Serum Interferon Gamma (IFN-ɣ ) Levels and Hematological Indices in Patients with HIV-MTB Co-Infection in North-Eastern Nigeria

: Introduction: The dual epidemic of human immunodeficiency virus (HIV) infection and Mycobacterium tuberculosis (MTB) poses significant health challenges, particularly in sub-Saharan Africa. Understanding the immune response and hematological changes in HIV-MTB co-infection is crucial for better management of affected individuals. Objectives: This study aimed to evaluate the serum levels of IFN-ɣ and hematological indices in patients with HIV-MTB co-infection in North-Eastern Nigeria, as well as explore any potential relationships between these factors. Methods: A total of 88 participants were enrolled in the study, including 44 antiretroviral therapy-naive patients with HIV-MTB co-infection (study group) and 44 HIV mono-infected individuals as controls. Data on personal biodata and clinical details were collected using an interviewer-administered questionnaire. Blood samples were obtained


Introduction
Human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (MTB) infections are major global health concerns, particularly in developing countries.The co-infection of HIV and tuberculosis (TB) is a significant challenge, especially in sub-Saharan Africa.TB ranks as the second leading cause of death from an infectious disease worldwide, following HIV.In Nigeria, the proportion of TB/HIV co-infected patients is considerable, and these individuals often experience poorer treatment outcomes.HIV infection significantly increases the susceptibility to and progression of TB, even before a decrease in CD4+ T-cell counts below a certain threshold.The increased susceptibility to active TB in HIV-infected individuals is multifactorial and extends beyond CD4 T-cell depletion.Understanding the immunological factors involved in this co-infection is crucial (Mohan et al., 2001;Maartens and Wilkinson, 2007;Daniel et al., 2006;WHO, 2005;Sonnenberg, 2005;Cooper et al., 2008;Schroder et al., 2004).
Interferon gamma (IFN-ɣ) plays a crucial role in the immune response against mycobacterial infections.It activates macrophages, enhances antigen presentation, and promotes the differentiation of CD4 T lymphocytes to the Th1 subpopulation.IFN-ɣ induces the transcription of various genes involved in antimicrobial functions, making it an essential effector mechanism against Mycobacterium tuberculosis (Cooper, 2009;Oberholzer et al., 2000).IFN-ɣ production is decreased in HIVinfected individuals, which can contribute to the impaired control of MTB infection and disease progression.The dysregulation of IFN-ɣ production in HIV-infected individuals is associated with chronic immune activation and the excessive production of type-1 interferon (IFN) by plasmacytoid dendritic cells.This dysregulation may further contribute to the impaired control of MTB infection and disease progression (Fitzgerald-Bocarsly et al., 2010).
The importance of IFN-ɣ in controlling M. tuberculosis infection has been demonstrated in both experimental and clinical studies.Mice with a disrupted IFN-ɣ gene show increased susceptibility to tuberculosis, and reintroduction of this gene into the lung confers resistance.Similar observations have been made in macaque monkeys and humans with high IFN-ɣ levels who are more likely to develop active tuberculosis (Moreira et al., 2000;Lin et al., 2009).Additionally, individuals with mutations in genes of the IL-12/IL-23/IFN-ɣ axis exhibit increased susceptibility to mycobacterial infections (Al-Musen and Casanova, 2006).
Anemia is a common complication in both TB and HIV infections and is associated with increased morbidity and mortality.Factors contributing to anemia in these infections include decreased red blood cell production, malabsorption syndrome, and nutritional deficiencies (Johnson et al., 2006;Belperio and Rhew, 2004).The affected hematological indices include packed cell volume (PCV), thrombocytes, total and differential counts, with neutropenia, lymphopenia, and thrombocytopenia as common features (Johnson et al., 2006).
The high burden of TB/HIV co-infections in African and Asian countries poses significant diagnostic and therapeutic challenges, placing immense pressure on healthcare systems.Therefore, understanding the host immune response to this co-infection is crucial for improving management strategies (Getahun et al., 2010).
Therefore the objective of this study was to evaluate the serum levels of IFN-ɣ and hematological indices in patients with HIV-MTB co-infection in North-Eastern Nigeria.Additionally, we aimed to explore any relationships between serum IFN-ɣ levels and hematological parameters in these patients.

