Association of interferon-gamma release assay and SARS-CoV-2 reverse transcriptase polymerase chain reaction test results in adults tested in a tertiary medical center

Introduction: For a country with high Tuberculosis (TB) prevalence, infection of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus with a covert co-infection of Mycobacterium Tuberculosis (MTB) is a real concern. The two pathogens being both intracellular infectious agents, share a common initial host immune response of innate immunity activation, signalling cascade of infl ammatory cytokines and chemokines, and eventual adaptive immunity activation in order to remove and/ or neutralize the microbes. Interferon-Gamma Release Assay (IGRA) is a TB diagnostic tool that indirectly tests for Latent TB Infection (LTBI) by measuring the interferongamma released by T cells in response against MTB-specifi c antigens. This study was conducted to determine if the T cell response to IGRA will be affected by the host immune response to coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 virus.

T helper lymphocytes and TB2 targets response from CD8+ T lymphocytes; mitogen tube serves as the positive control per sample to indicate immune status and check blood handling and incubation. Final results are calculated and interpreted as positive, negative or indeterminate based on the manufacturer's criteria [9].
This test is particularly helpful in TB endemic countries like the Philippines who hold the third highest prevalence of active tuberculosis in the world after South Africa and Lesotho [10]. With the recent outbreak of SARS-CoV-2, concern for COVID-19 infection among healthcare workers in TB wards and TB patients had been raised [11]. This concern is worsened by the fact that both diseases have a propensity to target the lungs and manifest as pulmonary symptoms. Being both obligate intracellular infectious agents, the natural course of infection would initiate activation of innate immune response to remove and/or neutralize the microbes. Following ingestion by alveolar macrophage for MTB and viral entry into alveolar epithelium for SARS-CoV-2, the host cell would undergo pyroptosis or lytic cell death to expel intracellular pathogens [12,13]. Neighboring Receptors (PRRs) like Toll-like receptors [14][15][16]. This will lead to a signaling cascade that activates the transcription factor NFB, driving the production of pro-infl ammatory cytokines (including tumor necrosis factor (TNF), Interleukin-1 (IL-1), IL-12, IL-6, granulocyte-colony stimulating factor (G-CSF), Monocyte Chemoattractant Protein-1 (MCP-1), Macrophage Colony Stimulating Factor (M-CSF) and interferon gammainduced protein-10) that will in turn stimulate neighboring T and NK cells to produce additional cytokines like IFN-ɣ [17].

Methods
This was an analytical, cross-sectional study that included all IGRA results with corresponding SARS-CoV-2 test results from March 10, 2020 to May 12, 2020.
IGRA tests were routinely run on Tuesdays except on STAT cases. The study period selected started from the second week of March until the second week of May2020, encompassing the start to end of enhanced community quarantine in NCR. The investigator accessed both the institution's Clinical Microbiology hard copy records of IGRA test results and Clinical Immunology's laboratory information system records of SARS-CoV-2 test results during the selected study period. Designated code served as patient identifi er to protect patient privacy and confi dentiality.

Inclusion criteria
All IGRA test results and their corresponding RT-PCR SARS-CoV-2 test results run during the study period were included. In case of repeat testing for RT-PCR SARS-CoV-2, the closest date between the time of specimen collection for both IGRA and RT-PCR SARS-CoV-2 tests were chosen. The maximum date for RT-PCR SARS-CoV-2 test included was 15 days from the extraction date for the IGRA test based on the symptom onset range derived by Lauer, et al. (2020) [18].

Operational defi nitions
• IGRA: 1 ml blood was placed into each of the four QFT-Plus blood collection tubes, incubated, and centrifuged to separate and harvest the plasma. This was then processed according to the manufacturer's instructions.

Sample size estimation
Assuming a level of signifi cance of 0.05, a power of 80%, a degree of freedom of 2, and a medium effect size (0.3), the required sample size to run a Chi-Square Test of Association is 108.

Data analysis
Data were analysed using proportion expressed in percentages using the following formulae: • Electronic data were stored in a password-protected laptop and were only accessible to the project and co-project leaders. The password will be the sole responsibility of the project leader.
• Study-related documents will be stored in a cabinet with lock and key. The key is kept by the project leader and the cabinet, which is located in a secure room, will only be accessible to members of the research team.
• All documents and electronic data will be kept by the project leader for a standard storage period of at least fi ve years after completion of study and shall be securely shredded and/or erased.

Demographic data
A total of 37 patients were included in the study, 21 (57%) of which were male and of these, 14 (14/23, 60.87%) were COVID-19 confi rmed cases. The patients were all adults, the youngest at 21 years old while the oldest at 89 years old. The mean age was 57.5 years old (61.13 years old for confi rmed COVID-19 cases; 51.57 years old for non-COVID-19 cases).
Most patients were clustered at age range of 60-69 years old, while the least was at 40-49 years old (Figure 1).

Association of IGRA and SARS-CoV-2 RT-PCR test results
There was a statistically signifi cant association between IGRA and SARS-CoV-2 RT-PCR test results (p=0.006) ( Table 1).
Majority of the IGRA results were indeterminate (60%) and where most of the detected SARS-CoV-2 cases were found (49%) Figure 2.

Discussion
Demographic data of the study was similar to reported cases during the initial outbreak where SARS-CoV-2 detected cases were elderly with slight male predominance [19][20]. Notably, most of these detected cases had indeterminate results in IGRA. Indeterminate IGRA results can be attributed to preanalytical, analytical and immunological sources of variability [6]. Pre-analytical and analytical variability like blood volume, tube shaking, incubation duration and manufacturing defects, although not completely eliminated, were presumed minimized due to the stringent quality control practices in place from specimen receiving to processing. As previously mentioned, the increased indeterminate results prompted a repeat run with additional inclusion of prior known positive specimen to ascertain validity of the previous run. This then leaves immunological variability as the most likely cause of indeterminate results.
Immunological variability may either be due to immune boosting or immunomodulation [6]. Immune boosting can be from excessive levels of circulating IFN-ɣ or the presence of heterophile antibodies; this will be translated computationally as a Nil value higher than the recommended cut-off [9]. Immunomodulation, on the other hand, may occur with insuffi cient lymphocytes or the functional defi ciency of lymphocyte to produce IFN-ɣ; this then will be represented computationally as Mitogen value below the recommended cut-off [9]. In this study, all indeterminate results were of the latter case (data not shown). Majority of positive IGRA results do not progress to active TB disease [6]. In case of SARS-CoV-2 and LTBI co-infection though, the sustained depressed T cell mediated response may increase risk of reactivation of active TB disease.
Studies of the immune response to SARS-CoV-2 virus emphasized peripheral lymphopenia, mainly CD4 and CD8 T cells, associated with symptomatic and/or severe form of the disease [21][22][23][24][25]. Sequestration of cytokine and chemokinerecruited immune cells from the blood into the infected site, particularly the lung; lymphocytic infi ltration of the airway; T cell exhaustion; and viral-initiated/induced lymphocytic  apoptosis were posited as possible reasons for the lymphopenia [13,21,24,26]. These evidences support

Conclusion
IGRA can serve not only as an indirect tool for LTBI detection, but can also implicitly refl ect the state of a patient's adaptive immune response, particularly the T lymphocytes.
Therefore, IGRA can indirectly assess the adequacy of T cell response to two highly infectious pulmonary pathogens, that is, MTB and SARS-CoV-2. (Appendix)