Frequency and Significance of Coinfection in Patients with COVID-19 at Hospital Admission.

Objective Viral pneumonia is not rare in community-acquired pneumonia (CAP). Mixed or secondary pneumonia (coinfection) can be seen in viral pneumonia; however, its frequency in coronavirus disease 2019 (COVID-19) has only been investigated in a few studies of short duration, and its significance has not been fully elucidated. We investigated the frequency and significance of co-infection in patients with COVID-19 over a 1-year study period. Methods Coinfection was investigated via multiplex polymerase chain reaction (PCR), culture of respiratory samples, rapid diagnostic tests, and paired sera. We used logistic regression analysis to analyze the effect of coinfection on severity at admission and Cox proportional-hazards model analysis to analyze the effect of coinfection on need for high-flow nasal cannula, invasive mandatory ventilation use, and death, respectively. Patients We retrospectively investigated 298 patients who suffered CAP due to severe acute respiratory syndrome coronavirus-2 infection diagnosed by PCR and were admitted to our institution from February 2020 to January 2021. Results Primary viral pneumonia, and mixed viral and bacterial pneumonia, accounted for 90.3% and 9.7%, respectively, of COVID-19-associated CAP, with viral coinfection found in 30.5% of patients with primary viral pneumonia. Influenza virus was the most common (9.4%). Multivariable analysis showed coinfection not to be an independent factor of severity on admission, need for high-flow nasal cannula or invasive mandatory ventilation, and mortality. Conclusion Viral coinfection was common in COVID-19-associated CAP. Severity on admission, need for high-flow oxygen therapy or invasive mandatory ventilation, and mortality were not affected by coinfection.

Introduction

Viral infection is a major component of community-acquired pneumonia (CAP) (1). A recent study investigating the etiology of CAP found that viruses accounted for about 20% of the infections (1). Another study in Japan showed a viral etiology of CAP in 23.1% of cases (2).

In November 2019, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection became pandemic resulting in a large number of severe cases and deaths, and since then, the importance of viral pneumonia has been recognized. To date, coinfection with not only bacteria but also viruses has been reported in viral pneumonia (1), and some reports have shown coinfection with viruses in coronavirus disease 2019 (COVID-19). However, studies investigating coinfection with COVID-19 have been performed for only a short duration, e.g., for a few weeks. As some coinfecting pathogens, typically viruses, show seasonal development, we thus thought it best to investigate coinfection for a complete year. In addition, the significance of coinfection on clinical courses of COVID-19, such as mortality and the requirement for high-grade pulmonary care, also has not been investigated (3,4). Therefore, the present study aimed to investigate the frequency of coinfection and whether coinfection influences severity, the clinical course during hospitalization, and mortality of patients with COVID-19.

Materials and Methods

We retrospectively analyzed patients who were admitted to Saitama Cardiovascular and Respiratory Center over the 12 months from February 2020 to January 2021 for CAP caused by COVID-19. Data were extracted from medical records. Informed consent was obtained in the form of opt-out on both the hospital web-site and information posted in the hospital. Nursing home residents and patients with non-resected lung cancer were excluded, as were those who declined to participate in the study. SARS-CoV-2 infection was confirmed using polymerase chain reaction (PCR) methods with nasopharyngeal swabs. Swabs were stored at -70℃ and used for the detection of respiratory pathogens on a Rotor-Gene Q instrument (Quiagen, Hilden, Germany) with a multiplex, real-time PCR (RT-PCR) using an FTD Resp 21 Kit (Fast Track Diagnostics, Silema, Malta) (5). The kit detects the following respiratory pathogens: influenza A and B viruses; coronaviruses (NL63, 229E, OC43, and HKU1); human parainfluenza viruses (HPIV) 1, 2, 3, and 4; human metapneumovirus A/B (hMPV); rhinovirus; respiratory syncytial virus (RSV) A/B; adenovirus; enterovirus; human parechovirus; bocavirus; and Mycoplasma pneumoniae. An EZ1 Virus Mini Kit v2.0 was used for nucleic acid extraction (Quiagen). Results of RT-PCR were considered positive with a threshold cycle value of <33 as indicated in the instruction manual. Paired sera included antibody titers of M. pneumoniae, Legionella spp., Chlamydophila psittaci, C. pneumoniae, influenza virus, RSV, HPIV, and adenovirus. Disease onset was defined as the day on which initial symptoms (e.g., fever, sore throat) developed. Coinfection was surveyed by multiplex PCR, culture, urinary antigen tests, paired sera, and rapid influenza diagnostic tests as reported previously (6). Pneumonia was classified into primary viral pneumonia, mixed viral and bacterial pneumonia, and secondary bacterial pneumonia based on a previous report (7). Severe pneumonia was defined when at least one major criterion or three minor criteria of the Infectious Diseases Society of America/American Thoracic Society guidelines (8) were present. Outcomes used in this study included severity at admission and time to need for high-flow nasal cannula (HFNC), invasive mandatory ventilation (IMV) use and death during the period from admission to final follow-up. The study protocol was approved by the Ethical Committee of Saitama Cardiovascular and Respiratory Center.