Study Design
This study employed a cross-sectional design.Participants were enrolled from the antiretroviral therapy (ART) clinic of FMC Yola, Adamawa, Nigeria.Two groups were included: a study group consisting of antiretroviral therapynaive patients with HIV-MTB co-infection, and a control group comprising HIV mono-infected individuals.A total of 88 participants were enrolled, with 44 participants in each group.
Data Collection and Laboratory Analysis: Personal biodata and clinical details were collected using an interviewer-administered questionnaire.Five milliliters of venous blood samples were collected from each participant, with 4 milliliters collected in EDTA BD Vacutainer tubes for hematological analysis.The remaining samples in plain containers were separated, aliquoted into vials containing aprotinin, and stored at -70°C until batch analysis of IFN-ɣ using ELISA.Hematological indices, including white blood cells (WBCs), platelets, PCV, and neutrophils, were determined using an automated hematology analyzer.
Ethical Considerations: This study obtained ethical approval from the Health Research Ethical Committees of Federal Medical Center, Yola.Informed consent was obtained from all participants, ensuring confidentiality and the freedom to withdraw from the study.
Statistical Analysis: The collected data were subjected to normality tests.Haematological data were summarized using mean and standard deviation, while IFN-ɣ levels were summarized using median and interquartile range.The Mann-Whitney U test was used to compare IFN-ɣ levels between the study group (HIV-MTB coinfection) and the control group (HIV monoinfection).Independent samples t-test was employed to compare hematological parameters (WBCs, platelets, PCV, and neutrophils) between the two groups.
Furthermore, Pearson's correlation analysis was conducted to examine the relationships between serum IFN-ɣ levels and hematological parameters within the study group.This analysis aimed to explore any potential associations between IFN-ɣ levels and hematological indices in HIV-MTB co-infected individuals.
The statistical analysis was performed using appropriate software, and the significance level was set at p < 0.05 to determine statistically significant results.
There is significant decrease in values of WBC, HGB, HCT, MCV and increase Eosinophil among the subjects compared to controls (p≤0.05)while other parameters were insignificant (p≤0.05).
White blood cell count (WBC) is significantly lower in the study group compared to the control group (p=0.038*).
Mean corpuscular hemoglobin concentration (MCHC) and platelet count (PLT) do not show a significant difference between the study and control groups.
The percentages of lymphocytes (LYMP), monocytes (MONO), and neutrophils (NEU) are similar between the study and control groups.
The percentage of eosinophils (EOS) is significantly higher in the study group compared to the control group (p=0.002*).IFN-ɣ levels in the study group have a positive correlation with white blood cell count (WBC) (r = 0.007, p = 0.962), indicating a weak positive association.

Table1. Haematological Indices in
In the control group, IFN-ɣ levels also show a positive correlation with WBC, although the correlation coefficient is slightly higher (r = 0.066, p = 0.670).
These correlations suggest that IFN-ɣ levels do not strongly correlate with the measured hematological indices in both the study group and the control group.In the study group, the mean WBC count was 4.855 x 10^9/L, which was significantly lower than the control group (5.405 x 10^9/L) with a p-value of 0.038.This finding suggests a potential alteration in the immune response of the study group compared to the control group.Similar observations have been reported in other studies conducted by Olaniyi et al. (2018) in Nigeria which investigated hematological parameters in HIV-positive individuals and found a significant decrease in WBC count compared to HIV-negative individuals.This reduction in WBC count could be attributed to the immunosuppressive effects of HIV, which impairs the body's ability to fight infections effectively.
Regarding other hematological indices, no significant differences were observed between the study and control groups for RBC count, HGB level, and PLT count.However, there were significant differences in HCT, MCV, MCH, and EOS percentages.
The HCT values were significantly lower in the study group (30.80%) compared to the control group (38.59%) with a p-value of 0.001.This finding suggests a lower packed cell volume in the study group, indicating a potential issue with red blood cell production or anemia.Anemia is a common condition observed in various populations, including Nigeria.It can be caused by nutritional deficiencies, chronic diseases, or other underlying factors.
Similar findings were reported in a study by Oguanobi et al. (2015) in Nigeria, where they observed a significantly lower HCT level in individuals with sickle cell disease compared to healthy controls.This highlights the importance of considering local studies when interpreting results within a specific population.
The lower HCT, MCV, and MCH values in the study group indicate the presence of microcytic and hypochromic red blood cells, which are characteristic of iron deficiency anemia.Iron deficiency is a prevalent nutritional disorder globally and can be caused by inadequate dietary intake, poor iron absorption, or chronic blood loss.Studies conducted within Nigeria, such as the one by Nwagha et al. (2011), have reported a high prevalence of iron deficiency anemia among certain population groups, including women of reproductive age.Therefore, it is crucial to evaluate the study for potential iron deficiency and implement appropriate interventions to address this condition.
The elevated eosinophil percentage in the study group suggests an increased presence of eosinophils, which are associated with allergic conditions or parasitic infections.Allergies, particularly respiratory allergies, are prevalent worldwide, including in Nigeria.Studies conducted both within and outside Nigeria have reported a high prevalence of respiratory allergies, such as allergic rhinitis and asthma.For instance, a study by Desalu et al. (2013) in Nigeria found a significant burden of allergic rhinitis among adults.Therefore, it is important to explore whether allergic conditions or parasitic infections contribute to the higher eosinophil percentage observed in the study group and implement appropriate diagnostic and management strategies.The observed differences in hematological indices between the study and control groups may have several implications.Firstly, the lower WBC count in the study group suggests a potential compromise in the immune response.This could be attributed to various factors, such as underlying infections, immune system disorders, or the influence of certain medications.Further investigation is warranted to identify the specific causes and assess the clinical implications of this finding.

Conclusion
Despite these limitations, this study contributes to the understanding of the immune response and hematological changes associated with HIV-MTB co-infection.The findings may have implications for the development of therapeutic strategies and interventions aimed at improving the management of co-infected individuals.

Figure 1 .
Figure 1.Serum IFN-ɣ Level in Both Study and the Control Groups

Table 2 . Correlation of IFN-ɣWith Haematological Indices of Study and Control Group
Data presented as Mean ± SD; Statistical tool: Two-tailed Un-paired t-test and *= p≤0.05.WBC White blood cells; RBC Red blood cells; HGB haemoglobin; HCT Packed cell volume; MCV Mean corpuscular volume; MCH Mean corpuscular haemoglobin; MCHC Mean corpuscular haemoglobin concentration; PLT Platelet