Introduction

In December 2019, a new coronavirus was associated with cases of pneumonia of unknown etiology in a group of individuals in Wuhan, Hubei, China (WHO 2020). This new coronavirus was named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the pneumonia it caused, COVID-19. Subsequently, the COVID-19 pandemic has spread to every continent. As of January 02, 2024, 773,449,299 confirmed cases of COVID-19 infection and 6,991,842 deaths have been reported worldwide [1]. COVID-19 pneumonia shares common symptoms and mode of transmission with other respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), rhinoviruses, enteroviruses, adenoviruses, and human coronaviruses [2,3]. In many countries, the circulation of respiratory viruses other than SARS-CoV-2 is endemic, with occasional seasonal peaks [4,5]. In viral respiratory infections, coinfections and superinfections are frequent [6,7] and generally cause increased severity in co-infected patients [8]. Thus, during the COVID-19 pandemic, several studies were carried out to assess the proportion of COVID-19 patients coinfected simultaneously with other respiratory viruses, and to describe co-pathogens. These studies have been carried out mainly in Asia, Europe and North America, and their analysis has resulted in a pooled prevalence of respiratory viral coinfections of 5.01% [9]. In patients with COVID-19, influenza A virus, rhinovirus and adenovirus are the most frequently identified co-pathogens. However, a marked decrease in the incidence of these respiratory co-circulating viruses has been noted overall [10].

In Senegal, the first case of COVID-19 was reported on February 26, 2020. Within a month of circulation, the disease spread slowly throughout the country, with an absence of severe cases, a high cure rate and a low incidence [11]. This slow spread of the virus possibly resulted from the non-pharmaceutical measures put in place early such as social distancing, compulsory wearing of face masks, border closures and a ban on intercity travel. This evolutionary dynamic was disrupted by the appearance of SARSCoV-2 variants of concern [12] and the attenuation of the measures put in place. This led to an increase in severe cases and deaths [13]. As of January 02, 2024, Senegal has experienced 4 epidemic waves of variable intensity, with 89,033 confirmed cases and 1,971 deaths [1].

In Senegal, respiratory viruses other than SARS-CoV-2 are widely implicated in influenza-like illnesses and acute respiratory infections. They circulate endemically with different patterns depending on the viral type [14]. It has also been shown that a large proportion of the population are asymptomatic carriers of these viruses [15]. During the COVID-19 pandemic, the implementation of social distancing and other physical barriers was associated with a decline in rates of influenza and RSV during 2020 to 2021 [16]. However, in the following year, respiratory viruses re-emerged and circulated in the population. To date in Senegal, data on the circulation of other respiratory viruses during the COVID-19 pandemic are scarce. The aim of our study was to assess co-pathogens and the prevalence of respiratory viral coinfections in SARS-CoV-2-positive patients during the first and second epidemic waves of COVID-19 in Senegal.

Materials and methods

Sampling

In this study, we used a panel of 437 residual aliquots of respiratory specimens (nasopharyngeal or oropharyngeal) from original respiratory specimens initially tested positive for SARS-CoV-2 by Real Time Polymerase Chain Reaction (RT-PCR) (Table 1). A SARS-CoV-2 positive sample was defined as any sample in which viral RNA was detected by a SARS-CoV-2 RT-PCR technique with a threshold cycle (Ct) lower than 35. This COVID-19 diagnosis had been carried out at the molecular biology platform of the Institut de Recherche en Santé, de Surveillance Epidémiologique et de Formation (IRESSEF), Diamniadio, Senegal. Sociodemographic data were provided by the patients themselves, using a questionnaire that they completed at the time of sampling [17]. The samples came mainly from the Dakar and Thiès regions, the 2 cities in Senegal most affected by COVID-19 [18]. Tested samples had been taken from suspected cases, confirmed cases and contact cases, as well as from travellers leaving Senegal. This systematic screening was part of the obligatory measures taken by the Senegalese government. For the search for other respiratory viruses, sample aliquots were transferred to the Institut Hospitalo-Universitaire Méditerranée Infection (Marseille, France) in biohazard containers (DHL, Dakar, Senegal) [17].

Detection of respiratory viruses other than SARS-CoV-2 by multiplex RT-PCR

Nucleic acids were extracted using a King Fisher extractor and the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation kit (Thermofisher Scientific, Illkirch, France). In accordance with the manufacturer’s instructions, viral nucleic acids were extracted from 200 µl of the sample and eluted in 80 µl. During extraction, the FTD Respiratory pathogens 21 kit (Siemens Healthineers, Courbevoie, France) internal control was added to each sample and to the negative control to check the quality of the extraction and any inhibition during PCR.

