Interferon alpha (IFN-ɑ) among Human Immunodeficiency Virus (HIV) patients exposed to Plasmodium falciparum infection in selected Hospitals in Niger State, Nigeria

Both Human Immunodeficiency Virus (HIV) and Plasmodium falciparum ( P. falciparum ) infection are associated with production of several cytokines such as type I Interferons which may be beneficial to the host during acute infection. The plasma level of type I interferons could vary among HIV patients co-infected with P. falciparum . This study therefore, aims to quantify the level of IFN-α, a type 1 interferon, among HIV patients exposed to P. falciparum infection in selected Hospitals in Niger State, Nigeria. This was a cross-sectional study. Blood samples were collected from 300 HIV patients for blood film microscopy using Giemsa staining technique and CD 4 count using flow cytometer. Of these, 65 were selected for IFN-α analysis (22 controls and 43 HIV/ P. falciparum patients) determined using enzyme-linked immunosorbent assay (ELISA). (Chi-square test, P < 0.05). Higher levels of IFN-α were observed in patients with mild P. falciparum infection and in those with low CD 4 below 200 cells/µL. Conclusively, mild P. falciparum infection in HIV patients is characterized by up-regulated IFN-α activity.


Introduction
Plasmodium falciparum and Human Immunodeficiency Virus (HIV) infections continues to be a public health concerns in the tropical and sub-tropical regions of the world, accounting for massive morbidity and mortality in sub-Saharan Africa (Alaofin et al., 2019).It is estimated that about 241 million people suffers from malaria worldwide and 37.7 million were infected with HIV in 2020 (WHO, 2021: UNAIDS, 2021).Due to overlap in the geographical distribution of these infections in the sub-Saharan Africa, HIV infection is very common in most areas where P. falciparum infection is endemic, and these infections may induce interferonemia (Cai et al., 2017).Also the host immune defenses of HIV individuals which is being weaken by HIV infection may promotes occurrences of co-infection especially with malaria in regions where it is endemic (HIV/gov.2019; Roberds et al., 2020).
Innate immunity is the first line of host defense against an invading pathogen.Interferons (IFNs) are a multigene family of inducible cytokines produced by the host immune system which are among the key molecules in the early defense against infections and are in many cases the first line of resistance to many viral and protozoan infections (McNab et al., 2015;Silva-Barrios and Stager, 2017).IFN-Is, particularly IFN-α, appear to be protective in human infections of P. falciparum (Subramaniam et al., 2015) and HIV infections (Nuvor et al., 2016).Studies have also shown associations between high levels of IFN-α produced early during an infection with protection (Subramaniam et al., 2015).On the other hand, excess production of IFN-α have been shown to cause immunopathology leading to immunosuppression (Davidson et al., 2014;Lukhele et al., 2019).
Many regulatory mechanisms of IFN-α production in Plasmodium and virus infections are similar because the IFN-α responses during both infections shared the same pathways.However, Plasmodium species are more complex organisms, and as a result, IFN-α responses and regulation during Plasmodium infections are likely more sophisticated (He et al., 2020).Furthermore, in what way do co-infection with HIV influences IFN-α responses during P. falciparum infection is still only partly known and none to our knowledge has been investigated in the study area.Thus, the aim of this study was to quantify the levels of IFN-α among HIV patients exposed to P. falciparum infection attending selected hospitals in Niger State, Nigeria.

Study Area
The study was conducted in three major hospitals in the three senatorial zones of Niger State, Nigeria.These are General Hospital Bida in Niger East (Zone A), General Hospital Minna in Niger South (Zone B) and General Hospital New Bussa in Niger North (Zone C) respectively.Niger state is located in the North-Central part of the country and lies between the latitude of 8 -11.3⁰N and longitude 3.30 -7.40⁰E covering a total area of 76,363 sqkm².The climate of the area is tropical with an average annual rainfall of 1090 mm³ and temperature ranging 27.38 -36.9⁰C as reported by Niger State Ministry of Land and Housing, (2020).Majority of the population are farmers.

