Ethical approval was obtained from the Institutional Review Boards of the National Institute of Allergy and Infectious Diseases (NIAID), USA, and the National Institute for Research in Tuberculosis (NIRT), India (NCT04813328, NCT04526613, NIRT-IEC 2020–033 and NIRT-IEC 2020 005). Written informed consent was obtained from all study participants.

We enrolled 38 individuals with TB infection (TBI) from Tiruvallur and Kanchipuram Districts, Tamil Nadu, South India, between September 2022 and January 2023: 15 with Ss co-infection (TBI+Hel+) and 23 without Ss co-infection (TBI+Hel−). Participants were aged 22–63 years. These 38 individuals were prospectively enrolled specifically for this study and were not selected from a larger pre-existing cohort. TBI diagnosis was based on positive QuantiFERON-TB Gold Plus (QFT-Plus) results (QIAGEN), absence of pulmonary symptoms, and normal chest radiographs. None of the participants were known contacts of active TB cases. Pregnant or lactating women were excluded from the study. All participants were HIV-negative and anti-TB treatment–naïve. TBI was identified for study enrolment; TBI-positive individuals were referred to TB clinics per public health protocols.

QFT-Plus testing followed manufacturer guidelines. Heparinized whole blood was incubated at 37°C for 18 hours in TB1, TB2, mitogen, and Nil tubes. In the QFT-Plus assay (Qiagen), the TB1 tube contains long ESAT-6 and CFP-10 peptides that predominantly stimulate CD4⁺ T cell responses, whereas the TB2 tube contains additional short ESAT-6 and CFP-10 peptides designed to activate both CD4⁺ and CD8⁺ T-cell subsets. Tubes were centrifuged at 3,000 g for 15 minutes, and supernatants were collected for IFN-γ measurement (IU/mL). Remaining supernatants were stored at −80°C.

Strongyloides stercoralis (Ss) infection was diagnosed by IgG antibodies to the recombinant NIE antigen (12, 13). A single stool sample was examined by Kato-Katz; stool microscopy was negative for other intestinal helminths. Ss infection was further confirmed by nutrient agar plate culture (14). Filarial infection was excluded by negative circulating filarial antigen tests. S. stercoralis was the only helminth species detected in this study population. All Ss-positive participants received ivermectin (12 mg, single dose) and albendazole (400 mg, single dose). Follow-up blood draws and repeat parasitological examinations were performed six months later to confirm clearance.

M.tb H37Rv was cultured in a Middlebrook 7H9 liquid medium (Sigma-Aldrich). The mycobacterial inoculation volume for each batch was titrated to achieve a time to positivity (TTP) of 7 days in MGITs in a BACTEC MGIT 960 machine. The inoculum was titrated to achieve a 7-day time to positivity (TTP) in MGITs; inocula ranged between approximately 10^2–10^3 CFU/mL.

Human peripheral blood mononuclear cells were isolated from peripheral blood and resuspended at an approximate concentration of 2–3 × 10⁶ cells per mL of RPMI (containing 10% fetal bovine serum and 2 mM l-glutamine) and benzonase was added to a final concentration of 25 U/mL. Cells were rested at 37°C for 2 hours with 5% CO₂. Six hundred microliters of RPMI (containing 2 mM L-glutamine and 25 mM HEPES) seeded with 1×10⁶ PBMC and M.tb H37Rv was added to duplicate 2-mL screw-cap tubes. The co-cultures were incubated on a 360° rotator at 37 °C for 96 hours, after which tubes were micro-centrifuged at 15,300 g for 10 minutes, and the supernatant was carefully removed by pipetting. Cells were lysed with the addition of 500 µL sterile water and the tubes pulse-vortexed at 0, 5 and 10 minutes. Five hundred microliters of lysate were then transferred directly to a BACTEC MGIT tube supplemented with PANTA antibiotics (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) and OADC enrichment broth. On day 0, duplicate direct-to-MGIT inoculum control tubes were prepared with the same M. tuberculosis input as samples. In the end, MGIT tubes were placed on the BACTEC 960 machine and incubated at 37 °C until the detection of positivity by fluorescence. TTP was converted to log10 CFU using standard curves generated by inoculating MGIT tubes with 10-fold mycobacterial dilutions and plating aliquots on 7H11 agar. Linear regression (GraphPad Prism v10) provided equations for TTP-to-CFU conversion. Normalized mycobacterial growth was defined as log10 CFU (sample) − log10 CFU (growth control). The difference between the medians of respective groups is described as Δ X log and was calculated by subtracting the median of the test group from the median of the control group.

