Mycobacterium tuberculosis (MTB) is the leading global health problem and a major infectious cause of morbidity and mortality (1). The WHO Global Tuberculosis Report 2024 estimated a total of 11 million people infected with TB in 2023 and identified an increasing trend in the TB incidence rate and achieving the WHO End TB strategy milestone of a 50% reduction by 2025, far from reach (2). The COVID-19 pandemic during 2019–22 was one of the major contributors to the increase in new cases due to setbacks in case identification and treatment (3). Latent TB infection (LTBI) is characterized by an enduring immune response to MTB antigens, despite the lack of gross clinical, radiological, and microbiological indicators (4). While one-third of the population is infected with MTB, there is an estimated 5–15% risk of reactivation, mostly within the first five years of initial infection (5). Attributing to the global TB burden and a large reservoir of LTBI-infected individuals, the WHO devised policy guidelines and recommendations on the management of LTBI within the framework of the WHO End TB Strategy underscores the need for early detection and timely intervention.

The reactivation of a latent infection to an active TB disease involves a gamut of underlying immunological responses leading to incipient and subclinical TB disease states. Currently, the diagnosis of LTBI relies on the WHO-recommended assays, including the Tuberculin Skin Test (TST) and the Interferon-Gamma Release Assay (IGRA), which suffer from several limitations. This includes a requirement of at least two visits, reduced sensitivity among immunocompromised individuals, and false-positivity among vaccinated individuals with TST, high-cost infrastructure, and uncertainty in the results due to low predictability with IGRA and the discordance between the results of TST and IGRA. The tests for LTBI must therefore be carefully interpreted along with other clinical and radiological evidence. Apart from the lack of a gold standard assay for LTBI detection, there is a lack of understanding of host-based diagnostic biomarkers such as hematological markers, metabolites, and other proteins associated with LTBI and indicators that could predict progression to active TB (6, 7). It is of paramount importance to identify potential diagnostic biomarkers across diverse TB-infected populations (8). This would facilitate the development of improved point-of-care (POC) tests applicable in resource-limited settings (9). Because TB tends to impact the economically weakened sections of society significantly, it is imperative to develop accessible and affordable tests for use in endemic areas with resource-constrained laboratories (10–12).

The decreasing levels of RBCs, WBCs, and platelet count, and elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) that indicate acute inflammation (13) also acted as inflammatory diagnostic markers in pulmonary TB and correlated with disease severity and prognosis (14, 15). Furthermore, many hematological abnormalities, viz., increased levels of fibrinogen and D-dimer, have been reported to correlate with clinical TB (16) and help in distinguishing it from community-acquired pneumonia (17). TB in a systemic hypercoagulable state involves a homeostatic mechanism activating procoagulant factors (18) with elevated thrombin–antithrombin complexes, D-dimer, and fibrinogen (19). However, to date, studies investigating the biochemical and hematological parameters among close contacts of active TB patients in an endemic setting are scarce. Hence, addressing the knowledge gaps in LTBI biomarker research is key to the development of diagnostic tools with improved accuracy and feasibility for use in resource-limited clinical settings. In this study, the biochemical and hematological profiles of HHCs of active TB disease were evaluated and correlated with their IGRA status. We also explored the likelihood of clinical laboratory analytes and platelet-associated parameters for use as surrogate markers of subclinical inflammation in LTBI.

A total of 111 HHCs and 61 healthy controls were recruited for the study. Of 111, 58 (52.3%) were positive for IGRA, 52 (46.8%) were negative, and one sample yielded an indeterminate result. In this study, a randomly chosen HHC/IGRA+ve (n=30), HHC/IGRA-ve (n=30), and healthy controls (n=30) were processed for further analysis. The median (IQR) age of HHC/IGRA+ve was 42 (31 – 54.8), HHC/IGRA-ve was 39.5 (33.5 – 46.3), and healthy controls were 36 (29.5 – 43) in years. All participants were evaluated for biochemical and hematological parameters following confirmation using the IGRA test. The frequency of males in each group was 11 (36.7%), 9 (30%), and 17 (56.7%), respectively. The control group had volunteers who had no HHCs with active TB cases. Further, the frequency of TB exposure duration per day in the HHC/IGRA+ve group was 12 to 18 hrs (56.6%), 6 to 12 hrs (26.6%), and more than 18 hrs (16.6%).

