology.^4,9

Recent advances in ophthalmic research have attempted to refine the role of these biomarkers. Recent studies highlighted the expanding role of diagnostic biomarkers and multimodal evaluation in ocular inflammatory disorders, emphasising the need for integrated diagnostic approaches rather than relying on isolated parameters.⁹ Their review underscored the limitations of single biomarkers such as ACE in differentiating granulomatous uveitis and advocated for combined diagnostic strategies. Similarly, studies such as those by De Fauw et al¹⁶ and Ting et al¹⁷ have demonstrated the growing importance of data-driven and multimodal approaches in improving diagnostic accuracy in ophthalmology, although their focus has largely been on imaging and artificial intelligence rather than biochemical markers.

Clinical studies evaluating serum ACE have reported variable sensitivity ranging from 40% to 83% in sarcoid uveitis, with specificity often affected by conditions such as tuberculosis, diabetes, and other granulomatous diseases.^18,19 Additionally, Mantoux test interpretation is influenced by prior Bacillus Calmette–Guerin (BCG) vaccination, endemicity of tuberculosis, and host immune status.¹² Lymphopenia, although recognized as a supportive marker in sarcoidosis, lacks sufficient sensitivity when used independently.²⁰ While individual studies have explored these parameters, their combined diagnostic utility remains inadequately defined, particularly in real-world clinical settings.^4,18 A critical review of existing literature reveals several lacunae. First, there is a paucity of prospective studies evaluating the combined diagnostic performance of serum ACE, Mantoux test, and lymphocyte count in uveitis.⁴ Second, most available studies are retrospective or conducted in non-endemic regions, limiting their applicability to countries like India where tuberculosis is highly prevalent.⁹ Third, there is limited evidence comparing these biomarkers across a broad spectrum of uveitis etiologies within a single cohort.⁴ Finally, there remains a lack of standardized diagnostic algorithms incorporating these readily available and cost-effective investigations.⁴ This study aims to address these gaps by evaluating the diagnostic utility of serum ACE levels in conjunction with Mantoux test and lymphocyte count in patients with uveitis presenting to a tertiary eye care center. By analyzing their individual and combined diagnostic performance, this study seeks to provide clinically relevant insights into differentiating ocular sarcoidosis from ocular tuberculosis. The findings are expected to contribute to the development of a more practical, evidence-based diagnostic approach, particularly in resource-limited and tuberculosis-endemic settings, thereby improving patient outcomes and guiding appropriate therapeutic interventions.

Methods

This prospective observational study was conducted at a Aravind Eye Hospital, Pondicherry in South India over a period of 18 months, from December 2020 to June 2022. The study adhered to the tenets of the Declaration of Helsinki and received approval from the Aravind Eye Hospital, Pondicherry’s Institutional Ethics Committee (IRB No-AEH (Aravind Eye Hospital)/PDY (Pondicherry)/EC (Ethics Committee)/THESIS/68/2020). Written informed consent was obtained from all participants prior to inclusion in the study. The overall study workflow, including patient recruitment, clinical evaluation, and laboratory investigations, is illustrated in Figure 1. A total of 95 consecutive patients diagnosed with active uveitis, involving 118 eyes, were enrolled. Patients older than 18 years of age belonging to both genders presenting with clinical features of anterior, intermediate, posterior, or panuveitis were included. Patients with inactive uveitis, history of recent intraocular surgery (within 3 months), or those already on systemic immunosuppressive therapy, Anti-tubercular therapy, ACE inhibitors and corticosteroids prior to evaluation were excluded to avoid confounding of laboratory parameters. All patients underwent a comprehensive ophthalmic examination including best-corrected visual acuity (BCVA) assessment using Snellen charts, slit-lamp biomicroscopy, intraocular pressure measurement, and detailed fundus examination using indirect ophthalmoscopy with +20 D lens and slit-lamp biomicroscopy with a +90D lens. Ancillary investigations such as fundus photography, optical coherence tomography (OCT), fundus fluorescein angiography (FFA), or ultrasonography were performed as indicated to characterize the extent and pattern of intraocular inflammation. A detailed systemic history was obtained, including symptoms suggestive of tuberculosis (chronic cough, weight loss, fever), sarcoidosis (respiratory symptoms, skin lesions), and other autoimmune conditions. Relevant systemic examinations and referrals to physicians or pulmonologists were undertaken when indicated. All enrolled patients underwent standardized laboratory evaluation, including measurement of serum angiotensin-converting enzyme (ACE) levels, Mantoux test, and complete blood count with differential leukocyte count to assess lymphocyte levels. Serum ACE levels were measured using an enzymatic assay, with values ≥52 IU/L considered elevated based on laboratory reference standards. The Mantoux test was performed by intradermal injection of 5 tuberculin units (TU) of purified protein derivative (PPD), and induration was measured after 48–72 hours. An induration of ≥10 mm was considered positive, taking into account regional epidemiological factors. Lymphopenia was defined as an absolute lymphocyte count below the lower limit of normal as per institutional laboratory reference ranges. Additional investigations, including chest radiography, high-resolution computed tomography (HRCT) of the chest, interferon-gamma release assays (IGRA), or biopsy, were performed selectively based on clinical suspicion to support the diagnosis of ocular tuberculosis or sarcoidosis. Final etiological diagnosis was made by application of pertinent international diagnostic criteria based on a combination of clinical findings, laboratory investigations, and systemic evaluation as per standard of care. Revised International Workshop on Ocular Sarcoidosis (IWOS) criteria was used for diagnosing Ocular Sarcoidosis.²¹ Ocular Tuberculosis was diagnosed using the ocular TB diagnostic criteria by Gupta et al²² Other uveitis entities were diagnosed using pertinent international diagnostic criteria.^23–29 Patients were categorized into etiological groups such as ocular sarcoidosis, ocular tuberculosis, idiopathic uveitis, and other specific causes.