Cytokine levels in culture supernatants were measured using a Human Magnetic Luminex Assay (R&D Systems) on the Bio-Rad MAGPIX platform (xPONENT 4.2; Bio-Plex Manager 6.1). Analytes included Interferon gamma (IFNγ), interleukin-2 (IL-2), Tumor Necrosis Factor alpha (TNFα), IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-6, IL-12p70, IL-18, IL-4, IL-5, IL-10, and IL-13. The lowest detection limits for cytokines were as follows: GM-CSF, 18.4 pg/mL; IFN-γ, 5.7 pg/mL; IL-1α/IL-1F1, 10.6 pg/mL; IL-1β/IL-1F2, 3.5 pg/mL; IL-2, 3.6 pg/mL; IL-4, 1.1 pg/mL; IL-5, 6.2 pg/mL; IL-6, 9.0 pg/mL; IL-10, 32.2 pg/mL; IL-12p70, 18.5 pg/mL; IL-13, 31.8 pg/mL; IL-17/IL-17A, 9 pg/mL; IL-18/IL-1F4, 2.5 pg/mL; and TNF-α, 12.4 pg/mL.

Data were summarized as medians with interquartile ranges (IQR). Between-group comparisons used the Mann–Whitney U test with Holm–Bonferroni correction for multiple comparisons. Correlations were assessed using Spearman’s rank correlation. Ss-specific IgG (OD values) and all cytokines measured in QFT or PBMC supernatants were entered as independent variables in a multivariate linear regression model to assess their combined association with MGIA (Δ log10 CFU). Analyses were performed in GraphPad Prism v10.0; significance was set at p ≤ 0.05.

The demographic, hematological, and biochemical characteristics of the study participants are summarized in Table 1. There were no statistically significant differences in age, gender, or body mass index (BMI) between the Tuberculosis infected (TBI) individuals with helminth infection (TBI+ Hel+) and those without helminth infection (TBI+). As only Strongyloides stercoralis infection was identified among helminth-positive participants, the comparisons presented here specifically reflect the impact of S. stercoralis co-infection on immune responses in TBI. However, notable differences were observed in certain hematological parameters. TBI+ Hel+ individuals exhibited significantly decreased levels of hemoglobin (TBI+ Hel+ 11.25 Vs TBI + 13.10 g/dl, p=0.022), Red Blood Cells (RBC) (TBI+ Hel+ 4.75 Vs TBI + 4.05 (10³/ml), p=0.034) and lymphocytes compared to TBI+ individuals. In contrast, eosinophil levels (TBI+ Hel+ 0.75 Vs TBI + 0.44 (10³/ml), p=0.028 were significantly increased in TBI+ Hel+ individuals compared to TBI+ individuals.

