Conclusions
The similarity in clinical presentation for both COVID-19 and Influenza
makes it difficult to assess their impact on ICU admission and
mortality. Our study highlights the importance of screening for
co-infecting viruses in COVID-19 patients, given the high prevalence of
Influenza viruses. The detection of co-infections in COVID-19 cases
shows the importance of flu vaccination and warrants its increased
coverage to reduce the hospitalization and associated mortality.
Keywords: SARS-CoV-2, COVID-19, Influenza A H1N1, co-infection,
ICU, comorbidities, mortality.
1. Introduction
The
newly emergent Severe Acute Respiratory Syndrome Coronavirus 2
(SARS-CoV-2) continues to circulate outside of Wuhan, China since
December 2019, and now exported to different countries all over the
world [1]. At the time of writing this report, there were nearly
quarter of a million of Coronavirus Disease-19 (COVID-19) confirmed
cases ranking Saudi Arabia as the 14th highest in the world [2].
Most hospitalized patients needed admission to intensive care unit (ICU)
and mortality reaches up to 50% in some cases [3]. Until now,
twenty-two studies have reported co-infection in COVID 19 and of these
16 have evidence of viral co-infection [4]. The prevalence of
critical cases with viral co-infection has been reported up to 35%
[5]. Early literature reported that 50% of the patients who died
had coexisting bacterial infection [6]. This is higher than what was
previously seen during influenza pandemic in 2009 when 25% of patients
with influenza infection had secondary bacterial co-infection [7].
SARS-CoV-2 is a single stranded RNA Betacoronavirus and belongs to the
corona virus family [8]. Phylogenetic analysis has revealed that
SARS-CoV-2 is closely related to SARS-CoV-1 and genetically distinct
from MERS-CoV [9]. SARS-CoV-2 utilizes ACE-2 receptors in the lower
airways which are also cellular receptors for other viruses in this
group i.e. SARS-CoV and MERS-CoV [10]. Despite similar expression of
ACE-2 receptors in different organs of the body, the most affected site
is the lung tissue [11]. Influenza strains also cause lung damage by
ACE-2 receptor mediated effects
[12]. On the other hand, since
the ACE-2 receptor used by SARS-CoV-2 is an interferon‐stimulated gene,
it was hypothesized that type I and III interferons produced after
bacterial infection may facilitate SARS‐CoV‐2 attachment [13].
During pandemics, the detection of the novel virus may lead to
underreporting of other pathogens that could be the etiological agent
contributing to the disease severity. Indeed, during the influenza A
(H1N1) pdm09 pandemic, 44.3% of patients had unreported respiratory
viruses [14]. Earlier studies indicated that common viral
co-infections reported in COVID-19 patients include Influenza viruses,
RSV and adenovirus [5,15]. Bacterial co-infection is more frequent
than viral co-infection and it is homogeneously distributed in mild,
moderate or severe illness [16]. The
commonly known COVID-19 co-infecting bacteria are Mycoplasma pneumoniae,
Pseudomonas aeruginosa, Hemophilus influenzae and Chlamydia pneumoniae
[17]. These findings clearly emphasize on the importance of
screening for other clinically important co-circulating respiratory
pathogens contributing to the etiology of the disease.
The novelty of SARS-CoV-2 and the complicity of profound etiology of
co-infection urged for consideration of comorbidities. COVID-19 patients
with an underlying condition such as hypertension, diabetes, chronic
kidney disease, and heart failure have been associated with COVID-19
disease severity [18]. Cardiovascular disease has a strong
association with COVID-19 pneumonia (14.4%) [7,18] and other common
comorbidities found in patients with SARS-CoV-2 include hypertension
(18.6%) and diabetes (11.9%) [19]. Comorbidities were also linked
with increased hospitalization, prolonged stay in ICU, and mortality.
Hypertension was more prevalent in severe cases (47%) compared to
diabetes (24%) and Respiratory diseases (10%) among other underlying
conditions [18].