Statistical analysis

Risk factors for severity on admission was evaluated by univariate and multivariable logistic regression analysis. Risk factors for need for HFNC or IMV, and mortality from CAP accompanying COVID-19 were evaluated by univariable and multivariable Cox proportional-hazards model. Variables showing significance in the univariable analysis (p<0.05) were included in the multivariable regression analysis, considering factors which had been reported to be significant for severity or mortality of COVID-19. The 95% confidence intervals (CIs) were also reported. In all instances, a 2-tailed p value of <0.05 was considered to indicate statistical significance. All statistical analyses were performed with SAS version 9.4 (SAS Institute, Cary, USA).

Results

Patient characteristics

During the study period, 452 patients with laboratory-confirmed COVID-19 were admitted to our institution. A total of 154 patients were nursing home residents, and there were no patients with non-resected lung cancer or patients declined to participate in the study, then, 298 patients were enrolled. All patients admitted in February 2020 were transferred from a cruise ship. Results are presented as frequency and percentage or mean ± standard deviation or median (range) unless otherwise indicated. Patient age was 61.1±14.6 years old and 205 (68.8%) were men (Table 1). The median number of disease days (range) from onset to admission was 7 (0-19). There were no underlying diseases in 109 (36.6%) of the patients. Chronic obstructive pulmonary diseases were the most common among the underlying pulmonary diseases, and bronchiectasis was found in only 1 (0.3%) patient. Hypertension and diabetes mellitus were common as non-pulmonary underlying diseases. Laboratory tests on admission showed lymphopenia (<500/mm3) in 21 patients, elevated D-dimer values (≥2 μg/mL) in 40 (13.4%), and elevated serum ferritin value (≥500 ng/mL) in 146 (49.0%).

Table 1.

Patients’ Characteristics, n=298.