Figure 1 Flowchart depicting the patient recruitment, clinical evaluation, and diagnostic investigations including serum ACE, Mantoux test, and lymphocyte count.

Statistical Analysis

Data were recorded and analysed using statistical software. Continuous variables were expressed as mean ± standard deviation, while categorical variables were presented as frequencies and percentages. The diagnostic performance of serum ACE, Mantoux test, and lymphocyte count was evaluated by calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Receiver operating characteristic (ROC) curve analysis was performed to assess the discriminative ability of serum ACE levels in diagnosing ocular sarcoidosis. A p-value of <0.05 was considered statistically significant.

The required sample size for the study is calculated to be 95, based on a precision of about 10% and a 95% confidence interval, using the formula given below.

Where:

p = Sensitivity of the new test

d = Precision

Z_{1-\alpha/2} = Desired confidence level

Results

The study cohort comprised 95 patients (118 eyes) with a mean age of 44.52 ± 13.77 years (range: 20–78 years). The gender distribution was nearly equal, with males accounting for 50.5% and females 49.5% of the population. Left eye involvement was most frequent (45.3%), followed by right eye (30.5%) and bilateral disease (24.2%). Among systemic comorbidities, hypertension (18.9%) and diabetes mellitus (13.7%) were the most commonly observed associations (Table 1). All patients included in the study had history of BCG vaccination at birth.The median visual acuity was 0.4 logMAR (UCVA) and 0.3 logMAR (BCVA), with an overall range of 0–1.7. Based on SUN classification, anterior uveitis was the predominant anatomical subtype (38.1%), followed by intermediate (18.6%) and posterior uveitis (16.9%) (Table 2).37 out of 95 patients (38.94%) had granulomatous uveitis and 58 out of 95 patients (61.05%) had non granulomatous uveitis. Defective vision (67.6%) and ocular pain (44.1%) were the most common presenting complaints. Fine non-pigmented keratic precipitates were the most frequently observed morphology (41.5%) (Table 2). Comparative analysis between ocular tuberculosis and sarcoidosis revealed a mean age of 38.0 ± 15.57 years and 44.63 ± 12.0 years, respectively, without statistical significance (p=0.192). Posterior uveitis was significantly more prevalent in the tuberculosis group (53.3% vs 10.7%, p=0.002), whereas intermediate uveitis was more common in sarcoidosis (46.4% vs 6.7%, p=0.008) (Figure 2). No statistically significant differences were observed with respect to gender distribution, laterality, or presenting clinical features (Table 3). 3 out of 12 patients with ocular TB also had coexisting pulmonary TB whereas no patient of biopsy proven systemic or ocular sarcoidosis was encountered in this study duration specifically. Serum ACE levels ≥52 IU/L were elevated in 89.5% of sarcoidosis cases compared to 33.3% of tuberculosis cases. The mean ACE level was highest in sarcoidosis (76.21 ± 25.44 IU/L) relative to tuberculosis (50.58 ± 34.16 IU/L) and other uveitic etiologies (Figure 3). Statistically significant differences in ACE levels were noted between sarcoidosis and tuberculosis (p=0.008), viral uveitis (p=0.0009), autoimmune uveitis (p=0.017), toxoplasmosis (p=0.006), and idiopathic uveitis (p=0.0006) (Table 4). Mantoux test positivity (≥10 mm) was observed in 58.3% of tuberculosis cases compared to 15.8% of sarcoidosis cases, while strong positivity (≥20 mm) was present in 33.3% of tuberculosis cases. The mean Mantoux induration was significantly higher in tuberculosis (12.67 ± 8.75 mm) than in sarcoidosis (2.95 ± 6.09 mm) (Figure 4). Statistically significant differences were observed between tuberculosis and sarcoidosis (p=0.0005), autoimmune uveitis (p=0.003), and idiopathic uveitis (p=0.001) (Table 5). The mean lymphocyte count ranged from 1.70 ± 0.53 ×10⁹/L in leptospirosis to 3.18 ± 1.08 ×10⁹/L in viral uveitis. Lymphopenia was identified exclusively in sarcoidosis (10.5%) and was not observed in any other diagnostic subgroup, with an overall prevalence of 2.1% in the study population (Table 6). In diagnostic performance analysis, serum ACE >52 IU/L demonstrated high sensitivity (89.5%) and good discriminative ability (AUROC 0.819) for sarcoidosis. In contrast, lymphopenia exhibited 100% specificity but low sensitivity (10.5%). For tuberculosis, Mantoux test ≥10 mm showed superior diagnostic performance, with sensitivity 58.3%, specificity 85.5%, and AUROC 0.835, outperforming serum ACE (Table 7). AUROC comparison further demonstrated that serum ACE had significantly higher discriminative ability than Mantoux (p<0.001) and lymphopenia (p=0.001) in diagnosing sarcoidosis. Conversely, Mantoux testing significantly outperformed ACE in tuberculosis (AUROC 0.835 vs 0.466, p=0.001), highlighting the complementary diagnostic utility of these biomarkers (Table 8) and (Figure 5–6).

Table 1 Depicts the Baseline Demographic and Clinical Characteristics of the Uveitis Patients

Table 2 Depicts the Clinical Characteristics and Presentation of Uveitis Patients

Table 3 Depicts the Demographic and Clinical Characteristics in Ocular Tuberculosis and Ocular Sarcoidosis

Table 4 Depict the Comprehensive Analysis of Serum ACE Levels Across Different Diagnoses of Uveitis

Table 5 Depicts the Comprehensive Analysis of Mantoux Test Results Across Different Diagnoses of Uveitis

Table 6 Depicts the Comprehensive Analysis of Lymphocyte Count Across Different Diagnoses of Uveitis

Table 7 Depicts the Comparative Diagnostic Performance of Biomarkers in Ocular Sarcoidosis and Ocular Tuberculosis

Table 8 Depicts the Comparative Area Under the Receiver Operative Curve (AUROC) Analysis of Various Diagnostic Tests in Ocular Sarcoidosis and Tuberculosis

Figure 2 Bar diagram depicting the spectrum of clinical diagnoses.

Figure 3 Bar diagram depicting the proportion of elevated serum ACE (>52 IU/L) across different uveitis etiologies.

Figure 4 Bar diagram depicting the distribution of Mantoux test positivity (≥10 mm) across uveitis subtypes, demonstrating higher positivity in ocular tuberculosis.

Figure 5 Receiver operating characteristic (ROC) curve analysis in ocular sarcoidosis demonstrating diagnostic performance of (A) serum ACE (>52 IU/L; AUC 0.819), (B) Mantoux test (<10 mm; AUC 0.408), and (C) lymphopenia (AUC 0.459).

Figure 6 Receiver operating characteristic (ROC) curve analysis in ocular tuberculosis showing diagnostic performance of (A) serum ACE (>52 IU/L; AUC 0.466) and (B) Mantoux test (≥10 mm; AUC 0.835).

