Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) has become a worldwide public health emergency since its appearance in December 2019 1. Optimal treatment for COVID-19 remains challenging but some progress has been made 2. In severe COVID-19, the use of corticosteroids has been associated with improvement in treatment outcomes. Other immunosuppressive and biological agents have been used empirically or in clinical trials for severe and critical COVID-19. The risk of opportunistic infections and reactivation of tuberculosis (TB) in patients undergoing these treatments remains unclear but some TB cases have been reported 3–5. The use of Interferon gamma release assays (IGRAs) has become a routine practice for screening and diagnosis of latent tuberculosis infection (LTBI) in high-income and low-TB incidence settings 6.

IGRAs measure the release of interferon gamma (IFN-γ) in response to stimulation with specific M. tuberculosis (Mtb) antigens (i.e. ESAT-6 and CFP- 10) in peripheral blood samples. QuantiFERON TB Gold Plus™ (QFT-Plus) is the latest version of ELISA-based IGRA platform that contains two Mtb-antigen tubes that combined elicits both CD4 and CD8 antigen-specific T cell responses. This ex vivo diagnostic platform includes a negative control tube and a positive control tube (mitogen), the latter of which allows measuring IFN-γ release in blood in response to a non-specific lymphocyte activator (i.e. phytohemagglutinin [PHA], a lectin, a type of glycoprotein).

The QFT-Plus assay is currently the most commonly utilized IGRA test in Asturias, Spain. Since December of 2019, and especially in the winter and early spring of 2020, we have observed an unusual increase in the number of QFT-Plus test results with low IFN-γ responses in the mitogen control tube and indeterminate QFT-Plus test results. This period coincides with the time of the COVID-19 pandemic in our region in Spain (Asturias). In this context, we hypothesized that this unprecedented increase in the number of low IFN-γ responses to PHA was related to an unidentified biological effect of COVID-19 or its treatment. To investigate further, we measured the frequency of this unusual IGRA testing result and studied the potential factors associated with low IFN- γ responses to PHA. Specifically, we prospectively reviewed charts and measured sequential PHA responses in blood T cell subsets in a cohort of hospitalized patients with suspected or confirmed COVID-19 pneumonia during the peak of the COVID-19 pandemic.

Since December 2019, we observed an unprecedented increase in the rate of low IFN-γ responses to PHA in the QFT-Plus assay. These abnormal immune response findings were especially pronounced during the first four waves of the COVID-19 epidemic in our region from March to May 2021 (Figure 1A), suggesting that the findings were related to biologic effects of COVID-19. We have also observed that this low mitogen response in the QFT assay is more often present in patients with confirmed COVID-19 pneumonia compared with patients with suspected COVID-19 pneumonia but with negative RT-PCR test for SARS-CoV-2 virus, further supporting the association between COVID-19 and low mitogen responses. Further, subgroup analysis showed that the presence of chest x-ray abnormalities were associated with abnormal QFT mitogen response in our univariate analysis, again suggesting that the low IFN-γ responses to PHA were associated with true COVID-19 pneumonia.

Other studies have also reported a similar increase frequency of low IFN-γ response to mitogen and indeterminate QFT results in hospitalized patients with COVID-19, including patients with severe and critical COVID-19 and hyperinflammatory syndrome 12,13. Interestingly, indeterminate QFT results were more frequent among COVID patients who died during hospitalization in a retrospective study from Spain (29.1% vs. 64.7%) 13, but these abnormal QFT results were not associated with increased mortality in a study from the United States 12. However, these studies did not adjust these survival analyses to other known factors associated with increased mortality in COVID-19, including older age, male sex, and various comorbidities 14.