Characteristics		Value		Characteristics		Value
Male sex		205 (68.8)		Laboratory data
Age, years		61.1±14.6		Arterial blood gas analysis
<65		168 (56.4)		PaCO2, Torr
65-74		79 (26.5)		Unknown		8 (2.7)
75≤		51 (17.1)		<35		160 (53.7)
Body mass index (BMI), kg/m2		25.5±4.54		35-45		124 (41.6)
30≤ BMI		44 (14.8)		45≤		6 (2.0)
BMI <18		7 (2.3)		Lactate, mmol/L
BMI 18≤, <30		233 (78.1)		Unknown		36 (12.1)
BMI, unknown		14 (4.7)		<2		221 (74.2)
Days from onset to admission		7 (0-19)		2≤		41 (13.8)
Antibiotics prior to admission, yes		39 (13.1)		WBC, /mm3		6,484±3,049
Smoking history, yes		145 (48.7)		Plt, /mm3		20.9±7.7
Underlying diseases, none		109 (36.6)		Neutrophils, /mm3		4,971±2,993
Pulmonary diseases				Lymphocytes, /mm3		1,073±508
COPD		17 (5.7)		Unknown		0
Bronchial asthma		14 (4.7)		<500		21 (7.0)
Bronchiectasis		1 (0.3)		500≤		277 (93.0)
Pulmonary nontuberculous mycobacteriosis		1 (0.3)		D-dimer, μg/mL		1.74±3.36
Old tuberculosis		2 (0.7)		Unknown		3 (1.0)
Interstitial lung diseases		8 (2.7)		<2		255 (85.6)
Post lung cancer operation		4 (1.3)		2≤		40 (13.4)
Pneumoconiosis		1 (0.3)		AST, IU/L		41±38
Chronic pulmonary artery thromboembolism		1 (0.3)		ALT, IU/L		35±33
Non-pulmonary diseases				LDH, IU/L		281±116
None		120 (40.2)		CK, IU/L		150±424
Hypertension		100 (33.6)		BUN, mg/dL		16±9
Congestive heart failure		3 (1.0)		BUN ≥20
Ischemic heart diseases		19 (6.4)		Cre, mg/dL		0.88±0.34
Diabetes mellitus		92 (30.9)		Na, mmol/L		137±8
Valvular diseases		1 (0.3)		CRP, mg/dL		5.6±5.8
Arrythmias		9 (3.0)		KL-6, U/mL		337±321
Cardiomyopathy		2 (0.7)		Unknown		8 (2.7)
Cerebrovascular diseases		7 (2.3)		<500		251 (84.2)
Dementia		4 (1.3)		500≤		39 (13.1)
Neuromuscular diseases		4 (1.3)		Ferritin, ng/mL		743±703
Post upper digestive system surgery		4 (1.3)		Unknown		8 (2.7)
Chronic liver diseases		5 (1.7)		<500		144 (48.3)
Connective tissue diseases		3 (1.0)		500-1,000		76 (25.5)
Systemic steroids or immunosuppressants		7 (2.3)		1,000≤		70 (23.5)
Psychiatric diseases		2 (0.7)		Procalcitonin, ng/mL		0.235±1.625
Malignancy		9 (3.0)		Unknown		11 (3.7)
Heavy drinker		1 (0.3)		<0.5		275 (92.3)
Chronic kidney disease		6 (20.1)		0.5≤, <1		8 (2.7)
Long-term oxygen therapy		1 (0.3)		1≤		4 (1.3)
Vaccination history, pneumococcus		25 (8.4)		Complications
Vaccination history, influenza virus		73 (24.5)		Deep vein thrombosis		4 (1.3)
Premorbid performance status				Acute pulmonary thromboembolism		1 (0.3)
0		262 (87.9)		Pneumothorax		1 (0.3)
1-2		29 (9.7)		Pulmonary hemorrhage		1 (0.3)
3-4		6 (2.0)		Acute kidney injury		16 (5.4)
Viral coinfection, yes		91 (30.5)		qSOFA, 2≤		2 (0.7)
Bacterial coinfection, yes		29 (9.7)		Severity, severe		46 (15.4)
				Treatment during hospital stay
				Antibiotics, yes		114 (38.3)
				Neuraminidase inhibitors, yes		112 (37.6)
				Corticosteroids, yes		100 (33.6)
				Day from onset to start corticosteroid		8 (0-18)
				HFNC		46 (15.4)
				IMV		30 (10.1)
				Continuous renal replacement therapy		1 (0.3)
				ECMO		6 (2.0)
				Days from onset to HFNC		9 (3-15)
				Days from onset to IMV		11 (4-19)
				Days from admission to IMV		2.5 (-1-18)
				Follow-up period, from onset		42 (6-398)
				Mortality		23 (7.7)

qSOFA: quick Sequential Organ Failure Assessment Score, IMV: invasive mandatory ventilation, HFNC: high-flow nasal canula, ECMO: extracorporeal membrane oxygenation

Pneumonia Subtypes and Microbiological Patterns

Among the pneumonia subtypes, primary viral pneumonia was present in 90.3% of patients, and no patients had secondary bacterial pneumonia. Pathogens coinfected with SARS-CoV-2 and methods used to identify the pathogens are listed in Tables 2 and 3. Bacterial coinfection was found in 10 patients (9.7%), with M. pneumoniae being the most common. Viral coinfection was found in 91 (30.5%) patients, with influenza virus being the most common followed by rhinovirus. The numbers of patients with viral infection for each month of the study are shown in Figure. SARS-CoV-2 showed an increase of patients in April, August, and December of 2020. None of patients who were transferred from the cruise ship in February 2020 showed coinfection. Coinfection with M. pneumoniae, influenza virus, and HPIV increased during the winter season. The number of viruses coinfecting with SARS-CoV-2 included 1 in 68 (22.8%), 2 in 14 (4.7%), 3 in 5 (1.7%), 4 in 2 (0.7%), and 5 in 2 (0.7%) patients, respectively.