Study Population
A hospital-based cross-sectional study was carried out among HIV patients attending these facilities from a period of February to June, 2021.Participants were recruited irrespective of their age, sex and with or without malaria-related signs and symptoms.Only those who agreed to participate in the study were enrolled, those that did not consent and non-HIV patients were excluded.Three hundred (300) HIV patients were selected out of which 22 that do not have malaria after microscopy were randomly selected as control group.

Sample Size Determination
A prevalence of 24.8% for P. falciparum by Daniel et al. (2016) was used to determine the sample size using the equation of Vinodh, (2018).

Ethical Approval
Ethical approval was obtained from the Ethical committee of Niger State Hospital Management Board/General Hospital, Minna, Niger State (HMB/GHM/136/VOL.III/590).Consent of participants or guardians of the children was also obtained prior to collection of samples.

Blood Sample Collection
Using sterile vacutainer needle/holder, 4 mL of blood samples were drawn aseptically by venipuncture from the patients and transferred into a sterile Ethylene Diamine Tetra Acetate (EDTA) anticoagulant bottle and mixed immediately (Yohanna et al., 2019).

Detection of Parasitaemia
Thick and thin blood films were made on glass microscope slides.Thin films were fixed using absolute methanol and both films were stained with 3% of Giemsa stain.The stained blood films were examined microscopically under x 100 objective lens for the detection and identification of malaria parasites (WHO, 2015).Malaria parasites were counted in all the positive thick blood films with an aid of a differential counter, the number of parasites seen (asexual stages) and the number of white blood cells (WBCs) were counted separately but simultaneously field by field.These was done till 200 WBCs were counted in all fields.The parasitaemia value was calculated and expressed as parasites per microliter of blood (WHO, 2015).The level of parasitaemia was graded as low (<1000 parasites/μL), moderate (1000-9999 parasites/μL) and high/severe (≥10,000 parasites/μL (Atroosh et al., 2015).The parasite density is calculated mathematically as: Malaria parasites/µL of blood = No. of observed parasites counted x estimated WBC (8000) / No. of WBC counted (200) ... (1)

Determination of CD4 Count
The CD4 count was determined using a two-color single platform flow cytometer (Partec Cyflow) within 6 hours of sample collection.Twenty microliter (20 µL) CD4 phytoerythrin (PE) antibody was dispensed into labeled Partec (Rohren) tubes and 20 µL of well mixed whole blood was added, mixed with a vortex mixer and incubated in the dark for 15 minutes at room temperature.The mixture was mixed at 5 minutes' interval during incubation.After incubation, 800 µL of CD4 diluting buffer was added to the mixture of antibody and samples mixed gently before analyzing.CD4 count was categorized according to WHO, 2018 as low or advanced stage (<200 cells/µL), moderate or chronic stage (200 -499 cells/μL) and high or asymptomatic stage (≥500 cells/μL).

Determination of Interferon alpha using Enzyme Linked Immunosorbent Assay
Interferon alpha was measured in plasma samples from 43 participants co-infected with P. falciparum (detected microscopically) and 22 participants as controls using ELISA (ABclonal Biotechnology Co., Ltd, USA).Plasma samples were diluted at 1 in 10 in sample diluents and used with their respective serially diluted standards on monoclonal antibody-coated 96-well plates.The plates were incubated at 25°C for 2 hours with shaking and then washed five times to remove the unbound antigens from the coating antibodies.This was followed by addition of Working Biotin-Conjugate Antibody to bind to the antigen-antibody complex.Plates were washed following 1 hour incubation at 25°C.After 5 washes, Working Streptavidin horse-radish peroxidase (HRP) was added, incubated for 1 hour, and then washed again four times before the substrate, tetramethyl-benzidine (TMB) was added.Development of a blue colour occurred within 15 minutes, the intensity of which depended on the extent of the antigen-antibody complex formed.The reaction was stopped by addition of an acid and the absorbance was read with the Microtitre plate Reader at 450 nm within 15 minutes.

Data analysis
All the data collected were entered into Microsoft Excel files and analyzed using STATA/MP 12.0 (StataCorp, College Station, TX) and Prism 5.0 (Graph pad Inc., San Diego, CA).Univariate analysis which includes descriptive statistics such as frequencies, percentages and exploration of the distribution of all variables were performed.Groups were compared with non-parametric Wilcoxon-matched paired and Kruskal-Wallis tests.Categorical variables were compared with Pearson's correlation co-efficient (Chi-square) and Odds ratio.Multiple logistic regression was performed to assess whether cytokine (IFN) levels were independently associated with the likelihood of P. falciparum infection.All tests were performed at 95% confidence interval (p < 0.05).