The FTD Respiratory pathogens 21 kit (Siemens Healthineers), a multiplex PCR assay for the simultaneous amplification and qualitative detection of influenza A virus, influenza A virus subtype H1N1, influenza B virus, human coronaviruses NL63, 229E, OC43 and HKU1, parainfluenza viruses (HPIV) 1, 2, 3 and 4, human metapneumoviruses (HMPV) A and B, human rhinovirus (HRV), human respiratory syncytial viruses (HRSV) A and B, human adenovirus (HAdV), enterovirus (EV), human parechovirus (HPeV), human bocavirus (HBoV) and Mycoplasma pneumoniae. The FTD Respiratory pathogens 21 assay was performed according to the manufacturer’s instructions (Siemens Healthineers). Briefly, each of the 5 primer and probe mixes contained in the kit was prepared by mixing 12.5 μl of 2x RT-PCR Buffer, 1.5 μl of Primer/ probe mix and 1 μl of 25x RT-PCR Enzyme mix. Then, in each well of the PCR plate containing this mix, 10 μl of nucleic acid extracts were added. Subsequently, multiplex real-time RT-PCR reactions were performed on a LightCycler^®480 thermal cycler (Roche Diagnostics, Meylan, France) using the following program: reverse transcription at 50°C for 15 min, initial denaturation at 94°C for 1 minute, primer hybridization at 94°C for 8 seconds and enzyme elongation at 60°C for 1 minute. Primer hybridization and enzyme elongation were repeated for 40 cycles.

Data analysis

Ct values from Respiratory pathogens 21 tests were analyzed using absolute quantification. Any sample showing a sigmoidal curve with a Ct value <35 was considered positive. Co-infection was determined by detecting SARS-CoV-2 with one or more additional respiratory pathogens in the same sample. RT-PCR test results and socio-demographic data were entered into a database using Microsoft Excel. All data were saved in a Microsoft Excel database. Descriptive analysis was performed using a series of formulas in Excel (Microsoft) and SPSS version 16.0 (Chicago SPPS Inc).

Ethical statement

The National Health Research Ethics Committee of Senegal (opinion 000159/MSAS/CNERS/SN) approved the protocol concerning the samples used in this study.

Results

Patient characteristics

Four hundred and thirty-seven patients were included in the study. Of these, 78.03% (341/437) lived in the Thiès region, 21.74% (95/437) in the Dakar region and 0.23% (1/437) in the Matam region. According to the epidemic waves in Senegal, 18.76% (82/437) of samples were collected between May and July 2020 during the first wave, and 81.24% (355/437) between February and May 2021, coinciding with the second wave during which the highest case-fatality rate was recorded [13]. The mean age was 46 years, with a range of 8 to 93 years. According to gender, 59.73% of patients (261/437) were males, 39.82% (174/437) were females and no information was available for 0.46% (2/437). Clinical data were only available for patients in the second wave. Of the 355 patients in the second wave, information on symptoms was available for 352. Of these, 287 (81.53%) were asymptomatic. For symptomatic subjects, cough was the most frequent symptom (64/65, 98.46%), followed by fever (29/65, 44.62%) and sore throat (28/65, 43.08%). Other symptoms were headache (25/65, 38.46%), myalgia (19/65, 29.23%), diarrhea (7/65, 10.77%), dyspnea (5/65, 7.69%) and vomiting (2/65, 3.08%) (Table 2).

Table 2: Characteristics of COVID-19 positive patients.

Detection of respiratory viruses other than SARS-CoV-2

Of 437 COVID-19-positive patients tested with the FTD Respiratory pathogens 21 kit, 15 (3.43%) were infected with at least 1 virus (Table 3). Of these 15 patients, 13 (86.67%) were infected with 1 additional virus, 1 (6.67%) with 2 additional viruses and 1 (6.67%) with 4 additional viruses.

Table 3: Co-infections in COVID-19 positive patients and other identified respiratory pathogens.

Among these 15 patients, rhinovirus was detected in 4 patients (26.67%), HCoV 229E in 2 patients (13.33%), HCoV-NL63 in 1 patient (6.67%), HCoV-OC43 in 1 patient (6.67%), HCoV-HKU1 in 1 (6.67%) patient, HPIV-4 in 2 patients (13.33%), bocavirus in 1 patient (6.67%), adenovirus in 6 patients (40.00%) and influenza B virus in 1 patient (6.67%) (Figure 1).

Figure 1: Number of other respiratory viruses detected in coinfected COVID-19-positive patients. Adenovirus was the most frequently detected, followed by rhinovirus, HPIV-4 and human coronavirus 229E. Influenza B virus, bocavirus and the 3 other human coronaviruses were each detected only once.

The COVID-19 positive patient with 2 additional viruses detected, was infected with rhinovirus and influenza B virus. The COVID-19 patient with 4 additional viruses detected was infected with all 4 human coronaviruses (HCoV 229E, HCoV NL63, HCoV OC43 and HCoV HKU1, Table 4). The threshold cycle (Ct) values of these detected respiratory pathogens ranged from 31 to 35.

Table 4: Respiratory virus combinations identified in COVID-19-positive patients.

Of the 15 patients with respiratory viral co-infections, 73.33% (n=11) were women, while 26.67% (n=4) were men. The median age was 40.5 years, ranging from 13 to 93 years. Of the 15 coinfected patients, clinical signs were available for only 9. Of these, 6 were asymptomatic and 3 symptomatic (Table 5). The most frequent symptom was cough (n=3), followed by fever (n=2 each), diarrhea and myalgias (n=1 each).