Table 2.

Etiology of Mixed Infection.

Pathogens		n (%)
Mycoplasma pneumoniae		23 (7.7)
Streptococcus pneumoniae		3 (1.0)
Legionella spp.		2 (0.7)
Escherichia coli		1 (0.3)
Influenza virus		28 (9.4)
Parainfluenza virus		27 (9.1)
Common cold coronavirus		18 (6.0)
Adenovirus		14 (4.7)
Bocavirus		10 (3.4)
Rhinovirus		9 (3.0)
Parechovirus		7 (2.3)
hMPV		6 (2.0)
RSV		6 (2.0)
Enterovirus		4 (1.3)

hMPV: human metapneumovirus, RSV: respiratory syncytial virus

Table 3.

Diagnostic Methods.

Methods	Number of positive diagnostic studies	Number of episodes studied
Urinary antigen test
Legionella spp., positive	2	291
Streptococcus pneumoniae, positive	3	291
Rapid influenza diagnostic test, tested	21	292
Paired sera, tested	2	123
Culture
Sputum	1	62
Bronchial toilet	1	8
Multiplex PCR
Nasopharyngeal swabs, sputum	91	298
BALF	2	2

PCR: polymerase chain reaction, BALF: bronchoalveolar lavage fluid

Figure.

The numbers of patients with SARS-CoV-2 infection and each co-infecting pathogen by month. The number of patients with COVID-19 increased in April, August, and December of 2020. Mycoplasma pneumoniae and influenza virus infections increased in winter.

Severity on admission, treatment, and clinical courses

Forty-six (15.4%) patients were in severe condition on admission. During the patients' clinical courses including before and after admission to our hospital, antibiotics and neuraminidase inhibitors (favipiravir) were administered in 114 (38.3%) and 112 (37.6%), respectively. Neuraminidase inhibitors were administered >72 h after onset in 108 patients. Corticosteroids were administered in 84 patients (including to 9 patients by local physicians before transfer) when they developed respiratory failure and required oxygen therapy and in 16 patients (all by local physicians before transfer) in non-respiratory failure without the requirement for O2. These 100 (33.6%) patients received corticosteroid therapy with dexamethasone 6 mg/day for 7-10 days. During the disease courses, HFNC and IMV were required in 46 (15.4%) and 30 (10.1%) patients, respectively. One day before their transfer to our hospital, 1 patient had been placed on HFNC and another patient on IMV by local physicians. One patient received continuous renal replacement therapy, 6 received extracorporeal membrane oxygenation, and 23 patients died.

Risk Factors for Severity on Admission

Results of the univariable and multivariable analyses are listed in Table 4. Multivariable analysis showed that the Odds ratio (OR) of age ≥75 years group was 5.61 (95% CI, 2.09 to 15.05) with age <65 years group as the reference, OR of elevated serum ferritin value of 500-1,000 ng/mL and ≥1,000 ng/mL were 2.62 (95% CI, 1.07 to 6.43) and 5.78 (95% CI, 2.33 to 14.33) with serum ferritin value <500 ng/mL as the reference, whereas coinfection with bacteria and viruses were nonsignificant factors.

Table 4.

Univariable and Multivariable Analysis of Severity on Admission.