Profile of Co-infected participants and Controls
Table 1 shows the profile of the fourty-three (43) study participants co-infected with P. falciparum and twenty-two (22) randomly selected study participants that acted as control.Out of the 43 co-infected participants, 15 (34.9%) of them had CD4 count > 500 cells/µL, a mean age of 38.8 years with a standard error of + 10.71, a mean CD4 Count of 1581 +430.56 cell/µL and a mean IFN-α of 44.4 + 18.31 pg/mL while the control group had a mean age of 31.36 years with a standard error of + 14.11, a mean CD4 Count of 1253 +682.32 cell/µL and a mean IFN-α of 39.03 +20.04 pg/mL.There is no significance difference in IFN-α levels between the two groups (p > 0.05).

Multiple logistic regression analysis with dependent variables of Malaria Parasitaemia
The logistic regression analysis shows a statistically significant relationship (p-value = 0.02) between the presence of malaria parasitemia (MP) and the independent variables of age, sex, CD4 count and IFN-α levels.The model explains 13.9935% of the deviance in MP, and the adjusted percentage is 1.97448%.
After adjustment for cofactors, the results of the likelihood ratio tests indicate that CD4 count (p-value = 0.0012) have a significant effect on the presence of malaria parasitemia.The p-value for age (p = 0.1694), sex (p = 0.9816) and IFN-α levels (p = 0.4065) are not statistically significant (Table 2).

Relationship between parasitaemia and Interferon alpha
Logistic regression correlation coefficient values and p values are shown in Fig. 2. Analysis for possible correlational relationship was done.There is a statistically moderate negative correlation between IFN-α and parasitaemia.

Relationship between parasitaemia and CD4 count
To determine whether cytokine levels in our study were associated with CD4 counts that are a standard marker of progression of HIV infection, we measured the IFN-α levels in the patients.IFN-α plasma concentrations showed a trend toward higher levels in patients with lower CD4 counts compared with those with higher CD4 counts (Kruskal-Wallis test; P = 0.06 for both comparisons) (Figure 2).When considering P. falciparum infection as a confounder, the trend toward a negative association between IFN-α levels and CD4 counts was confirmed only in patients who were parasitemic (not indicated here).The HIV infection has been shown to impair proinflammatory cytokine production (Roberds et al., 2021;Klatt, 2022) which could be considered as being beneficial for the persistence of malaria parasites.CD4 cells have been reported to play a critical role in HIV/malaria immune control.Earlier studies have shown that stimulation of an early IFN-ɑ response influence the direction of host immune responses such as CD4+ T cell activation (He et al., 2020).This study shows that the presence of infection with P. falciparum is associated with low CD4 counts, themselves primarily related to the concurrent HIV infection, which lead to a stronger likelihood that these participants have plasmodial infections.Though, it should be stressed in this context that our sample size is small, and that some of our data only revealed trends that show borderline significance: multivariate analyses adjusted for multiple confounders however confirmed most of the findings obtained in univariate analyses.
Another reason for increased levels of IFN-α could be low CD4 count which may be due to the fact that infection of HIV as well as P.falciparumhas been known to induce depletion of CD4 T-cells and to reduce CD8 T-cells causing downmodulation, reduction in T-cell sub-population and defective cell mediated immunity against any microbial infection (Kirinyet, 2019).This study however, contradicts the findings of Nuvor et al. (2016) which suggests that high levels of IFN-α may be supporting the persistently high CD4+ T cell numbers in HIV infection from an early stage of infection.
Whether and how IFN-α regulate host immune mechanisms, including T cell activation and antibody production require additional investigation.

Conclusion
High plasma levels of IFN-α were associated with mild falciparum malaria and low CD4 counts.IFN-α levels were also found to be correlated with falciparum malaria infection.

Table 1 :
Profile of Co-infected participants and Controls

Table 2 :
Multiple logistic regression analysis with dependent variables of Malaria Parasitaemia Key: IFN-α = Interferon alpha.