In conclusion, an extensive evidence revealed that viral infections
predispose patients to subsequent bacterial co‐infections [7]. This
knowledge gap is puzzling as limited number of reports have described
prevalence of bacterial and viral co-infections simultaneously. We
hypothesized that undetected co-infections might have severe clinical
implications associated with increased hospitalization, prolonged stay
in ICU, and mortality. Therefore, our aim was, to investigate the
presence of viral and bacterial co-infections in ICU and non-ICU
COVID-19 patients.
2. Materials and Methods
2.1. Patients
Forty-eight extracted RNA samples were collected from COVID-19 positive
patients, of which 14 were critical cases needing admission to the ICU,
and 34 were mild cases. Nine patients died, (all were admitted to the
ICU), and the rest survived. Thirteen patients were Saudi citizens and
the rest were non Saudi (Table 1). Samples were collected from King
Fahad Hospital, Medina, Saudi Arabia. This study was approved by the
Institutional Review Board at King Fahad Medical City (IRB Log No.
20-160). Informed consent to participate was waived or not required
since only remaining left‐over specimens were used for this study.
2.2. RNA Extraction and PCR
Nasopharyngeal swabs were collected from the patients and carried in a
suitable fluid viral medium. The RNA extraction of SARS-CoV-2 was
performed via a MagNA Pure 96 machine, using the MagNA Pure 96 DNA and
Viral NA small volume kit, (Roche, Germany). The amplification RT-PCR
was performed within a Roche LightCycler® 480 II instrument, using the
RNA Process Control Kit Trial Pack (Roche, Germany) with an internal,
positive, and negative controls.
2.3. Real time PCR panel for Co-infection
Eluted nucleic acid was stored at −80°C until use, and all reagents were
stored at −20°C. The quantitative RT-PCR assay for respiratory pathogens
was performed on 7500 Fast Real-Time PCR
System (Thermo Fisher scientific, USA). Extracted nucleic acid was
screened by RT/q-PCR with Fast Track Diagnostic (FTD) Respiratory
pathogens 21 plus kit (Biomerieux, Luxemburg) following the
manufacturer’s protocol using six multiplex PCR for respiratory viruses
and bacteria. The pathogens tested were influenza A (H1N1) virus
(swine-lineage); influenza B virus; human rhinovirus; human
coronaviruses NL63, 229E, OC43 and HKU1; human parainfluenza viruses 1,
2, 3 and 4; human metapneumoviruses A/B; human bocavirus; human
respiratory syncytial viruses A/B; human adenovirus; enterovirus; human
parechovirus; Mycoplasma pneumoniae; Chlamydia pneumoniae;
Staphylococcus aureus; Streptococcus pneumoniae; Haemophilus influenzae
B. Six positive controls were performed with every run (five for each
viral panel and one for bacterium) on multiplex PCR assay. Moreover, six
negative controls (NC), provided in the kit, were incorporated with each
run. Briefly, 10 µl of the extracted nucleic acid was used as a template
in each reaction for the FTD Respiratory pathogens 21 plus multiplex PCR
following the manufacturer’s instructions. The thermal cycle
amplification condition includes reverse transcription for 15 minutes at
42°C, denaturation for 3 minutes at 94°C followed by 40 cycles for 8
seconds at 94°C, and 34 seconds at 60°C. Specimens were determined to be
pathogen positive or negative based on the manufacturer’s interpretation
criteria, and 12 samples were randomly chosen and repeated to confirm
the results.
2.4. Data collection
Demographic and clinical data (Table 1) were collected, including the
following clinical laboratory results: age, gender, history of chronic
illness, Ct value, Dimar, CK, CK-MB, Trop, HB, Platalet, RBC, WBC,
Nutrophile, Lymph, CRP, Pro calciponin, Glucose, ESR, LDL, AST, ALT,
Ureae, Creatinine, LDH, Albumin, Total Protein and blood group.