		Univariable analysis						Multivariable analysis (final model)
		OR		95% CI		p value		OR		95% CI		p value
Body mass index (BMI)
30≤ BMI		1.08		0.45, 2.59		0.8728
BMI <18		0.92		0.11, 7.87		0.9401
BMI 18≤, <30		Ref
Sex, male		1.77		0.84, 3.74		0.1355
Age, years
<65		Ref
65-74		1.83		0.81, 4.11		0.1456		1.86		0.79, 4.36		0.1542
75≤		6.06		2.79, 13.17		<0.0001		5.61		2.09, 15.05		0.0006
Smoking history, yes		0.76		0.41, 1.44		0.402
Pulmonary diseases
Chronic obstructive pulmonary disease		0.72		0.16, 3.25		0.6678
Bronchial asthma		1.53		0.41, 5.71		0.5278
Bronchiectasis		>999.999		<0.001, >999.999		0.9875
Pulmonary nontuberculous mycobacteriosis		<0.001		<0.001, >999.999		0.9909
Old tuberculosis		5.58		0.34, 90.85		0.2271
Interstitial lung diseases		3.45		0.79, 14.95		0.0984
Post lung cancer operation		<0.001		<0.001, >999.999		0.9881
Pneumoconiosis		<0.001		<0.001, >999.999		0.9909
Chronic pulmonary artery thromboembolism		<0.001		<0.001, >999.999		0.9909
Non-pulmonary diseases
Hypertension		1.19		0.62, 2.30		0.5957
Congestive heart failure		<0.001		<0.001, >999.999		0.9897
Ischemic heart diseases		2.07		0.71, 6.07		0.1832
Diabetes mellitus		2.14		1.13, 4.07		0.0201		1.66		0.81, 3.39		0.1692
Valvular diseases		<0.001		<0.001, >999.999		0.9909
Arrythmias		0.68		0.08, 5.55		0.7175
Cardiomyopathy		<0.001		<0.001, >999.999		0.9916
Cerebrovascular diseases		2.25		0.42, 11.94		0.3427
Dementia		5.69		0.78, 41.42		0.0863
Neuromuscular diseases		1.84		0.19, 18.13		0.5996
Post upper digestive system surgery		5.69		0.78, 41.42		0.0863
Chronic liver diseases		3.77		0.61, 23.23		0.1522
Connective tissue diseases		11.41		1.01, 128.51		0.0488
Systemic steroids or immunosuppressants		4.33		0.94, 20.01		0.0609
Psychiatric diseases		<0.001		<0.001, >999.999		0.9916
Malignancy		0.68		0.08, 5.55		0.7175
Heavy drinker		<0.001		<0.001, >999.999		0.9909
Chronic kidney disease		11.90		2.11, 67.05		0.005
Long-term oxygen therapy		>999.999		<0.001, >999.999		0.9909
Vaccination history, pneumococcus		2.21		0.50, 9.71		0.2939
Vaccination history, influenza		1.98		0.84, 4.64		0.117
Premorbid performance status
0		Ref
1-2		2.48		1.02, 60.35		0.045		0.95		0.31, 2.89		0.9221
3-4		6.52		1.27, 33.58		0.025		3.27		0.41, 26.07		0.2638
Viral coinfection, yes		1.41		0.73, 2.72		0.3053		1.47		0.71, 3.06		0.2995
Bacterial coinfection, yes		1.16		0.42, 3.21		0.7772
Ferritin, ng/mL
<500		Ref
500-1,000		2.91		1.24, 6.82		0.0138		2.62		1.07, 6.43		0.0354
1,000≤		5.74		2.53, 13.05		<0.0001		5.78		2.33, 14.33		0.0002
Procalcitonin, ng/mL
<0.5		Ref
0.5≤, <1		5.98		1.44, 24.86		0.0139		2.65		0.49, 14.29		0.2579
1≤		1.99		0.20, 19.62		0.5549		2.65		0.24, 29.13		0.4265

