There is a high prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations which also have a high prevalence of tuberculosis (TB). ^{1– 4} Epidemiologic studies have shown that concomitant Mycobacterium tuberculosis (Mtb) infection worsens the prognosis of COVID-19. ^{5– 7} Moreover, there is concern that the temporary immunosuppressive effect associated with many viruses may lead to progression of subclinical or reactivation of latent mycobacterial infection. ^{8, 9} Coinfection may therefore have important implications for public health.

Murine studies seem to suggest that primary SARS-CoV-2 infection has little to no impact on the outcome of subsequent Mtb infection. ^{10– 12} Data in humans are very limited at present. In vitro and ex vivo studies have provided some evidence that primary infection with Mtb may not protect against SARS-CoV-2, but rather impair critical T cell responses to SARS-CoV-2. ^{8, 13– 15} Studies which indirectly examine the impact of primary infection with SARS-CoV-2 on the outcome of Mtb infection in humans often focus on cohorts with severe COVID-19 and these suggest that recent or concomitant SARS-CoV-2 may adversely affect the outcome of Mtb infection. ^{8, 15– 19}

Monocyte dysfunction is one potential mechanism underlying this interaction. Mtb-influenza coinfection is known to worsen TB outcomes through alteration of the anti-mycobacterial activity of monocytes/macrophages in the lung, and influx of Mtb-susceptible myeloid cells that serve as a reservoir for Mtb. ^{20– 22} A dysregulated peripheral blood myeloid compartment is a feature of both COVID-19 and TB. ^{23– 25}

In vitro growth inhibition of mycobacteria by peripheral blood mononuclear cells (PBMC), of which 15% - 20% are monocytes, is an objective measure of Mtb control by peripheral immune cells, and correlates with total pathology at necropsy, lung lesion count, and erythrocyte sedimentation rate after in vivo Mtb challenge in non-human primates. ^{26, 27} In human studies, a PBMC-based assay using Mycobacteria Growth Indicator Tube (MGIT) culture found that mycobacterial growth inhibition was better in people recently exposed to Mtb than in those with latent infection, and correlated with the presence of CD14 ^dim monocytes. ²⁸ A whole blood growth inhibition assay using BCG-lux found no difference between Tuberculin skin test negative or positive household contacts (HHC) though this observation may depend on local TB prevalence. ^{29, 30} These authors also showed a correlation between Mtb growth inhibition and peripheral blood neutrophil counts in HHC, which was absent in healthy controls. ²⁹ Lastly, altered proportions of intermediate and non-classical monocyte subsets correlated with differential mycobacterial control in whole blood from people with active TB disease compared to controls. ³¹

As most SARS-CoV-2 infections result in clinically mild disease, ³² we tested whether recent previous mild SARS-CoV-2 infection affects the ability of PBMC to inhibit Mtb growth, by adapting and implementing a mycobacterial growth inhibition assay (MGIA) previously optimised for BCG vaccine trials. ^{8, 9} We modified it for our in-house Mtb H37Rv strain, and compared the outcomes in healthy HHC (with known exposure to Mtb and therefore presumed recent Mtb infection) with evidence of previous SARS-CoV-2 infection, HHC without evidence of previous SARS-CoV-2 infection, and people with both active TB disease and previous SARS-CoV-2.

Our experiments show that previous mild SARS-CoV-2 infection does not affect the in vitro Mtb H37Rv growth inhibition by PBMC, including when autologous serum is added to test the additional mycobactericidal impact of non-cellular blood components. A linear model which adjusted for the available clinical covariates found that PBMC from people with current TB disease and previous mild SARS-CoV-2 infection demonstrated better Mtb growth inhibition than the group without CoV infection and without active TB disease, in keeping with evidence from whole blood MGIAs, possibly due to priming of immune cells against Mtb. ³⁴ These findings suggest that people in high SARS-CoV-2 prevalence areas, where most will manifest only mild or asymptomatic COVID-19, may not have impaired ability to eradicate new or existing Mtb infection.

There is strong epidemiologic evidence showing worse clinical outcomes – more severe disease and higher mortality – from concurrent Mtb/SARS-CoV-2 infection in humans, which suggested our hypothesis that SARS-CoV-2 impairs Mtb killing. Our results do not support such a conclusion for the long term effects of SARS-CoV-2 on Mtb infection. This may be because the outcome of Mtb infection is only affected by severe COVID-19, which is known to cause generalised T cell exhaustion, reduced circulating regulatory T cells, CD8 ⁺ T cells, natural killer cells, and CD4 ⁺ T cells, thereby possibly impairing the immune response to Mtb. ³⁵ This is supported by Riou et al. who showed that the frequency of Mtb-specific CD4+ T cells is 5-fold lower in hospitalised patients with mild-moderate and severe acute COVID-19, than in a pre-pandemic cohort with latent TB infection. ⁸ It may be that our cohort of people with recent mild SARS-CoV-2 had no residual effects on their immune system which were significant enough to impair in vitro Mtb growth control.