Reduced IFN-γ responses to PHA stimulation are common in certain clinical scenarios. Most often, they are observed in immunosuppressed patients, both those with underlying immunodeficiency and those on immunosuppressive treatments 15,16. Importantly, none of the patients in our cohort received immunosuppressive drugs for the treatment of COVID-19 prior to undergoing IGRA testing. Specific immune dysfunctions are also well-established to increase the rate of indeterminate IGRA tests, including the low CD4 counts of HIV and the T cell exhaustion exhibited among critically-ill patients and patients with active lupus erythematosus 17,18. However, in our study, patients with low IFN- γ responses did not have reduced CD4 counts, and we did not find an association between severity of COVID-19 and low mitogen response, which argues against T-cell exhaustion. However, T-cell exhaustion and complex immune activation are common in patients with COVID-19 pneumonia, and we cannot exclude these pathophysiological factors playing a role 19. In addition, in patients with inflammatory bowel disease, low serum albumin, lymphopenia, and corticosteroid therapy have been associated with increased odds to have indeterminate IGRA results with low mitogen response in a multivariate analysis 20. In our study, we did not analyze serum albumin levels but the presence of lymphopenia had a trend towards statically significant association with a low mitogen response in the QFT assay. Another study with critical COVID-19 patients found no direct association between lymphopenia, inflammatory markers, or severity of COVID-19 with QFT indeterminate results 12; however, some of those critically-ill COVID-19 patients received immunosuppressive treatments, which was not a cofounder in our study. Occasionally, indeterminate response can be seen related to technical problems inherent to the assay variability of any immunodiagnostic test (pre-analytical, analytical and post-analytical phases). However, we were unable to identify any analytical laboratory factor or patient-related immunosuppressive factors to explain these study laboratory findings.

In our prospective study cohort with T cell immune profiling data, a reduction in total CD8 T cell counts and differentiated CD8 EMRA T-cell subset were statistically associated with the observed reduction in IFN-γ responses to PHA stimulation in the QFT-Plus assay. Lymphopenia with preferential peripheral blood CD8+T-cell reduction has been described in COVID-19, but the mechanism of the observed frequent reduction in IFN-γ responses to PHA activation in the QFT-Plus assay is not entirely clear 21. Recent reports suggest the presence of T cell and NK cell dysfunction and exhaustion phenotypes associated with a hyperactivation immune state during COVID-19. This phenomenon is suggested by overexpression of CD69, a marker of activation, and TIM-3 on NK, CD4 and CD8 T cells. TIM-3 is a negative regulator of immune cell function and its ligands induces T and NK cell exhaustion in viral infections 22. Along these lines, another study showed that a small subset of critically-ill COVID-19 patients with indeterminate QFT results had increased IL-10 levels and reduced Th1-related cytokine levels, which the authors suggest a relationship with Th2 immune responses in this setting. However, recent immune profiling data in patients with COVID-19 pneumonia showed not only increase Th2 response can be observed but also a global immune and inflammatory activation process that includes complex immunoregulatory mechanisms and T-cell exhaustion that can affect Th1-immune response, including IFN-γ responses to activating factors 19. In addition, there is CD8 T cell dysfunction in elderly patients with COVID-19, and EMRA CD8+T-cell subsets also display a senescence-associated secretory phenotype that may contribute to age-related inflammation in COVID-19 23,24. Interestingly, there was a trend towards an older age in the study group with low mitogen response in the QFT compared with the group with normal mitogen response in our study (Table 1). In this context, there are increasing numbers of reported cases of COVID-19 and TB co-infection 25,26. In fact, there is also a concern that viral infections, including COVID-19, could have a pathophysiologic effect on TB progression and TB reactivation in LTBI 27–30. However, the clinical implications of these abnormal CD8+T cell responses associated with COVID-19 for other infections, including potential for TB reactivation in the future, are unclear at this time of the pandemic.