Discussion

This prospective observational study evaluated the diagnostic utility of serum angiotensin-converting enzyme (ACE), Mantoux test, and lymphocyte count in patients with uveitis, with particular emphasis on differentiating ocular sarcoidosis and ocular tuberculosis in a tertiary care setting.⁴ The study cohort included 95 patients (118 eyes), with a mean age of 44.52 ± 13.77 years, reflecting a typical working-age population affected by uveitis.² The comparable age distribution between ocular tuberculosis (38.0 ± 15.57 years) and sarcoidosis (44.63 ± 12.0 years) aligns with prior reports, although variability exists across studies, with Sahin et al¹⁹ reporting a mean age of ~41 years and Cotte et al³⁰ reporting ~57.3 years, suggesting demographic and geographic heterogeneity in disease presentation. A key clinical observation in this study was the differential anatomical pattern of uveitis. Posterior uveitis was significantly more common in ocular tuberculosis (53.3%) compared to sarcoidosis (10.7%, p=0.002), whereas intermediate uveitis predominated in sarcoidosis (46.4% vs 6.7%, p=0.008). These findings are consistent with established clinical paradigms, where tuberculosis tends to involve the choroid and retina, while sarcoidosis more commonly affects the vitreous and peripheral retina. Such distinctions reinforce the importance of integrating clinical phenotype with laboratory parameters.³¹ Serum ACE demonstrated the strongest diagnostic performance for ocular sarcoidosis in this study. Elevated ACE levels (≥52 IU/L) were observed in 89.5% of sarcoidosis cases compared to 33.3% in tuberculosis. This sensitivity (89.5%) is higher than that reported in most previous studies, where sensitivity ranges from 40% to 83%. Hunninghake et al³² reported ACE elevation in 93% of systemic sarcoidosis cases and 41% in tuberculosis, which closely parallels our findings.

The mean ACE level in sarcoidosis (76.21 ± 25.44 IU/L) was significantly higher than in tuberculosis (50.58 ± 34.16 IU/L, p=0.008) and other uveitic etiologies, confirming its discriminatory value. Notably, ACE also demonstrated strong negative predictive value (NPV 96.4%), suggesting that a normal ACE level effectively excludes sarcoidosis in most cases. Similar findings were reported by Niederer et al,¹⁸ who emphasized the high NPV (~97%) of ACE in ruling out sarcoidosis. However, increasing the diagnostic threshold to ≥104 IU/L resulted in a marked decline in sensitivity (10.5%) with improved specificity (97.4%), indicating a trade-off between screening and confirmatory roles. This highlights that ACE is best utilized as a screening biomarker rather than a standalone diagnostic tool. Interestingly, this study also evaluated ACE in ocular tuberculosis, an area rarely explored in prior literature. Elevated ACE showed limited diagnostic value in tuberculosis, with low specificity and PPV, reinforcing that ACE elevation is not disease-specific and may reflect granulomatous inflammation rather than etiology. The overlap between tuberculosis and sarcoidosis observed in ACE values supports the hypothesis of a “sarcoid–tuberculous spectrum.”³ Studies by Gupta et al⁹ demonstrated mycobacterial DNA positivity in up to 48% of sarcoid tissues, and Aggarwal et al³³ proposed a spectrum model (S, ST, TS, TB), suggesting immunological overlap between the two diseases. This may partly explain the diagnostic ambiguity in endemic regions. Mantoux test showed superior diagnostic utility for ocular tuberculosis. Positivity ≥10 mm was seen in 58.3% of tuberculosis cases compared to 15.8% in sarcoidosis, with sensitivity 58.3%, specificity 85.5%, and AUROC 0.835. These values are comparable to previous reports, such as Gupta et al⁹ (sensitivity 71%, specificity 66%) and Hong et al (PPV ~17%), indicating moderate diagnostic performance.