A second possible explanation is that the observed increase in mortality from coinfection is attributable to non-immune factors that affect both COVID-19 and TB outcomes, such as nutritional status, delayed presentation, or pandemic-related limitations in health care service provision. This would be in keeping with evidence from murine studies. Rosas Mejia et al. showed that Mtb-infected mice were resistant to the pathological consequences of secondary SARS-CoV-2 infection, but that SARS-CoV-2 infection did not affect Mtb burdens in the lungs or distant organs. ¹⁰ Baker et al. also found no difference in TB burden between mice with prior SARS-CoV-2 and those without, and that Mtb/SARS-CoV-2 coinfection did not affect the proportions of pulmonary Mtb-specific CD4+ and CD8+ T cells, but did significantly reduce the proportion of SARS-CoV-2-specific CD4+ and CD8+ T cells. ¹²

Lastly, the higher mortality from coinfection reported in epidemiologic studies might be explained by the dysfunction of an element of the immune system, or at an anatomical site, or in a TB disease state which is not captured by our PBMC-based MGIA. In an analysis of transcriptomic signatures of PBMC and bronchoalveolar lavage fluid from people with SARS-CoV-2 infection and people with Mtb infection across all levels of severity, integrative single-cell-RNAseq analysis identified FCN1- and SPP-expressing macrophages enriched in both SARS-CoV-2 BALF and TB blood. ¹³ Moreover, human macrophages cultured in the inflammatory milieu of Mtb-infected macrophages showed increased susceptibility to SARS-CoV-2 infection in vitro, which correlated with the induction of several proinflammatory genes. ¹³ The interaction may therefore be macrophage-specific, or reliant on the lung microenvironment to manifest. A PBMC-based assay also excludes granuloctyes, which are an important part of both the Mtb and SARS-CoV-2 innate immune response. ^{23, 36– 39} Additionally, PBMC contains no serum, so it must be manually added to the culture wells. Autologous serum may be superior to pooled human serum, as the experiment then captures the additive effect of the serum-soluble components of the immune response. ^{40, 41} However, this is not always feasible. Our results on a subset of participants suggest that serum-derived factors did not contribute to Mtb control by peripheral immune cells. Lastly, our infection model does not capture the full spectrum of TB disease, which often lasts months to years, traversing various levels of symptomatology and severity, and potentially eliciting different immune responses than those which are elicited by a four-day in vitro infection.

An interesting finding of this experiment was the >14.0% of samples which grew very small numbers of Mtb from uninfected PBMC in the COV+ groups, suggesting circulation of viable Mtb inside cells at the time the specimens were collected. CD34 ⁺ haemopoietic stem cells (HSCs) are likely one peripheral immune cell type harbouring these Mtb bacilli. Mtb DNA has been detected in circulating CD34 ⁺ PBMC from asymptomatic TB contacts irrespective of latent infection status, both in people with and without HIV. ⁴² In mice, Mtb from CD34 ⁺ long-term repopulating pluripotent HSCs begin to replicate and cause disease when transferred from donor mice without disease into immune-deficient mice. ⁴³ CD34+ HSCs express the cell surface receptor used by SARS-CoV-2, rendering them potentially susceptible to infection. ⁴⁴ Exposure to the SARS-CoV-2 S protein causes phenotypical and functional changes in CD34+ HSCs ex vivo. ⁴⁴ This suggests the possibility that prior SARS-CoV-2 infection results in the circulation of dysfunctional CD34+ HSCs, which may also be more susceptible to Mtb. Unfortunately, these positive cultures were not sequenced, so we were unable to definitively exclude contamination from the lab strain.

Our study may be limited by our sample collection spanning all four major waves of the pandemic (including wild type, beta, delta and omicron variants), introducing a possible confounder. Another limitation is that participants were classified into the SARS-CoV-2 groups based on serology only, and as most of them were asymptomatic, we were unable to reliably quantify the time since infection. According to the antibody kinetics of SARS-CoV-2 infection, infection could have occurred anywhere between 4 days and 6 months before the date of testing. This might explain the variability observed between the samples in the COV+ groups. Importantly, our findings do not address or exclude an interaction between Mtb infection and severe COVID-19.

Future work in the field of Mtb/SARS-CoV-2 coinfection should explore the immune responses of lung immune cells, including the effect of SARS-CoV-2 on cytokine production and the gene transcription in response to new Mtb exposure. An important target group is people with subclinical TB, to ascertain whether SARS-CoV-2 might be one of the factors determining their disease course.

In conclusion, we have provided the first direct evidence that the most prevalent form of COVID-19, mild SARS-CoV-2 infection, does not affect the efficacy of Mtb killing by circulating mononuclear immune cells.

This work was presented at the European Respiratory Society Congress in Vienna, Austria on 9 September 2024.

This research was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent. These studies were approved by the Stellenbosch University Health Research Ethics Committee, reference numbers: N19/10/150 and N20/04/052, and all participants provided written informed consent.

Associate Professor Jane A Shaw

janeshaw@sun.ac.za

Room 2020 BMRI South, Francie Van Zijl Drive, Tygerberg, 7505

Stellenbosch University Tygerberg Medical Campus|South Africa

+27 21 938 9953|+27 76 530 6682

JAS, NdP, GW, KU, STM conceived and designed the study; AH, AS, BS, IvR, FN, JAS collected and contributed to the data and samples; JAS and CP performed the experiments with assistance from CO and OE; MM and JAS performed the statistical analyses with assistance from OE; KU, IvR, BS, FN and DL contributed toward project management and execution, JAS wrote the paper, which was critically reviewed, edited and approved in the final draft by all authors.