The median BMI (IQR) of the HHC/IGRA+ve, HHC/IGRA-ve, and healthy controls were 26.3 (23.3-29.7), 24.8 (22.1- 28.2), and 25.6 (23.1-28.9), respectively. About 80% of the HHCs reported having BCG vaccination during childhood. The most common comorbid conditions observed were diabetes and hypertension among HHC+ve and HHC-ve individuals, respectively. Among HHC/IGRA+ve individuals, eight (26.6%) individuals presented diabetes, six individuals (20%) in HHC/IGRA-ve, and five (16.7%) in healthy controls, while the rate of hypertension was four (15.4%) in HHC/IGRA+ve, four (13.3%) in HHC/IGRA-ve, and six (20%) among healthy controls. The characteristics of the cohorts are presented in Table 1 . No significant difference in patient characteristics was observed between the groups. We did not adjust for potential confounders such as diabetes or hypertension, both of which can alter inflammatory and platelet parameters. However, the frequency of these conditions was low and comparable across study groups, reducing, but not eliminating, the risk of bias. Larger studies with higher prevalence of comorbidities are warranted to examine their potential modifying effects.

The global burden of LTBI was estimated to be 23% and the majority of them (80%) were from the WHO Southeast Asia, Western-Pacific, and African regions (20). There is an increasing need to identify novel approaches or host diagnostic biomarkers for improved diagnosis to prevent the risk of reactivation and subsequent transmission of the disease and to achieve the target of TB elimination by 2050. The biochemical and hematological indices serve as diagnostic and prognostic markers of several infectious diseases (21–23). These markers could aid in early clinical diagnosis and timely medical intervention. TB-infected patients undergo considerable immunohematological and biochemical changes during infection compared to healthy controls (24). Such parameters could potentially help in forecasting the onset of active TB and response to anti-TB treatment. However, the biological parameters of individuals with close contact with active TB patients who are either positive or negative for IGRA have not been investigated and largely remain ambiguous. To our knowledge, this is the first study to evaluate the biochemical and hematological parameters among HHCs of active TB patients. We previously reported the usefulness of plasma cytokines that could aid in diagnosing LTBI across HHCs/IGRA+ve (25). The study found an increased plasma CXCL8 and decreased MCP-1, TNF-α, and IFN-γ associated significantly with LTBI.

The study found ESR, D-dimer, PDW, MPV, and P-LCR to be significantly different from healthy controls and HHC/IGRA+ve individuals. The values obtained could potentially be used to establish reference ranges and to differentiate LTBI from healthy individuals, to aid clinical interpretation and decision-making.

The study found ESR levels among individuals with LTBI to be significantly higher in the HHC/IGRA+ve group. The diagnostic accuracy of ESR above the cut-off of 36.50 mm/hr, which indicates inflammation, although not reliable, is used to predict LTBI. High ESR (>40mm/hr) and thrombocytosis with platelet count (>500×10³/μL) have been associated with active TB infection; however, its use in LTBI has not been established (26). Despite low specificity due to increased levels observed across various conditions like cancer or traumatic ailments, ESR can provide valuable information on disease pathology involving inflammation and infection (27).

