Viral Etiological Agent(s) of Respiratory Tract Infections in Symptomatic Individuals during the Second Wave of COVID-19 Pandemic: A Single Drive-Thru Mobile Collection Site Study

One of the tools to contain the SARS-CoV-2 pandemic was to increase the number of performed tests and to improve the access to diagnostics. To this effect, mobile collection sites (MCSs) were established. This study was performed on samples collected at the MCS between November 2020 and March 2021. We aimed to confirm/exclude SARS-CoV-2, differentiate SARS-CoV-2 variants, and detect other respiratory pathogens. SARS-CoV-2 and other respiratory viruses were identified by RT-qPCRs. A total of 876 (46.35%) SARS-CoV-2 positive specimens in the diagnostic tests were identified. The wild-type variant was determined in 667 (76.14%) samples; the remaining 209 (23.86%) samples specimens were identified as Alpha variant. A total of 51 (5.6%) non-SARS-CoV-2 cases were detected in retrospective studies. These accounted for 33 cases of mono-infection including rhinovirus (RV), human adenovirus (HAdV), human metapneumovirus (HMPV), enterovirus (EV), and influenza virus, and 18 cases of co-infection (SARS-CoV-2 with RV or HAdV or HMPV, and RV with EV). Our research shows that the results obtained from the MCS have value in epidemiological studies, reflecting national trends on a micro scale. Although the spread of COVID-19 is a major public health concern, SARS-CoV-2 is not the only pathogen responsible for respiratory infections.


Introduction
Respiratory tract infections (RTIs) are the most common infections in patients who present themselves for medical consultation and constitute a major source of morbidity and mortality worldwide, accounting for approximately 3 to 5 million deaths annually [1,2]. RTIs cause a range of infections limited to the upper respiratory tract (rhinitis, sinusitis, pharyngitis, or tracheitis) and/or the lower respiratory tract (mainly bronchitis and pneumonia). The etiological factors of these infections may be various microorganisms including viruses, bacteria, and fungi [1][2][3]. Viruses are responsible for the majority (nearly 80%) of acute RTIs and until recently, the most common viruses were influenza, human respiratory syncytial virus, parainfluenza, and human adenovirus [2]. Over time, rhinoviruses and coronaviruses were discovered, and as methods for their detection improved, their importance in viral respiratory diseases was recognized. They are considered to be responsible for 50% to 75% of upper respiratory tract infections [4]. The most common bacterial pathogens in upper and lower respiratory tract infections are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila. Fungal RTIs are a major clinical problem, especially in

Specimen Collection
The clinical samples collected from individuals with suspected COVID-19 between November 2020 and March 2021 were analyzed. The specimens were not subject to selection; all patients were qualified for diagnostic study. The samples originated from a drive-thru MCS in Ostrow Mazowiecka and were sent to the NMI in Warsaw together with basic patient demographic data obtained through a laboratory form. The patients completed a laboratory form giving consent to the collection of material for testing and General Data Protection Regulation (GDPR) consent. All samples were nasopharyngeal cavity swabs transported in virus-dedicated media (all containing Hanks' balanced salt solution) of different producers (Biocomma Limited, Shenzen, China; Liofilchelm, Roseto degli Abruzzi, Italy; and ClinicScience, Nanterre, France). The collected specimens were assigned codes that prevented identification of personal information at the time of testing. After the diagnostic testing for SARS-CoV-2, both positive and negative specimens were stored at −80 • C, pending further studies.

Nucleic Acids Isolation and RT-qPCR Tests
The nucleic acids were extracted using the NucleoMag Pathogen kit (Machery-Nagel, Duren, Germany) according to the manufacturer's instruction. A manual procedure was applied with magnetic blocks, as described previously [22]. RT-qPCRs were carried out according to the program recommended by the test manufacturers (Sections 2.2.1-2.2.3) using the Applied Biosystems QuantStudio 6 Pro Real-Time PCR System (Life Technologies Holdings Pte Ltd., Singapore). The results were interpreted based on the quantification cycle value according to manufacturers' recommendations.

