Viral co-pathogens in COVID-19 acute respiratory syndrome – what did we learn from the first year of pandemic?

Open AccessPublished:January 13, 2022DOI:


      • During COVID-19 respiratory pathogen distribution had changed dramatically
      • COVID-19 occurrence of co-pathogen is varied
      • We describe the 1st pandemic year occurrence of respiratory pathogens
      • COVID-19 ARI patients had very low occurrence of co-pathogens



      : To describe the distribution of respiratory pathogens and the occurrence of co-pathogens during the first year of the COVID-19 pandemic.


      : We used a multiplex-PCR panel targeting 23 microorganisms to analyze oro-pharyngeal samples of patients admitted to our hospital with acute respiratory infection (ARI) between March 1st 2020 and February 28th 2021. We age and sex matched 40-50 SARS-CoV-2 positive and negative patients per month.


      : A total of 939 patients with multiplex-PCR test results included in the study. Respiratory pathogens where detected in only 8/476 (1.6%) COVID-19 versus 87/463 (18.7%) non-COVID-19 ARI patients. Diversity and rates of pathogens vastly differed from previous years yet showed seasonal variance.


      : SARS-CoV-2 patients presenting with ARI during the first COVID-19 pandemic year demonstrated paucity of respiratory co-pathogens.


      Acute infectious respiratory illness (ARI) is a substantial contributor to global morbidity and mortality (GBD 2016 Lower Respiratory Infections Collaborators, 2018). During COVID-19 pandemic, a major decrease in the incidence of respiratory infectious diseases was noted in countries where non-pharmaceutical measures as masks, school closure and social distancing were taken to mitigate the pandemic (Lee and Lin, 2020). Occurrence of bacterial co-pathogens or secondary infections is well described in the context of severe COVID-19 (Langford et al., 2020; Patel et al., 2021), contributing to grave prognosis of patients with severe disease. The co-occurrence of viral and atypical pathogens in COVID-19 was reported to be 11.6% (pooled prevalence) in one meta-analysis (Davis et al., 2020), but the studies addressing this topic are methodologically diverse. The effects of the introduction of the SARS-CoV-2 to the oral microbiota are only partially studied, yet bacterial composition does not seem to be substantially altered (Braun et al., 2021). As the pandemic progressed in both southern and northern hemispheres, an almost universal decrease of major viral pathogens was reported (Lee and Lin, 2020; Soo et al., 2020). The possible ramifications of co-infection on viral transmissibility and infectivity and the severity of co-infection are immense. Co-infections of SARS-CoV-2 and other common respiratory viral pathogens may impose additional challenges on the already stretched ambulatory and hospitalizing health systems. Thus, valid data regarding the rate of co-infection is of major importance for both public health authorities and infection control units. To assess background prevalence of respiratory pathogens and co-infections in COVID-19 patients we analyzed banked oro-nasopharyngeal specimens from patients admitted to the hospital with acute respiratory symptoms during March 1st 2020 and February 28th 2021.
      Setting - Sheba medical center (SMC) is the largest tertiary center in Israel, with 1600 hospitalization beds.
      Samples collection and patients’ inclusion (SupplementaryFigure 1) - all oro-nasopharyngeal respiratory specimens sent for either SARS-CoV-2 testing or other workup for respiratory viruses during 2020-2021 were biobanked in -80°C. A digital list of the biobank served as the main registry for the study. For each studied month, the first eligible 40-50 consecutive cases from the positive and negative cohorts were included. Patients’ files were manually reviewed by an infectious disease specialist, selecting cases with acute respiratory infection (ARI), either upper or lower, lasting less than 14 days. Patients where considered eligible for inclusion if they had one or more of the following signs and symptoms: cough, shortness of breath, tachypnea, rhinorrhea and sore throat, not attributed to other illness. We documented presence of oxygen desaturation (SpO2<94%), fever (≥38°C), and chest X-ray suggestive of lower respiratory tract infection. Excluded were patients with respiratory complaints or proved COVID-19 disease duration of more than 14 days, and patients hospitalized for more than 14 days during the testing time.
      Matching – patients positive and negative for SARS-CoV-2 were age (±5 years) and sex matched to create a similar number of matched monthly cohorts (40-50 consecutive, matched patients a month accommodating for the limited number of kits).
      Molecular assay - frozen oro-nasopharyngeal samples (both SARS-CoV-2 positive or negative) with sufficient residual volume were thawed and re-analyzed using the BioFire Filmarray® respiratory panel 2.1 (BioFire Diagnostics, UT, USA, "BioFire"). The BioFire is a sample-to-result, multiplex, nested-PCR platform allowing rapid syndromic-based diagnoses. The respiratory panel comprises a set of 23 pathogens, mostly viral, including seasonal and non-seasonal pathogens. Of note, although human rhinovirus (HRV) and enteroviruses are indiscriminate in this panel, data from the national surveillance sentinel clinics for respiratory pathogen, and data of hospitalized patients in SMC show profound activity of HRV, thus we refer to this signal as HRV.
      Statistics - after confirmation of a non-normal distribution, we used the non-parametric Mann-Whitney test to compare patients’ contiguous characteristics, and Chi-square test to compare dichotomous variables.
      Ethics - The study was approved by the local Institutional Review Board (SMC IRB approval number 7913-20-SMC).