To determine the influence of coexistent Ss infection on type 1, type 17, and proinflammatory cytokines in TBI, we measured the QFT supernatants levels of IFN-γ, TNF-α, IL-2, IL-17, IL-22, IL-1α and IL-1β in TBI+Hel+ and TBI+Hel- individuals. As shown in Figure 1, TBI+Hel+ co-infected individuals exhibited significantly decreased levels of Type 1, median (IFN-γ: TBI+Hel+, 63 pg/mL Vs TBI+, 268.1 pg/mL, p=0.0126, IL-2, TBI+Hel+, 195 pg/mL Vs TBI+, 265 pg/mL, p=0.0267; TNF-α: TBI+Hel+, 25.06 pg/mL Vs TBI+, 47.50 pg/mL, p=0.0177; Type 17 (IL-17, TBI+Hel+, 10 pg/mL Vs TBI+, 16 pg/mL, p=0.0035; IL-22, TBI+Hel+, 15.88 pg/mL Vs TBI+, 23.56 pg/mL, p=0.0306 and proinflammatory cytokines, IL1α, TBI+Hel+, 3.45 pg/mL Vs TBI+, 4.07 pg/mL, p=0.0233 and IL1β, TBI+Hel+, 15.74 pg/mL Vs TBI+, 17.42 pg/mL, p=0.0365 at baseline and upon TBAg1 stimulation, median (IFN-γ: TBI+Hel+, 267.5 pg/mL Vs TBI+, 415.1 pg/mL, p=0.0312, IL-2, TBI+Hel+, 175 pg/mL Vs TBI+, 352.9 pg/mL, p=0.0181; TNF-α: TBI+Hel+, 27.38 pg/mL Vs TBI+, 61.11 pg/mL, p=0.0395; and IL-22, TBI+Hel+, 98 pg/mL Vs TBI+, 124 pg/mL, p=0.0240; upon TBAg2 antigenic stimulation median (IL-2, TBI+Hel+, 230 pg/mL Vs TBI+, 381.2 pg/mL, p=0.0086; TNF-α: TBI+Hel+, 40.99 pg/mL Vs TBI+, 68 pg/mL, p=0.0217; Type 17 (IL-22, TBI+Hel+, 28.20 pg/mL Vs TBI+, 78.20 pg/mL, p=0.0084; proinflammatory cytokines, IL1α, TBI+Hel+, 4 pg/mL Vs TBI+, 5.5 pg/mL, p=0.0145 and IL1β, TBI+Hel+, 15.48 pg/mL Vs TBI+, 19.80 pg/mL, p=0.0220 in comparison with TBI individuals alone. In contrast, the upon mitogenic stimulation there was no significant difference between the two groups. Thus, coexistent Ss infection is associated with diminished systemic levels of type 1 type 17 cytokines and proinflammatory cytokines in TBI alone individuals.

We assessed the in vitro growth inhibition of Mycobacterium tuberculosis H37Rv in peripheral blood mononuclear cells (PBMCs) from individuals with latent tuberculosis infection (TBI) with and without Ss co-infection. As shown in Figure 2, TBI+Hel+ individuals exhibited significantly higher normalized mycobacterial growth (Δ log10 CFU 3.8) than TBI+Hel− individuals (Δ log10 CFU 2.8; p=0.0127), indicating reduced capacity to inhibit M. tuberculosis growth in the Ss co-infected group.

We evaluated cytokine responses following stimulation of PBMCs with Mycobacterium tuberculosis H37Rv to assess the impact of Ss co-infection in individuals with tuberculosis infection (TBI). As shown in Figure 3, TBI-helminth co-infected individuals exhibited significantly diminished secretion of Th1 cytokines, median concentrations were IFN-γ (35 pg/mL Vs 26.51 pg/mL), IL-2 (7.6 pg/mL Vs 6.8 pg/mL), and TNF-α (39.1 pg/mL Vs 33.8 pg/mL), compared to TBI+ individuals alone. Furthermore, median concentrations were IL-17 (139.2 pg/mL Vs 119.0 pg/mL) and GM-CSF (21.08 pg/mL Vs 18.2 pg/mL) —key cytokines involved in Th17-mediated immunity and granuloma integrity—were also significantly reduced in the helminth-infected group.

