Use of natural killer cell based assays to assess response to isoniazid preventive therapy among Type 2 Diabetes patients with latent tuberculosis

Abstract

Background: Despite the presence of latent tuberculosis treatment such as Isoniazid treatment therapy, a quarter of the global population remains latently infected with the numbers expected to increase. There is also an increase in the prevalence of type 2 diabetes in low and middle-income countries where tuberculosis is endemic leading to an increase in the number of latently infected individuals who are at risk of progressing to active TB disease. The type 2 diabetic patients with latent tuberculosis can be diagnosed and started on Isoniazid Preventive therapy but type 2 diabetes in this dual disease burden complicates treatment response and outcomes necessitating for treatment monitoring tools. Available treatment monitoring tools such as QuantiFeron TB test still show some positive results at end of treatment in some individuals. Natural killer cells frequencies fluctuate during different infection stages and these phenotypic and functional changes in Natural Killer cells can be used to monitor treatment response. Hypothesis: Natural Killer cell frequencies and function are elevated among type 2 diabetic patients with latent TB infection compared to active TB patients, and decrease upon isoniazid preventive therapy. Objectives: General objective: To determine the effect of Isoniazid Preventive Therapy on natural killer cell phenotype and function among type 2 diabetic patients with latent tuberculosis. Specifically, this study will: (i) determine longitudinal changes in NK cell frequencies among type 2 diabetic patients with latent TB infection before initiation of Isoniazid preventive therapy and at the end of 6 months of treatment (ii) assess the changes in degranulation function of natural killer cells among type 2 diabetic patients with latent tuberculosis at baseline and end of 6 months of treatment. Methodology: The study was a longitudinal retrospective laboratory-based study. PBMCs from LTBI +T2DM, ATB only individuals were stimulated with a combination of ESAT-6 and CFP-10 antigens and later cultured. The cells were surface and intracellularly stained to determine the frequencies and percentage cytokine release by NK cells respectively. Flow Cytometer Standard files were got from CYTOFLEX LX cytometer and analyzed with FlowJo v10.8.1 to obtain NK Cell frequencies and IFN-g, CDIO7a expressing NK Cells. Data was further analyzed with GraphPad Prism v8.0.1 to determine Analysis of Variants between the follow up samples. x Results: 9 of the 32 samples were studied, of these 6 LTBI + DM and 3 ATB only. Among the LTBI + DM group, the median NK Cell frequencies were highest at month 0 (onset of IPT treatment) and decreased at the end of 6-month treatment as we hypothesized (P= 0.0148). The reverse occurred for the ATB group only, at month 0 (onset of IPT treatment) the NK Cell frequencies were low and went on increasing up to end of treatment at month 6 (P = 0.0148). of the two study groups, NK Cell subset CD56 Dim which the mature subset dominated the NK Cell population. As expected IFN-g producing NK Cells were highest at month 0 and reduced at month 6 among the LTBI + DM group, the reverse occurred among the ATB group, were the IFN-g producing NK Cells were low at month 0 and increased at end of 6-month treatment. We further observed that NK Cells still remained cytotoxic even during LTBI + DM and ATB due to expression of CD107a a profound degranulation marker. Conclusion: The findings still reveal that even during LTBI + DM and ATB only. The NK Cell frequencies still vary from baseline to follow up among the different study groups. NK Cells can now be used as treatment response monitoring tools irrespective of the co-morbidity associated with LTBI or ATB since our findings are still in line with other studies carried out among LTBI + HIV. CD56 Dim NK Cell subset is the more abundant therefore it’s advisable to always trace for this subset during determination of NK Cell frequencies. IFN-g producing NK Cells are more abundant at baseline signifying that part of the IFN-g produced is from potent NK Cells showing that they play an important role as correlates of protection against Mycobacterium tuberculosis.