      Overall, 2050 patients’ electronic charts were reviewed, of which 939 (Supplementary Figure 1 and Supplementary Table 1) were included in the analysis with valid BioFire test results. Among the 476 COVID-19 patients, 468 tested positive for SARS-CoV-2 as a single pathogen, co-pathogens were found in only 8/476 (1.6%) (Supplementary Table 2). Of the patients with ARI who were negative for SARS-CoV-2, 87/463 (18.7%) were detected with other respiratory pathogens. Pathogens detected in SARS-CoV-2 negative patients temporally varied. In March-April 2020 (spring), during the first lockdown in Israel (Figure 1a), 25/90 (27.8%) of the non-COVID-19 patients with ARI tested positive for seasonal coronaviruses, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza and parainfluenza, while human rhinovirus (HRV) was detected only in 11/90 (12.2%). In May 2020, the first lockdown was lifted, and only few patients admitted with ARI (13 tested – both groups) (Figure 1b). During June – December 2020 we noted an almost exclusive recovery of HRV – 44/368 (11.7%), while other pathogens activity dropped to 13/368 (3.3%). During the summer and autumn months (June-November, 2020) only 11.0% (25/227) of COVID-19-negative patients with ARI were detected with respiratory pathogens. This rate nearly doubled in winter months (December 2020-February 2021), to 21.2% (30/141) with frequent HRV, yet zero cases of the usual seasonal coronaviruses, RSV and influenza that practically disappeared that winter (Figure 1a and 1b, Supplementary Table 3).
      Figure 1
      Figure 1a - Daily newly diagnosed COVID-19 patients, Israel 2020-2021
      b - Annual pathogen distribution of patients with acute respiratory infection, Sheba, 2020-2021
      In this study, we report the paucity of respiratory co-pathogens detected in COVID-19 patients presenting with ARI compared with the background rates of respiratory pathogens in non-COVID-19 ARI patients. We observed seasonal changes both in respiratory pathogens incidence and variety (Supplementary Table 3). While in early spring 2020 we detected influenza, RSV, HMPV and seasonal coronaviruses, later in the year we saw the disappearance of those viruses with almost exclusive detection of HRV (as implied from Supplementary Figure 2). Our findings are compatible with the 2020-2021 disappearance of influenza and RSV in Israel (Weinberger Opek et al., 2021).
      Unlike other reports addressing co-pathogen occurrence in COVID-19 patients (Chen et al., 2020; Kim et al., 2020, 2021; Thelen et al., 2021), our study was designed to circumvent several biases: i) We included only ED admitted patients with acute respiratory complaints and matched them by sex and age. ii) The study was annual and longitudinal with similar number of samples per group monthly. This highly selective design overcomes biases of either secondary/nosocomial complications of severe COVID-19 patients or merely a “virome map” from testing asymptomatic COVID-19 patients.
      Numerous factors presumably account for the disappearance of major respiratory viruses during COVID-19 pandemic year: social distancing, face masks, gloves and extensive hand and surface disinfection, lockdowns and flight halting. Never the less, SARS-CoV-2 incidence and prevalence were extremely high during disease surges despite these measures and the low activity of other viruses. Thus, did the new player in the "respiratory arena" displace other viral pathogens? Does these phenomena are related to intrinsic viral factors effecting epidemiological patterns of SARS-CoV-2 transmissibility compared to other respiratory viruses? studies worldwide report the resurgence of RSV through the pandemic second year (Foley et al., 2021; Weinberger Opek et al., 2021). However, the relatedness of the resurgence of other respiratory viruses to the actual presence of airways SARS-CoV-2 remains unanswered.
      A notable observation of our study is the paucity of respiratory co-pathogens detected in COVID-19 patients. Should this observation prove consistent, it may have a significant impact on the diagnostic flow of ARI patients in high-COVID-19 prevalence zones, questioning the immediate need to search for pathogens other than SARS-CoV-2. This observation may further influence infection control policies in terms of placement of ARI patients within the emergency departments and later, hospitalization units, especially those of vulnerable patients as immunocompromised and pregnant women.
      This study has several limitations. Although all attempts were made to create a calendric-balanced data set of SARS-CoV-2 positive and negative patients, in some disease nadir months we could not reach the 50 patients required. In addition, generalizability of the study is limited with regards to the younger age groups as matching criteria could not be fulfilled due to paucity of pediatric COVID-19 patients. Likewise, this is a single-center study, representing merely an annual local epidemiology of a unique year. Further surveillance is needed to assess the effects of SARS-CoV-2 on the personal and societal virome.
      In conclusion, the annual rates of co-pathogens in Israeli COVID-19 patients with ARI were low compared with the background rates of respiratory pathogens in SARS-CoV-2 negative ARI patients. The appearance patterns of the various pathogens diverged from previous years and along the study period, with human rhinovirus being the prominent non-SARS-CoV-2 pathogen. Further studies should address the impact of SARS-CoV-2 presence per-se on the co-occurrence of other respiratory pathogens seasonality and diversity. Such data are of major importance for policy makers with regards to surveillance, acute illness diagnostics and infection control.
      Transparency declaration: All authors report no conflict of interest
      Funding: This study received no external funding.
      Authors' contributions - OK: study concept and preparation, data management, analysis and interpretation, and manuscript preparation. SA, MM: study concept and design, data interpretation, and manuscript review. SGH, EL, GS, NB, and AE: manuscript review, and study supervision. RK, MO, AS, YB, OAH, RH, YA, JA, IN, LK and HS: manuscript review. All the authors have read and approved the final draft submitted.
      Declaration of interests
      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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      Appendix. Supplementary materials