In our study, significantly higher PDW, MPV, and P-LCR values were seen in HHC/IGRA+ve individuals. The ROC analysis showed that among the three platelet indices, PDW and P-LCR showed a significant association with LTBI. Recent studies suggest that platelets play a vital role in innate defense against TB (28). An elevated platelet count, CRP, and ESR but not MPV were shown to be significantly associated with pulmonary TB cases (29). The elevated PDW, MPV, and P-LCR values indicate platelet activation and hypercoagulability. The clinical importance of infection-associated thrombosis has recently been recognized and is still not completely understood (30). A recent study showed the risk of myocardial infarction among people with LTBI. The study also showed a higher risk of developing hypertension among untreated LTBI+ve individuals compared to healthy controls (31). Studies have found that PDW and MPV were elevated in pulmonary TB (32, 33). Platelets were identified within the granulomas, and high platelet counts have been found to be associated with TB disease severity (34). Despite clear evidence on the association between TB and platelet indices reported previously (35), information is scarce on the characteristics of these indices in LTBI individuals and their role in reactivation. P-LCR is a marker that indicates the proportion of circulating platelets of more than 12 femtoliters. It identifies platelets that metabolically and enzymatically exhibit more significant activity than smaller ones. The present study indicates the association between P-LCR and LTBI.

Platelets are now increasingly recognized as active players in immune modulation and inflammation. Even during latency, there is a low-grade immune activation to MTB antigens that drives compensatory hematopoietic responses such as increased megakaryopoiesis in the bone marrow (36). This could lead to more large or immature platelets and therefore higher P-LCR. The pattern recognition receptors including Toll-like receptors and C-type lectin receptors, produced by platelets in response to interaction with MTB antigens, release pro-inflammatory cytokines that sustain granuloma stability (37). Prior studies have shown that P-LCR can serve as a diagnostic tool for hematologic disorders (38), such as chronic myeloid leukemia, and has been proven as a biomarker of myelodysplastic syndromes (39, 40). Most existing studies on platelet indices and TB have focused on active TB patients or general inflammatory conditions, not specifically IGRA-defined LTBI in household contacts. Our study showed a significant association with P-LCR, indicating subtler but still detectable platelet activation patterns, potentially manifesting as an increased proportion of large platelets (higher P-LCR) rather than overt thrombocytosis or MPV changes noted in active disease (41). D-dimer, a fibrin degradation product, serves as an indicator of thrombosis. Increased D-dimer indicates hypercoagulability in pulmonary TB (18). In this study, we showed D-dimer levels to be significantly higher in LTBI. However, this is contrary to other findings that have reported an average D-dimer level in LTBI patients diagnosed using the PPD test (42).

The present study showed that when combining ESR and P-LCR, the efficacy of diagnosing LTBI was high, with an AUC of 0.906. Additionally, Spearman’s correlation analysis revealed a significant positive correlation between ESR and other parameters such as CRP, ALT, and fibrinogen in LTBI cases. It could hence be construed that ESR and P-LCR as good surrogate diagnostic markers of subclinical inflammation associated with LTBI among HHCs. The ESR that is measured using the Westergren method and P-LCR through automated blood cell counting acts as a valuable surrogate diagnostic marker and, when adequately validated, could prove the effect on the diagnosis and subsequent clinical outcome of interest (43). Although these inflammatory markers cannot independently replace IGRA and TST, these tests could aid in LTBI, especially among high-risk individuals where rapid diagnosis is critical. Our study showed that the HHC/IGRA -ve individuals, albeit IGRA negative, biochemical profiles were similar to those in HHC/IGRA+ve individuals, except for serum ferritin and MCHC levels, indicating that they may have been in contact with TB cases or LTBI individuals.

The present study indicated the possible association of serum ferritin and MCHC levels with IGRA-ve. MCHC indicates the average concentration of hemoglobin in the RBCs. In this study, the binary regression revealed that MCHC (OR 2.240, 95% CI: 1.042 – 4.814, p=0.039) was found to be significantly associated with males in the IGRA-ve group. Ferritin levels were found to be significantly low (OR 1.037, 95% CI: 1.001 – 1.079, p=0.03) in the females who were IGRA-ve. The study conceivably had sex-imbalanced sampling, which could lead to unintentional gender bias as the study population was not an age- and sex-matched cohort, which is one of the limitations of the study.