Risk factors for the need for HFNC or IMV

Risk factors for the need for HFNC or IMV were evaluated except for each one patient who had been placed on HFNC and another patient on IMV by local physicians. Results of the univariable and multivariable analyses are listed in Tables 5 and 6. Multivariable analysis for the need for HFNC showed severe condition on admission [hazard ratio (HR), 4.30; 95% CI (1.52, 12.14) with non-severe condition as the reference], elevated serum KL-6 value of ≥500 U/mL [HR, 3.29 95% CI (1.20, 8.99) with KL-6 value <500 U/mL as the reference], elevated serum ferritin value of 500-1000 ng/mL [HR, 6.01; 95% CI (1.57, 23.04) with serum ferritin value <500 ng/mL as the reference], corticosteroid treatment in non-respiratory failure [HR, 4.31; 95% CI (1.15, 16.17) with non-corticosteroid use as the reference], and corticosteroid treatment in respiratory failure [HR, 2.76; 95% CI (0.81, 9.35), with non-corticosteroid use as the reference] to be the independent factors (Table 5). These were also the independent factors for the need for IMV after admission: severe condition on admission [HR, 3.35; 95% CI (1.06, 10.58)], elevated serum ferritin value of 500-1000 ng/mL [HR, 17.45; 95% CI (2.09, 146.09)], and corticosteroid treatment in respiratory failure [HR, 4.39; 95% CI (1.11, 17.33)] (Table 6). Coinfection with bacteria and viruses were not associated with the need for HFNC or IMV.

Table 5.

Univariable and Multivariable Analysis of the Need for Nasal High-flow Oxygen Therapy during the Hospital Stay.

		Univariable analysis						Multivariable analysis (final model)
		HR		95% CI		p value		HR		95% CI		p value
Body mass index (BMI), kg/m2
30≤ BMI		1.61		0.77, 3.35		0.2073
BMI <18		0.98		0.13, 7.12		0.9801
BMI 18≤, <30		Ref
Sex, male		1.59		0.78, 3.21		0.2005		1.03		0.36, 2.98		0.9501
Age, years
<65		Ref
65-74		1.57		0.77, 3.21		0.2132		1.51		0.61, 3.76		0.3747
75≤		2.60		1.27, 5.30		0.0087		0.67		0.20, 2.19		0.506
Smoking history, yes		0.70		0.39, 1.28		0.245
Pulmonary diseases
Chronic obstructive pulmonary disease		1.61		0.58, 4.51		0.3623
Bronchial asthma		0.50		0.07, 3.62		0.4913
Bronchiectasis		14.05		1.89, 104.43		0.0098
Interstitial lung diseases		1.76		0.43, 7.29		0.4327
Non-pulmonary diseases
Hypertension		1.06		0.57, 1.97		0.8655
Congestive heart failure		2.59		0.36, 18.82		0.3468
Ischemic heart diseases		1.56		0.56, 4.36		0.3962
Diabetes mellitus		2.02		1.12, 3.66		0.0201		0.92		0.37, 2.27		0.8603
Arrythmias		0.74		0.10, 5.36		0.7649
Cerebrovascular diseases		0.97		0.13, 7.07		0.9795
Chronic liver diseases		1.42		0.20, 10.32		0.7287
Systemic steroids or immunosuppressants		1.02		0.14, 7.40		0.9856
Malignancy		1.43		0.35, 5.90		0.622
Chronic kidney disease		1.52		0.21, 11.01		0.6813
Long-term oxygen therapy		-		-		-
Vaccination history, pneumococcus		0.95		0.34, 2.642		0.9146
Vaccination history, influenza		2.12		0.90, 5.03		0.0864
Severity on admission, severe		10.45		5.71, 19.14		<0.0001		4.30		1.52, 12.14		0.0059
Premorbid performance status
0		Ref
1-2		1.47		0.62, 3.48		0.3795
3-4		-		-		-
Viral coinfection, yes		1.07		0.57, 2.01		0.8399		0.80		0.34, 1.91		0.6149
Bacterial coinfection, yes		0.44		0.11, 1.82		0.2587
PaCO2, Torr
<35		1.44		0.77, 2.70		0.2508
35-45		Ref
45≤		1.23		0.16, 9.31		0.8414
Lactate, mmol/L
<2		Ref
2≤		1.95		0.96, 3.95		0.0634
Lymphocytes, /mm3
<500		2.38		1.01, 5.64		0.0483		1.37		0.36, 5.15		0.6415
500≤		Ref
D-dimer, μg/mL
<2		Ref
2≤		2.35		1.16, 4.76		0.0176		0.78		0.25, 2.47		0.6757
KL-6, U/mL
<500		Ref
500≤		5.34		2.90, 9.84		<0.0001		3.29		1.20, 8.99		0.0205
Ferritin, ng/mL
<500		Ref
500-1,000		5.62		2.39, 13.21		<0.0001		6.01		1.57, 23.04		0.0089
1,000≤		5.28		2.17, 12.83		0.0002		2.91		0.76, 11.17		0.1202
Procalcitonin, ng/mL
<0.5		Ref
0.5≤, <1		2.22		0.54, 9.17		0.272
1≤		1.89		0.26, 13.71		0.5311
qSOFA, 2≤		4.38		0.60, 31.83		0.1447
Treatment during hospital stay
Antibiotics, yes		2.226		1.23, 4.04		0.0086		1.29		0.66, 2.52		0.4523
Corticosteroids, no		Ref
Corticosteroid use in non-respiratory failure		20.86		7.51, 57.96		<0.0001		4.31		1.15, 16.17		0.0304
Corticosteroid use in respiratory failure		9.46		3.72, 24.05		<0.0001		2.76		0.81, 9.35		0.1035
Neuraminidase inhibitors		0.84		0.45, 1.57		0.5855

