IMPACT OF COVID-19 ON PEOPLE LIVING WITH HIV IN MADAGASCAR: A SARS-COV2 SERO-PREVALENCE SURVEY

Background: In Madagascar, no study has reported the impact of COVID-19 on people living with HIV (PLHIV). The present work aimed to analyze the seroprevalence of SARS-CoV-2 in Malagasy PLHIV before and during the three waves of COVID-19 pandemic.This is a retrospective study. Materials and Methods: We conducted a retrospective serological survey in PLHIV followed up for HIV viral load (VL) monitoring at the Centre d’Infectiologie Charles Mérieux Madagascar (CICM) between June 2019 and April 2022. The presence of IgM and/or IgG antibodies against SARS-CoV-2 nucleoprotein was detected using a rapid diagnostic test (COVID-PRESTO®). Results: A seroprevalence of 2.5% was found in the 877 patients tested before March 2020, compared to 25.4% (512/2,011) between March 2020 and April 2022. This seroprevalence was 21.7%, 22.3% and 60.1% after the first, second and third waves of COVID-19, respectively. We observed a marginally significant difference (p = 0.043) in SARS-CoV-2 seroprevalence between patients on highly active antiretroviral therapy (HAART) (27.5%) and those who were not (23.7%). No statistically significant difference was observed between PLHIV with undetectable HIV VL (27.4%) and the different detectable VL categories (p>0.05). Conclusions: Our data show the presence of antibodies to SARS-CoV-2 among PLHIV as early as December 2019 in Madagascar. At least 25.4% (512/2,011) of Malagasy PLHIV have been in contact with SARS-CoV-2 since March 2020. There is no significant relation between HIV-1 VL and SARS-CoV-2 seroprevalence. Additional studies with more robust assays in the general population are needed for a detailed knowledge of SARS-CoV-2 impact in Madagascar.


Introduction
In Madagascar, the first nine cases of COVID-19 were detected on March 22 nd , 2020 (Randremanana et al. 2021).As of November 22 nd , 2023, 67,849 cases and 1,421 deaths related to this disease have been reported, which is probably underestimated in this country with its fragile health system.In addition, only about 6 % of the Malagasy population is fully vaccinated against COVID-19 as of February 22 nd 2023 (WHO 2023).A population-based study conducted in Fianarantsoa, one of the six Malagasy provinces, between February and June 2021 revealed a SARS-CoV-2 seroprevalence of 41.2% (Struck et al., 2022).Another recent study in the Malagasy blood donors registered a SARS-CoV-2 seroprevalence of 40% in 2020 (Schoenhals et al., 2021).The COVID-19 pandemic adds up to other infectious diseases conditions such as HIV/AIDS.By the end of 2021, the United Nations Programme on HIV/AIDS (UNAIDS) has estimated that there were 60,000 people living with HIV (0.2% of a total population of 27 million) in Madagascar ("UNAIDS" 2022).The objective of the present work was to determine the seroprevalence of SARS-CoV-2 among Malagasy PLHIV, which has not been documented so far.

Materials and Methods
This is a retrospective serological analysis of PLHIV from 5 (Antananarivo, Antsiranana, Mahajanga, Toamasina and Toliara) out of 6 Malagasy provinces.The study materials were plasma samples of distinct PLHIV collected as part of their virological follow-up between June 2019 and April 2022.The plasma samples were stored at -80°C in the biobank of Centre d'Infectiologie Charles Mérieux (CICM).We detected the presence of IgM and/or IgG antibodies to the SARS-CoV-2 nucleoprotein in 10 µL of plasma of the patient using a rapid diagnostic test (COVID-PRESTO®) (Charpentier et al., 2020).In accordance with the manufacturer's recommendations, a sample was considered SARS-CoV-2 positive if it showed an IgM or IgG or both IgM and IgG bands.Chi-square tests were performed, with a significance level of p ≤ 0.05, for i) the calculation of positivity rate per period and their 95% Confidence Intervals (95% CI); ii) the identification of the differences between SARS-CoV-2 seroprevalence in PLHIV on HAART and untreated PLHIV; iii) the identification of the differences between SARS-CoV-2 seroprevalence in PLHIV who have an RNA HIV viral load undetectable versus on PLHIV who have an RNA HIV viral load detectable; and iv) the comparison of the differences between SARS-CoV-2 seroprevalence in PLHIV for the 5 provinces of Madagascar.