Serum ferritin, a test of iron status, originates from damaged cells, reflecting cellular damage and therefore serves as an important inflammatory disease marker (44). Iron plays a crucial role as a cofactor in mycobacterial infection. The production of acute-phase proteins (APPs) in response to inflammation can stimulate or inhibit iron metabolism (45). Elevated serum ferritin (hyperferritinemia) is associated with different inflammatory diseases and their severity. A possible explanation lies in the differing iron metabolism profiles between infected and non-infected individuals. In LTBI (IGRA-positive), immune activation can induce hepcidin-mediated iron sequestration, leading to elevated ferritin as part of the anemia of inflammation. In contrast, IGRA-negative participants, particularly females, may reflect baseline physiological or nutritional iron status, with lower ferritin likely attributable to menstrual blood loss or dietary iron insufficiency rather than infection-related inflammation. Similarly, the association of lower MCHC in IGRA-negative males may relate to underlying hematological or nutritional variations rather than a direct protective effect against LTBI. A previous study showed decreased serum ferritin levels in active TB cases compared to the HHCs, suggesting the downregulation of NRAMP1 gene expression in TB cases (46). Other studies showed Hepcidin but not ferritin to be associated with TB disease severity and progression to active TB (47). However, a recent study among children infected with active and latent infection indicated elevated ferritin levels as a reliable marker for discriminating LTBI from active infection (48). This could possibly be due to the maintenance of iron balance that is critical for effective regulation in MTB, as excess iron can enhance survival, replication, and virulence (45, 49). The analysis was adjusted for sex as a covariate, and previous evidence indicates minimal sex-related variation in hematological and ferritin values within the studied population, suggesting the imbalance is unlikely to have influenced the results (50). Several confounding factors could influence the serum ferritin level, including diet, other causes of inflammation, underlying comorbidities, menstrual cycle, other hormonal changes, and environmental determinants. These factors were not considered in our analysis, and the association with the identified variation in the ferritin level is not known. Given the cross-sectional nature of our study, these findings should be interpreted cautiously and may reflect population-specific factors or residual confounding rather than causal relationships. We did not observe significant differences in hematological parameters like TC, neutrophils, lymphocytes, monocytes, basophils, eosinophils, platelet counts, or hemoglobin and hematocrit levels. This was in corroboration with other studies that found no association of blood cell counts with LTBI (51).

Our findings suggest that simple, routinely available hematological indices, such as ESR, PDW, MPV, and P-LCR, could serve as cost-effective adjunctive markers for identifying HHCs at higher risk of LTBI in resource-limited settings, potentially enabling earlier targeted screening and preventive interventions. While these markers cannot replace IGRA, their integration into clinical workflows could help prioritize individuals for confirmatory testing, thereby improving case detection efficiency.

The present study, however, suffered certain limitations. Firstly, the small sample size appears to limit the statistical power and generalizability of the findings to a broader population, which is particularly paramount given the diversity and variability within a larger population. Secondly, the lack of longitudinal follow-up restricts our ability to assess the long-term outcomes and deviations in the parameters studied over time. Thirdly, confounding factors that could influence the elevation of these inflammatory markers have not been studied. Fourthly, the cross-sectional design precludes establishing causal relationships, and the relatively small, single-center sample limits the generalizability of the findings to other populations. Furthermore, lack of external validation, unmatched controls and the assessed parameters that are closely linked to infection status at the time of sample collection present significant limitations. The findings are likely population-specific, and differences in genetic background, nutritional status, comorbidities, and TB exposure patterns across other demographic, geographic, or clinical settings may yield different associations. Therefore, this warrants future studies to validate the findings using larger and more diverse cohorts to ensure that the results are extrapolated across diverse demographics and clinical settings. Incorporating longitudinal follow-up is key to monitoring long-term outcomes and an improved understanding of the sustainability of the diagnostic biomarkers identified herein.

The current study identified ESR and P-LCR as prominent surrogate diagnostic biomarkers of subclinical inflammation associated with LTBI. The hematological profile, especially ESR and P-LCR of IGRA-ve HHCs of active TB, was similar to that of LTBI, indicating that they might have been infected with MTB. The serum ferritin and MCHC were found to be associated with IGRA-ve. Additional investigations are warranted to arrive at more accurate and definitive explanations of the significant findings observed herein.