
Type and place of the study
The Day Care Unit of the Yaoundé Central Hospital served as a framework for this study given its geographical location and its ability to receive a large number of People living with HIV (PLHIV) because it represents the largest management unit for these patients. We conducted a two-months cross-sectional analytical study from October 28 to December 27, 2020 on HIV-infected patients followed at the above-mentioned unit.
Eligibility and recruitment
Given that the epidemic form of Kaposi disease is an opportunistic disease upon HIV infection, all participants of this study were HIV positive. All patients were recruited consecutively at the level of the management unit (MU) of PLHIV at the day hospital. Included in our study were any HIV-infected patient followed at the Day Hospital of the Yaoundé Central Hospital having freely consented to participate in our study. This study did not include any HIV positive Patient undergoing any other therapy than ARV as well as any patient who withdrew consent during the study. Based on these criteria, we obtained 87 participants of both sexes aged between 26 and 72 years.
Ethical consideration
The study was conducted in patients whose identity and address remained confidential. The target population was informed in advance and we retained only those who had freely gave their consent. Ethical clearances were issued by the Centre Regional Ethics Committee for Human Health Research (CE-1567/CRERSHC/2020), Institutional Ethics Committee (295/UYI/FMSB/VDRC/DAASR/CSD) and Research as well as research authorizations from the Directorate of the Yaoundé Central Hospital (179/20/AR/DHCY/CM/SM) and the Directorate of the Imagery Centre and Biological Analysis EXACT (0001/21/CIAB EXACT/RH/DG) that permitted us to carry out this study as designed.
Data collection and processing methods
All patients coming to the management unit of PLHIV during our collection period were informed of our study through an information leaflet followed by a proposal for informed consent. Prior to the deduction, those who freely consented to participate were subject to a questionnaire (Marital status, Educational level, Age, Sex…).
Sample collection and retention
Venous blood was collected from each subject in a dry/EDTA tube while respecting the general rules of asepticism, hygiene and safety in the laboratory. These samples were centrifuged (1500 g for 5 min) and the serum/plasma aliquoted in triplicates and transported in a cooler containing ice accumul ators to the Biochemistry laboratory of the Faculty of Medicine and Biomedical Sciences (FMSB) or frozen at -20 ̊C until the time of the analyses. These biological analyses were carried out in the immune-serology laboratory of the Imagery Centre and Biological Analysis EXACT and the Biochemistry Laboratory of the Faculty of Medicine and Biomedical Sciences (FMSB) of Yaoundé I University.
Determination of immunological parameters
Determination of HHV-8 antigen (HHV-8 ELISA kit mybiosource Cat.No MBS167838)
It was done according to the principle of the indirect ELISA method wherein anti-HHV-8 antibodies are adsorbed on microwells [13]. Positive and negative samples or controls are added to the wells, the HHV-8 antigens in the sample bind to the antibodies adsorbed in the wells, unfixed antigens are eliminated during washing. An enzyme-coupled detection antibody (HRP) is added to wells and incubated. Excess enzyme is eliminated during a washing step and then added a chromogenic substrate. The reaction is then stopped and the optical densities measured at 450 nm. To express the results, the cutoff value is calculated by averaging the optical densities of the negative control + 0.15 and then any sample OD cutoff is a positive result.
Determination of IL-6 according to the principle of the sandwich ELISA method (IL-6 ELISA Kit ElabscienceR Cat.No E-EL-h0102)
In this technique, a specific anti-IL-6 antibody is adsorbed on microwells which binds to the IL-6 present in the sample or standard [14]. A monoclonal anti-IL-6 antibody conjugated to biotin is then added that binds to IL-6 captured by the first antibody. After incubation, the unbound conjugate is removed during a washing step. Streptavidin-HRP is added and binds to the anti-IL-6 biotin conjugate. After incubation, the unfixed streptavidin-HRP is removed during a washing step, and the HRP-reactive substrate solution is added to the wells. A colored product is formed in proportion to the amount of IL-6 present in the sample. The reaction is stopped by adding the stop solution and absorbance is measured at 450 nm. A standard curve is prepared from seven IL-6 standard dilutions and the concentration of the IL-6 sample is obtained from the equation of this curve. In order to have the homogeneous data, we divided the population into two groups with 37 as the threshold.
Determination of oxidative stress parameters
Assessment of serum lipids oxidation
The determination of malondialdehyde (MDA) was done by the Wilbur et al. method [15]. Carbonyl compounds such as malondialdehyde resulting from the decomposition of fatty acid hydroperoxides react with thiobarbituric acid (TBA) to give pink chromophores whose concentration is determined by reading absorbance at 532 nm.
Determination of serum total antioxidant capacity
The principle is based on the FRAP method (Ferric Reducing Antioxidant Power) of Benzie et al. [16]. This method measures the ability of plasma to reduce ferric iron (Fe3+) to ferrous iron (Fe2+) in an acid environment pH (3.6). The intensity of the blue color formed by ferrous tripyridyltriazine and measured at 593 nm is proportional to the antioxidant power of the sample.
Determination of reduced glutathione (GSH)
The principle of this method is based on 2,2-dithio-5,5’-dibenzoic acid (DTNB) reacting with the SH groups of glutathione in the samples and standard forming a yellow colored complex that absorbs light at a wavelength of 412 nm [17].
Determination of HIV Viral load
HIV Viral load was determined using ABI PRISM 7500 Fast Real Time PCR (Applied Biosystems) with a threshold of 40 Copies/mL.
Data analysis
All data in this study was analyzed using the SPSS 12.0 software. This consisted of expressing the results as mean ± standard error. All comparisons were done using the student t-test and ANOVA. On the other hand, correlations between Kaposi’s disease, immunological and biochemical parameters were done using the Pearson test. The threshold of significance was set at 5%.