Results
The median age of the 2,888 PLWH (sex ratio: 1.2) was 32 years.Among them, 74.8% (2,161/2,888) received HAART and 82.1% (1,773/2,161) were in virological success, that is, they had a plasma HIV VL below 1,000 copies/ml.An overall SARS-CoV-2 seroprevalence of 2.5% was found in the 877 patients tested before March 2022 compared to 21.7% of the 1056 patients tested during the first wave of the COVID-19 epidemic in Madagascar.During the second wave, 22.3% of the 770 patients tested were SARS-CoV-2 positive compared to 60.1% of the 185 patients tested during the third wave (Table 1).
We compared the SARS-CoV-2 seroprevalence in PLHIV on HAART (n = 2,161) and those who were not (n = 727).We observed a marginally significant difference between the two groups (p = 0.043) with prevalence of 27.4% and 23.6% in those on HAART and those who were not, respectively.When we stratified the prevalence of antibodies to SARS-CoV-2 by HIV-1 viral load, no statistically significant difference was observed between PLHIV with undetectable HIV RNA viral load (27.4 %) and the different categories of detectable viral loads (p>0.05).
Samples from this study were collected in 5 different provinces of Madagascar.SARS-CoV-2 seroprevalence stratified by province varied from 0% in Toamasina during the pre-pandemic period to 60.1% (112/185) in Antananarivo during the 3 rd wave.This distribution of seroprevalence by province is significantly different (p = 0.01) (Table 1).

Discussion
Our data first indicate the presence of SARS-CoV-2 among PLHIV, although at low frequency, below 3%, as early as December 2019 in Madagascar.This observation is in line with the hypothesis that COVID-19 was circulating at low and unnoticed level in human population already between October and November 2019 as determined by molecular clock analysis (Pekar et al., 2022).This prevalence before official COVID-19 declaration can also be due to insufficient assay performance.
Our data show that at least 25.4% (512/2,011) of Malagasy PLHIV have been in contact with SARS-CoV2 since the epidemic ignition in March 2020.A study carried out between March 2020 and November 2020 among Malagasy blood donors reported a high proportion of contamination of around 40% in this population group (Schoenhals et al., 2021).This is a rather high seroprevalence rate in this time frame of the epidemic, whatever the country considered.A plausible explanation is the quality of the assay used (first generation of SARS-CoV-2 serological assays).Else, one could also consider cluster contamination in blood donors, although this hypothesis is less likely.
Although we observed a marginal significant difference in SARS-CoV-2 seroprevalence between PLHIV on HAART and those who were not, we observed no significant relation between HIV-1 viral load and SARS-CoV-2 seroprevalence, contrarily to what was reported in a study from South Africa (Lambarey et al., 2022).This discrepancy can be explained by the sample size because the report from South Africa was from 150 patients on HAART while ours was on 2,161 PLHIV on HAART.
The explanation may also lay in other parameters/factors like covid-19 prevalence, study period, type of PLHIV targeted handled differently in the two works.
The present work also has some limitations.A first limitation of our study is the use of a single test, not a combination of tests, for our sero-survey.Another limitation is the use of a single antigen which can lead to incorrect results because of possible cross-reactions with antigens of seasonal beta-coronaviruses like OC43 and 229E (Guo et al., 2021).
Further analyses using more robust assays targeting additional SARS-CoV-2 antigens (e g, the Spike), like Luminex assay as reported elsewhere are necessary (Ayouba et al., 2020).Another perspective to this work is to perform population-based surveys as per WHO recommendation to have a global view of the pandemics in Madagascar.

Table 1 :
SARS-CoV-2 seroprevalence per province in Malagasy people living with HIV during the successive waves