Tuberculosis represents a chronic infectious disease whose clinical presentation becomes increasingly complex and atypical in HIV-infected individuals (1). Laboratory detection of Mtb often proves challenging, necessitating the integration of multiple diagnostic techniques to directly or indirectly identify evidence of Mtb infection — a critical step for ensuring timely diagnosis and treatment. Meta-analysis has demonstrated that the aggregated sensitivity of the IP-10 assay reaches 85% (95% CI: 80%–88%), with a specificity of 89% (95% CI: 84%–92%) (13), indicating the substantial potential of IP-10 as a biomarker for auxiliary TB diagnosis. However, evidence supporting effective diagnosis using this method in HIV-infected individuals, particularly those with severe immunosuppression, remains extremely limited.
In this study, we evaluated the diagnostic performance of the IP-10 mRNA release assay in 101 HIV-infected individuals suspected of having TB, categorizing them into 57 cases of pulmonary TB and 29 cases of non-TB based on final diagnosis. When compared with the commonly used immunological assay TB-IGRA, our results demonstrated the sensitivity and specificity of IP-10.TB for diagnosing PTB were 68.4% and 69.0%, respectively. Notably, the sensitivity of IP-10.TB was significantly superior to that of TB-IGRA (P<0.01), while no statistically significant difference in specificity was observed (P=0.07). Meta-analysis has revealed that the aggregated sensitivity of the IP-10 assay reaches 85% (95% CI: 80%–88%), with specificity at 89% (95% CI: 84%–92%) (13), indicating the substantial potential of IP-10 as a biomarker for auxiliary TB diagnosis. However, evidence supporting effective diagnosis using this method in HIV-infected individuals, particularly those with severe immunosuppression, remains extremely limited.
Our findings demonstrate that the IP-10 is predominantly secreted by monocytes and myeloid dendritic cells (14), with its release process being minimally affected by CD4+ T cell depletion. Furthermore, previous multicenter studies have indicated that the sensitivity of IP-10 mRNA as a biomarker remains largely uninfluenced by CD4+ T cell counts (15). Our results align with these conclusions, demonstrating that IP-10.TB exhibits high diagnostic sensitivity even in severely immunosuppressed populations. This enhanced performance can be attributed to the fundamental differences in detection targets between TB-IGRA and IP-10.TB. The former detects IFN-γ at the protein level, whereas the latter quantifies the transient expression of IP-10 mRNA, a downstream molecule in the IFN-γ signaling pathway (16), thereby substantially improving detection sensitivity.
In our study, IP-10 mRNA release assays demonstrated lower specificity compared to TB-IGRA, although this difference was not statistically significant. This reduced specificity may be attributed to the fact that most participants had CD4+ T-cell counts below 200 cells/μL, and this compromised immunity likely increased the prevalence of latent Mtb infection (17). Additionally, the limited sample size of the non-TB group may have further amplified this difference. Furthermore, false-positive cases in the non-TB group consisted mainly of NTM-infected individuals, with a negative detection rate of 52.6% (10/19). The ESAT-6, CFP-10, and PPE antigens utilized in IP-10.TB are derived from the region of difference-1 (RD-1) of Mtb. This region exhibits no cross-reactivity with antigens from BCG or the vast majority of NTM strains, except for a few species such as M. kansasii, M. marinum, and M. szulgai (18). Therefore, it effectively avoids interference from most NTM strains. However, when infected with NTM strains containing ESAT-6, CFP-10, or PPE antigens, these antigenic components may stimulate the host immune system, inducing a T-cell immune response similar to that elicited by Mtb infection. This phenomenon may result in positive IP-10 release assay results, thereby causing false-positive outcomes. False positives may also result from differences in the TB-specific antigens employed: TB-IGRA utilizes ESAT-6 and CFP-10 antigens, whereas IP-10.TB employs ESAT-6, CFP-10, and PPE antigens. However, the precise mechanisms underlying these differences require further investigation. In the present study, concordance between IP-10.TB and TB-IGRA results were poor, which may be attributed to three factors. Firstly, the differences in assay targets and their expression levels (mRNA versus protein). Secondly, a greater variability in HIV-infected individuals with severe immunosuppression. Thirdly, the lower sensitivity of TB-IGRA. Consequently, IP-10.TB may represent an attractive alternative diagnostic method for HIV-infected individuals.
Compared with traditional IGRAs, the IP-10.TB method requires a shorter incubation time, with results available in as little as six hours. Moreover, the linear detection range of PCR technology is broader than that of ELISA. By leveraging the amplification curves to calculate the results, the variability in result interpretation is reduced, enhancing the objectivity of the detection results. The findings of this study suggest that IP-10.TB has great potential for use in the early diagnosis of tuberculosis in HIV-infected individuals. By optimizing and integrating specific tests, patients with tuberculosis can be identified earlier, thus facilitating early initiation of treatment and potentially reducing the mortality rate associated with the disease.
This study also has some limitations. First, it was a single-center, small-sample study, and the extrapolation of conclusions are challenging. Moreover, the enrolled hospitalized patients were predominantly severely immunocompromised, which may have resulted in selection bias. Lastly, it might be more objective and convincing to use a third alternative method to validate the inconsistency between the IP-10 mRNA release assay and the TB-IGRA test results. This part of our work is in progress.
Therefore, multicenter studies with larger and more diverse samples are necessary to validate the diagnostic performance of IP-10 mRNA release assay. Additionally, although IP-10.TB has a high sensitivity, it is not effective in differentiating between latent infection with Mtb and active tuberculosis, and cannot predict the transition from latent infection to active tuberculosis. Besides, the IP-10.TB results could not reflect the relationship with the Mtb bacterial load, thus limiting its application in evaluating the efficacy of anti-tuberculosis treatment.
IP-10 mRNA release assays have significant clinical value and potential as an auxiliary tool for diagnosing TB in HIV-infected individuals. Furthermore, the combined use of IP-10.TB and Xpert MTB/RIF can increase the diagnostic efficacy of TB. In the cases of EPTB with insufficient diagnostic evidence, IP-10 mRNA release assays provide complementary and auxiliary diagnostic benefits.