Detection of SARS-CoV-2
Identification of SARS-CoV-2 for diagnostic testing was performed using the commercial MutaPLEX ® Coronavirus (SARS-CoV-2) kit (Immundiagnostik AG, Bensheim, Germany) which detects 3 viral genes: SARS-CoV-2-specific S and RdRP genes, and E gene that is characteristic for both known SARS viruses. Re-detection of SARS-CoV-2 verifying quality of deposited specimens after thawing for retrospective studies was carried out using the MutaPLEX ® RespiraScreen 1 kit (Immundiagnostik AG, Bensheim, Germany) which identifies only the E gene.

Differentiation of SARS-CoV-2 Variants
All SARS-CoV-2 positive samples were tested for differentiation of the most common variants present at the time of collection using two assays: the Bosphore ® SARS-CoV-2 Variant Detection Kit v1 (Anatolia Geneworks, Istanbul, Turkey), which identifies lineage B.

Statistical Analysis
The statistical analysis was performed with STATISTICA Software (Version 9.0, Stat-Soft Inc., Tulsa, OK, USA). The statistical significance of differences between groups was determined by the two-sided chi-square test. A p-value <0.05 was considered statistically significant. The figures were created using GraphPad Prism 7.0 software (GraphPad Software, Inc., San Diego, CA, USA).

Distribution of SARS-CoV-2
The results of the SARS-CoV-2 detection are shown in Table 1

Distribution of SARS-CoV-2
The results of the SARS-CoV-2 detection are shown in Table 1

Distribution of SARS-CoV-2
The results of the SARS-CoV-2 detection are shown in Table 1    The SARS-CoV-2 mono-infection was detected in 859 samples (45.45% of all tested, 94.40% of viral positive cases, and 98.06% of SARS-CoV-2 positive). The study group consisted of 454 female (52.85% of SARS-CoV-2 positive) and 405 male (47.15% of SARS-CoV-2 positive) (p = 0.1722). The most frequent cases were identified in the 36-64 age group with n = 491 (48.0% of all tested and 94.79% of viral positive in this age group) and 19-35 with n = 164 (47.95% of all tested and 93.71% of viral positive in this age group), followed by those aged ≥65 with n = 173 (41.0% of all tested and 96.65% of viral positive in this age group), 6-18 with n = 24 (38.1% of all tested and 88.89% of viral positive in this age group), and 0-5 with n = 7 (17.5% of all tested and 63.64% of viral positive in this age group). The co-infection of SARS-CoV-2 with other respiratory pathogens was found in 17 specimens (0.9% of all tested, 1.87% of viral positive, and 1.94% of SARS-CoV-2 positive).

Changing Epidemiology of SARS-CoV-2
All 876 SARS-CoV-2 positive samples were screened for differential Alpha, Beta, Kappa, and Delta variants. In general, the wild-type (WT) variant, was determined in 667

Co-Infection Rates
The results of the co-infection distribution (except the month of virus identification) are shown in Tables 1 and 2 and Figure 1. Eighteen co-infections were identified among the studied cases, which constituted 0.95% of all tested and 1.98% of positive cases. A positive result for SARS-CoV-2 co-infection with other respiratory viral agent(s), as mentioned previously, was found in 17 samples (0.9% of all tested, 1.87% of viral positive, and 1.94% of SARS-CoV-2 positive). The most frequent cases were identified in the 36-64 age group with n = 10 (0.98% of all tested and 1.93% of viral positive in this age group), followed by those aged 19-35 with n = 2 (0.58% of all tested and 1.14% of viral positive in this age group), ≥65 with n = 2 (0.47% of all tested and 1.12% of viral positive in this age group), 0-5 with n = 2 (5.0% of all tested and 18.2% of viral positive in this age group), and 6-18 with n = 1 (1.59% of all tested and 3.7% of viral positive in this age group). The highest rate of SARS-CoV-2 co-infections was observed with RV (n = 13; nine males and four females) identified in each month except January (n = 8, n = 2, n = 1, and n = 2 per month, respectively) and every age group except 6-18. Two SARS-CoV-2 co-infections with HAdV (males in age groups: 6-18 and 36-64) in December and HMPV (female and male in 36-64 age group) in March and December were found. In addition, one RV and EV co-infection was detected in March at female over 65 years old.