KL-6: Krebs von der Lungen-6, qSOFA: quick Sequential Organ Failure Assessment Score

Table 6.

Univariable and Multivariable Analysis of the Need for Invasive Mandatory Ventilation during the Hospital Stay.

		Univariable analysis						Multivariable analysis (final model)
		HR		95% CI		p value		HR		95% CI		p value
Body mass index (BMI)
30≤ BMI		1.99		0.85, 4.68		0.1156
BMI <18		-		-		-
BMI 18≤, <30		Ref
Sex, male		1.70		0.69, 4.20		0.2489		1.40		0.38, 5.11		0.6123
Age, years
<65
65-74		2.19		0.95, 5.05		0.0660		1.50		0.50, 4.51		0.4714
75≤		1.97		0.73, 5.33		0.1817		0.52		0.12, 2.34		0.3917
Smoking history, yes		0.60		0.28, 1.27		0.1801
Pulmonary diseases
Chronic obstructive pulmonary disease		1.23		0.29, 5.19		0.7762
Bronchial asthma		0.80		0.11, 5.85		0.8223
Bronchiectasis		26.55		3.43, 205.66		0.0017
Non-pulmonary diseases
Hypertension		1.12		0.52, 2.43		0.7706
Congestive heart failure		-		-		-
Ischemic heart diseases		2.69		0.93, 7.75		0.0671
Diabetes mellitus		2.07		0.98, 4.35		0.0553		0.84		0.29, 2.48		0.7579
Valvular diseases		-		-		-
Arrythmias		-		-		-
Cardiomyopathy		-		-		-
Cerebrovascular diseases		1.64		0.22, 12.10		0.6252
Systemic steroids or immunosuppressants		1.66		0.23, 12.22		0.6191
Malignancy		1.12		0.15, 8.27		0.9088
Chronic kidney disease		2.55		0.35, 18.75		0.3592
Vaccination history, pneumococcus		0.79		0.24, 2.60		0.6913
Vaccination history, influenza		1.53		0.58, 4.04		0.3858
Severity on admission, severe		8.35		3.97, 17.60		<0.0001		3.35		1.06, 10.58		0.0399
Premorbid performance status
0		Ref
1-2		0.33		0.045, 2.46		0.2815
3-4		-		-		-
Viral coinfection, yes		1.48		0.69, 3.16		0.3107		1.02		0.39, 2.65		0.9753
Bacterial coinfection, yes		0.71		0.17, 3.01		0.6466
PaCO2, Torr
<35		1.37		0.63, 2.97		0.4245
35-45		Ref
45≤		-		-		-
Lactate, mmol/L
<2		Ref
2≤		1.05		0.37, 3.03		0.9255
Lymphocytes, /mm3
<500		1.86		0.56, 6.17		0.3086		1.19		0.22, 6.32		0.8416
500≤		Ref
D-dimer, μg/mL
<2		Ref
2≤		3.12		1.37, 7.08		0.0066		0.72		0.21, 2.43		0.5907
KL-6, U/mL
<500		Ref
500≤		6.03		2.85, 12.76		<0.0001		3.11		0.90, 10.71		0.0721
Ferritin, ng/mL
<500		Ref
500-1,000		13.06		2.97, 57.45		0.0007		17.45		2.09, 146.09		0.0083
1,000≤		13.87		3.10, 61.99		0.0006		5.30		0.63, 44.83		0.1255
Procalcitonin, ng/mL
<0.5		Ref
0.5≤, <1		6.45		1.94, 21.46		0.0024
1≤		3.18		0.43, 23.49		0.2575
Treatment during hospital stay
Antibiotics, yes		2.64		1.22, 5.71		0.0139		1.13		0.49, 2.58		0.7807
Corticosteroids, no
Corticosteroid use in non-respiratory failure		18.32		4.92, 68.28		<0.0001		3.49		0.71, 18.25		0.1254
Corticosteroid use in respiratory failure		13.01		4.14, 40.88		<0.0001		4.39		1.11, 17.33		0.0349
Neuraminidase inhibitors		0.91		0.42, 1.96		0.8012