Importantly, some experts suggested that the use of IGRA to screen for TB infections in patients with severe COVID-19 has limited value11,31. Our study results show the overall number of patients with a positive QFT result remained unchanged before and during the pandemic (13.95% vs. 12.2%, respectively). We also did not find there any significant difference in rates of “positive” response to the QFT Mtb specific antigens in those patients with low/very low PHA response in the mitogen tube before and during the pandemic (6.2% vs. 7.9%; and 1.3% vs. 2.1%; for low and very low response to PHA, respectively). Moreover, some of our hospitalized COVID-19 patients showed “positive” Mtb antigen-specific responses (≥0.35 IU/mL) despite transiently or persistent low response to mitogen in repeat QFT testing (Table 3). Overall, our results suggest considering repeating QFT testing in 3-7 days in patients with COVID-19 pneumonia with risk factors for Mtb infection, particularly in those patients with “positive” Mtb specific antigen response.

Of note, the first confirmed COVID-19 case in Spain was officially reported in February 2020 but we observed an unexpected high rate of abnormal QFT mitogen response since December 2019 in our study. If our hypothesis holds true, our study laboratory findings also suggest that the COVID-19 epidemic may have started earlier in our region (Figure 1 A). This study finding also suggests that monitoring the rate of low mitogen response in the QFT-Plus assay can be utilized as an epidemiological screening tool for early detection of COVID-19 epidemics that could trigger more specific testing of suspected cases and undiagnosed patients with pneumonia in the community. The complexity (logistics and cost) inherent to a mass COVID-19 screening strategy is unquestionable. In our region, the results observed (at least with regard to the evolution of the number of deaths due to COVID-19 and the temporal correlation with the different pandemic waves) were, surprisingly, identical to those registered with about 2 million diagnostic tests performed for COVID-19 between March 2020 and October 2021, compared to 12,837 QFT assays (mean 642 test/month). Moreover, in contrast with previous epidemic waves, during the fifth wave, the number of cases with RT-PCR SARS-CoV-2 positive increased in our region, while severe cases and mortality were significantly reduced, and also the number of patients with low IFN-gamma response to PHA in the mitogen tube. This situation could be explained because in most cases were young people with mild symptoms, as well as by the high degree of vaccination coverage in our region. We think that these findings support the hypothesis of a potential population-based marker of COVID-19-related mortality but additional studies would be needed to determine if this marker is sensitive and specific for early detection and prediction of COVID-19-related mortality at the population based level.

Our study has some potential limitations, including the observational nature of the study design and the relatively limited number of hospitalized patients studied in the cohort part of the study. A small number of subjects can lead to suboptimal power to differentiate true differences in the study subgroups. Multiple comparison analysis can also increase the risk of false discovery rates. However, we believe that our study cohort was representative of patients suspected of and confirmed COVID-19 pneumonia in our region during the first peak of the pandemic given the referral nature of our laboratory and hospital services. Our study also has several strengths, including the centralized nature of our laboratory services that allow us to capture the high frequency of QFT testing utilization and LTBI screening results in our entire region during the pandemic. In addition, our study benefited from the excellent epidemiological tracking of COVID-19 cases and their laboratory results in our region in Spain. In addition, the prospective nature of our multifactorial and immune profiling investigations is another important strength of our study.

In conclusion, our study findings suggest that low IFN-γ responses in the mitogen control tube of the QFT-Plus assay often occur in COVID-9 pneumonia, which can be utilized as a surveillance tool to track the COVID-19 pandemic waves. These abnormal QFT-Plus results were likely related to a suboptimal number and response of a subset of differentiated CD8 T cells to PHA associated with SARS-CoV-2 infections. The clinical implications of these abnormal immune responses in COVID-19 for other infections, including the potential for an increase in TB reactivation rates in the future, are unclear at this time of the pandemic. However, this abnormal mitogen response does not seem to affect the IFN-γ response to specific Mtb antigens in COVID-19 patients, and thus, we propose to revisit and redefine the QFT-Plus indeterminate test results criteria in this setting.

Ethical clearance was approved by the Comité de Ética de la Investigación del Principado de Asturias, Spain

The authors have no conflicts of interest to declare.

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