Discussion
Since the first identification of SARS-CoV-2 in late 2019, the WHO has recorded more than 410 million confirmed cases of COVID-19, including almost 6 million deaths [24]. The COVID-19 pandemic spread worldwide causing health, economic, and social distresses. It represents not only a serious diagnostic/therapeutic problem, but also a major epidemiology and public health challenge, straining the resources of healthcare systems. To contain the pandemic, it is crucial to monitor and detect every infected person in order to apply the isolation and treatment. Therefore, the rapidity and accessibility of SARS-CoV-2 diagnostics is of great importance. The establishment of drive-thru MCSs was intended to relieve the burden on health-care facilities and increase the availability of detect virus tests. This testing strategy has improved and provided direct and easy access to diagnostic testing to reach the broader population. The drive-thru testing formats have previously been promoted as a safe and effective method for large volume testing initiatives. It allows direct detection the pathogen during pandemic situations and has the benefit of reducing the number of infectious persons entering and contaminating healthcare establishments as well as promoting social distancing [25]. Our research shows that the results obtained from the drive-thru are also of value in epidemiological studies, reflecting national trends on a micro scale.
In Poland, the first SARS-CoV-2 infection was registered on 4 March 2020, and by 14 February 2022, nearly 5.5 million people had contracted the disease and almost 110,000 people had died [26]. The present study includes clinical specimens from patients with suspected COVID-19 who presented for testing during the second wave of the pandemic at a drive-thru mobile collection point located in Mazovia. Most epidemiological studies on COVID-19 to date have rarely focused on samples taken from individuals at MCSs; the majority of investigations have involved hospital patients whose parameters could be followed during hospitalization [25,[27][28][29]. The number of SARS-CoV-2 positive cases identified in the NMI reflected a nationwide trend [26], with a peak in November, then a decline until February and another increase in March (Figure 2), which was covered by the global data [24]. A total of 876 SARS-CoV-2 positive specimens in the diagnostic tests were identified. This corresponded to 859 mono-infections and 17 co-infections. SARS-CoV-2 was statistically significantly (p < 0.0001) more frequently responsible for respiratory tract infections than other viruses, which was in line with national data [30,31]. No gender differences in virus infection were observed; SARS-CoV-2 was detected in 466 (51.21%) females and 410 (45.05%) males. The majority (96.12%) of cases were identified in adults, that was statistically significant (p < 0.0001). Our observations on the population data are similar to those of other studies [15,[32][33][34].
The vaccination is one of the most effective means to prevent infectious diseases. The introduction of effective vaccines against SARS-CoV-2 was expected to prevent COVID-19 cases [35]. Nowadays, the vaccines are widely available, but of limited durability in vaccine-induced immunity. The inability of subgroups of the population to be vaccinated and increased infectious and/or vaccine-insensitive variants of concerns, VOCs (Alpha, Beta, Gamma, Delta, and Omicron variants), have fueled recurring global infection waves [36][37][38][39]. The Alpha variant (B.1.1.7 lineage) was first observed in the UK and, then, soon began to spread rapidly around the world [40]. The current study showed a change in the epidemiology of the virus during the 5-month period in favor of the Alpha variant ( Figure 3). The wild-type virus dominated initially, which gradually began to decline to the B.1.1.7 lineage in the following months. In March, it represented more than 96% of all identified cases. This result reflects national data where the Alpha variant dominated until May 2021 and then began to be displaced by the Delta variant [41].