In addition to the suppression of adaptive immune responses, innate inflammatory cytokine production was also markedly impaired. Notably, levels of median concentration were IL-1α (179.7 pg/mL Vs 144.9 pg/mL) and IL-6 (201.2 pg/mL Vs 132.3 pg/mL), were significantly decreased in LTBI-helminth co-infected individuals following M. tuberculosis stimulation. Whereas IL-1β (16.8 pg/mL Vs 13.88 pg/mL), IL-12p70 (49.2 pg/mL Vs 43.8 pg/mL) and IL-18 (5.7 pg/mL Vs 5.3 pg/mL) levels were decreased in TBI- Ss co-infected individuals but did not show any significant difference when compared with TBI+ individuals alone. These cytokines are critical for early-phase immune activation and effective initiation of host defense mechanisms against TB. Together, these results indicate that Ss co-infection leads to broad immune suppression encompassing both innate and adaptive arms of the immune response to M. tuberculosis.

Next, we evaluated the Th2 and regulatory cytokines following stimulation of PBMCs with Mycobacterium tuberculosis H37Rv to assess the impact of Ss co-infection in individuals with tuberculosis infection (TBI). In contrast, Ss infected individuals exhibited elevated levels of type 2 and regulatory cytokines, median concentrations were IL-4 (5.1 pg/mL Vs 7.1 pg/mL), IL-5 (21.2 pg/mL Vs 26.4 pg/mL), IL-10 (142.9 pg/mL Vs 204.3 pg/mL), and IL-13 (47.9 pg/mL Vs 50.6 pg/mL), following M. tuberculosis stimulation (Figure 4). These findings are consistent with a Ss induced immunological milieu that favors Th2 and regulatory responses, potentially at the cost of effective antimycobacterial immunity.

Principal component analysis (PCA) was performed to identify patterns of immune variation associated with helminth coinfection in TBI individuals (Figure 5). The first two components (PC1 and PC2) accounted for approximately 44.6% of the total variance (PC1: 28.6%, PC2: 16%), capturing the major immunological differences between the study groups. The PCA biplot showed distinct clustering between TBI and TBI⁺+Hel⁺ group, indicating that Ss infection significantly altered the cytokine response network. Cytokines such as IL-4, IL-5, IL-10, and IL-13 loaded strongly on the positive axis of PC1, aligning with MGIA, and reflected a Th2/regulatory-dominant profile in helminth-infected individuals. In contrast, IL-17, IFN-γ, IL-2, TNF-α, IL-1α, and IL-1β clustered negatively along PC1, representing Th1/Th17-associated inflammation characteristic of helminth-negative TBI cases. This segregation highlights that helminth infection promotes a systemic shift from Th1/Th17 to Th2/regulatory dominance, consistent with reduced mycobacterial control.

Correlation analysis between cytokine profiles and Mycobacterial Growth Inhibition Assay (MGIA) responses in tuberculosis-infected (TBI) individuals with Ss co-infection (TBI⁺Hel⁺) revealed distinct immunological associations. As shown in Table 2 3II, among the measured cytokines, IL-17 (r = –0.3884, p = 0.0110), IL-1α (r = –0.3537, p = 0.0371), and IL-1β (r = –0.3950, p = 0.0189) displayed significant negative correlations with MGIA, suggesting that elevated levels of these proinflammatory mediators were associated with reduced mycobacterial growth control. Other Th1 cytokines such as IFN-γ, IL-2, and TNF-α showed negative but non-significant trends with MGIA, while Th2 and regulatory cytokines (IL-4, IL-5, IL-10, IL-13) showed weak positive correlations, none reaching significance. Although some correlations reached statistical significance, the Spearman rho values were low, indicating that these associations are relatively weak and should be interpreted with caution. To further evaluate the independent contributions of cytokines and Ss-specific IgG to mycobacterial growth inhibition, all measured cytokines along with Ss-IgG were included in a multivariate regression model (Table 3). The model demonstrated that Ss-IgG, IL-17, IL-1α, and IL-1β remained negatively associated with MGIA values, while selected Type 2 cytokines such as IL-5, IL-10, and IL-13 showed positive associations. These results indicate that despite the presence of inflammatory mediators, the functional ability to restrict mycobacterial growth is impaired in Ss -TBI co-infected individuals, pointing toward a qualitative imbalance rather than quantitative deficiency in cytokine responses.