Increasing the threshold to ≥20 mm improved specificity (94.0%) but reduced sensitivity (33.3%), consistent with known trade-offs in tuberculin testing. The high NPV (93.4%) observed in this study suggests that a negative Mantoux test effectively reduces the likelihood of tuberculosis, although clinical correlation remains essential. The relatively low sensitivity may be attributed to factors such as prior Bacillus Calmette–Guérin (BCG) vaccination, immunosuppression, malnutrition, and endemic tuberculosis exposure.³⁴ Additionally, false-negative results may occur in active disease due to anergy, particularly in sarcoidosis and severe tuberculosis. Interestingly, the study also evaluated negative Mantoux test (<10 mm) as a potential marker for sarcoidosis. The sensitivity was high (84.2%) but specificity was low (22.4%), indicating that while a negative Mantoux may support sarcoidosis, it lacks diagnostic specificity. Similar findings have been reported in pulmonary studies, where negative tuberculin reactivity is frequently observed in sarcoidosis due to immune dysregulation.³⁵ Lymphopenia demonstrated very high specificity (100%) but extremely low sensitivity (10.5%) for sarcoidosis. Only 2 patients (10.5% of sarcoidosis cases) exhibited lymphopenia, limiting its diagnostic utility. These findings are consistent with previous studies; Cotte et al³⁰ reported sensitivity of ~15.3%, while Jones et al reported ~31.6%, indicating poor standalone performance.²⁰ The low sensitivity in our study may be explained by delayed presentation, as lymphopenia is more prominent in early or active systemic disease. Additionally, coexisting infections and the COVID-19 pandemic period may have influenced lymphocyte counts, reducing diagnostic reliability. A novel aspect of this study is the evaluation of combined biomarkers. The combination of elevated ACE, negative Mantoux, and lymphopenia demonstrated 100% specificity and PPV, albeit with low sensitivity (5.26%). This indicates that while rare, the presence of all three markers strongly confirms sarcoidosis. Previous studies have explored biomarker combinations. Baarsma et al³⁶ evaluated ACE and lysozyme, while Cotte et al³⁰ demonstrated improved specificity (99%) when ACE and lymphopenia were combined. However, the addition of Mantoux in this study provides a more comprehensive diagnostic framework, particularly relevant in tuberculosis-endemic settings. This study has several notable strengths. It is a prospective study conducted in a single tertiary center with standardized evaluation by a trained observer, ensuring consistency. It is among the first studies to evaluate the combined diagnostic utility of ACE, Mantoux test, and lymphocyte count in uveitis. Additionally, the diagnostic performance of ACE in ocular tuberculosis was explored, which has not been systematically studied previously. The use of updated diagnostic criteria further enhances the reliability of findings. Despite its strengths, the study has certain limitations. The observational design and consecutive sampling may introduce selection bias. Ocular sarcoidosis cases were not biopsy-proven, limiting diagnostic certainty. Serum ACE levels can be influenced by confounding factors such as diabetes, liver disease, and smoking, which could not be fully controlled. The study period overlapped with the COVID-19 pandemic, potentially affecting lymphocyte counts. This study highlights the importance of a multimodal diagnostic approach in uveitis. Serum ACE serves as a useful screening tool for sarcoidosis, while Mantoux test remains more relevant for tuberculosis. Lymphopenia, although specific, has limited sensitivity. The combined use of biomarkers enhances diagnostic confidence, particularly in ambiguous cases. In the South Indian context, where tuberculosis prevalence is high, distinguishing sarcoidosis from tuberculosis is particularly challenging. This study provides a practical, cost-effective diagnostic framework that can be applied in routine clinical practice, especially in resource-limited settings.³⁷ However the conclusions should be interpreted cautiously given the relatively modest sample size, subgroup analyses, and single-center design, which may limit generalizability.Future research should focus on larger multicentric studies, incorporation of advanced diagnostics such as interferon-gamma release assays and molecular testing, and development of standardized diagnostic algorithms integrating clinical, laboratory, and imaging data.³⁸

Conclusion

Serum ACE is a valuable screening biomarker for ocular sarcoidosis, particularly at a cut-off of ≥52 IU/L, owing to its high sensitivity and negative predictive value. Mantoux test demonstrates superior diagnostic utility for ocular tuberculosis, especially at a threshold of ≥10 mm. Lymphopenia, while highly specific, has limited standalone utility due to low sensitivity. A combined biomarker approach improves diagnostic specificity and may aid in differentiating granulomatous uveitis. These findings are particularly relevant in tuberculosis-endemic regions and support a multimodal, context-specific diagnostic strategy.