The time when specimens were collected for SARS-CoV-2 testing was also the peak season for respiratory infections caused by other viruses. Therefore, a retrospective study was conducted to identify non-SARS-CoV-2 viruses in mono-and co-infections. It should be noted that the introduction of various responses to the COVID-19 pandemic, ranging from temporary room closures to the wearing of masks, social distancing, increased personal hygiene, and travel restrictions, was intended to limit the spread of SARS-CoV-2, but also affected the occurrence of other common seasonal respiratory viruses. Seasonal influenza is an acute respiratory infection caused by influenza viruses that circulate in all parts of the world. It represents a year-round disease burden. It causes illness of varying degrees of severity, which sometimes leads to hospitalization and death. As mentioned previously, before the COVID-19 era, the influenza virus was one of the most common viral etiological agents of acute RTIs [2]. During the 2019/2020 flu season, more than 4.8 million cases of influenza illness or suspected illness were reported in Poland and a total of 65 deaths were recorded [30]. In contrast, in 2020/2021, 2 million fewer reported cases and no deaths were registered [31]. Only one case of influenza virus (3.03%) was identified in this study. Our result differs from those described in other papers, where influenza virus was a higher proportion [14,15,18,[42][43][44].
Rhinovirus-dependent infections occur throughout the year, with a seasonal peak in incidence in early autumn and spring. During these periods, up to 80% of colds can be associated with a documented rhinovirus infection [45]. Among viruses other than SARS-CoV-2 detected in this study, RV was the most frequently represented in mono-infections (66.67%). Our observations are similar to those of other studies [16][17][18][19]44,46].
HMPV infections can occur throughout the year, but seasonality has been described in several studies [47]. In our study, HMPV was found to be the agent of respiratory infection in 10 patients (30.30%) and was the second most commonly identified non-SARS-CoV-2 virus fallowing RV, which is consistent with other reports [16,18,44].
Before COVID-19 achieved pandemic status, reports from Wuhan, China, described a very low number of co-infections with other respiratory pathogens during SARS-CoV-2 infection [9][10][11]. At present, there are increasing reports on the integration of SARS-CoV-2 into existing circulating infection patterns [48,49]. Co-infections, compared with single infections, may lead to changes in transmission of the pathogen, progression of clinical symptoms, and the adverse effects associated with any given infection, which ultimately determines the management of infectious diseases. Co-infection (two respiratory viruses) was detected in 18 (0.95% of all tested and 1.98% positive cases) individuals, which is statistically significantly compared to mono-infections (p < 0.0001). As mentioned previously, a positive result for SARS-CoV-2 co-infection with other respiratory virus was found in 17 samples (94.44%). The highest rate of SARS-CoV-2 co-infections was observed with RV (76.47%), followed by HMPV and HAdV (11.76% each). In addition, one RV and EV co-infection was identified. These results are in line with data from other researchers [14][15][16][17][18][19][44][45][46][47][48][49].
A limitation of our study was that the laboratory questionnaire did not detail specific symptoms, so we cannot conclude which complaints/symptoms were most commonly reported by patients. In addition, unlike hospital-based studies, where various patient parameters are tracked, we could not follow the RTIs and their outcomes.

Conclusions
This study analyzed the viral etiological agents responsible for respiratory tract diseases. The most prevalent identified virus was SARS-CoV-2; it was statistically significantly more common in adults, regardless of gender. Although the spread of COVID-19 is a major public health concern, SARS-CoV-2 may not be the only pathogen responsible for respiratory infections. Other viruses, such as adenovirus, rhinovirus, metapneumovirus, enterovirus, and influenza, have also been detected, more frequently in mono-infections, but also in co-infections (mainly with SARS-CoV-2). It should be noted that RTI co-infections, depending on the patient's immune system status and comorbidities, usually result in a worse prognosis for the patient. Funding: This work was financed by internal funding from the National Medicines Institute.

Institutional Review Board Statement:
The study did not require bioethics committee approval. The study was part of routine diagnostics in a medical laboratory. All patients completed a laboratory form giving consent to the collection of material for testing and General Data Protection Regulation (GDPR) consent.

Informed Consent Statement:
The study was part of routine diagnostics in a medical laboratory. The samples used for scientific research were analyzed anonymously. The genetic material of the viruses was the object of interest and human material was not analyzed.