KL-6: Krebs von der Lungen-6

Risk factors for mortality

Results of the univariable and multivariable analyses are listed in Table 7. Age ≥75 years old [HR, 8.49; 95% CI (1.79, 40.36) with age <65 years old as the reference], lymphopenia <500/mm3 [HR, 9.07; 95% CI (1.79, 46.01) with lymphocyte count ≥500/mm3 as the reference], D-dimer value of ≥2 μg/mL [4.67; 95% CI (1.16, 18.77) with D-dimer value of <2 μg/mL as the reference], serum ferritin value of 500-1000 ng/mL [HR, 15.65; 95% CI (1.70, 144.31) with serum ferritin value <500 ng/mL as the reference], and corticosteroids in non-respiratory failure [HR, 15.62; 95% CI (1.99, 122.68) with non-corticosteroid use as the reference] and in respiratory failure [HR, 10.66; 95% CI (1.57, 72.18) with non-corticosteroid use as the reference] were the factors associated with death. Coinfection with viruses or bacteria was not associated with mortality (Table 7).

Table 7.

Univariable and Multivariable Analysis of Mortality.

		Univariable analysis						Multivariable analysis (final model)
		HR		95% CI		p value		HR		95% CI		p value
Body mass index (BMI)
45≤		1.57		0.53, 4.65		0.4172
BMI <18		-		-		-
BMI 18≤, <30		Ref
Sex, male		1.25		0.49, 3.18		0.6357		1.07		0.285, 3.983		0.9244
Age, years
<65		Ref
65-74		1.65		0.44, 6.16		0.4531		0.48		0.07, 3.17		0.4438
75≤		12.33		4.42, 34.41		<0.0001		8.49		1.79, 40.36		0.0072
Smoking history, yes		0.59		0.25, 1.36		0.2113
Pulmonary diseases
Chronic obstructive pulmonary disease		1.39		0.33, 6.93		0.6567
Bronchial asthma		0.99		0.13, 7.38		0.9952
Bronchiectasis		40.49		4.98, 329.11		0.0005
Interstitial lung diseases		3.42		0.80, 14.60		0.0970
Non-pulmonary diseases
Hypertension		1.67		0.73, 3.80		0.2262
Congestive heart failure		6.21		0.83, 46.27		0.0748
Ischemic heart diseases		2.63		0.78, 8.88		0.1187
Diabetes mellitus		4.69		1.99, 11.06		0.0004		3.72		0.82, 16.77		0.0878
Arrythmias		1.14		0.15, 8.49		0.8967
Cerebrovascular diseases		2.02		0.27, 15.02		0.4906
Neuromuscular diseases		8.89		2.07, 38.28		0.0034
Post upper digestive system surgery		3.89		0.52, 28.97		0.1852
Chronic liver diseases		2.76		0.37, 20.52		0.3202
Systemic steroids or immunosuppressants		2.31		0.31, 17.14		0.4136
Malignancy		1.23		0.17, 9.09		0.8430
Chronic kidney disease		3.00		0.40, 22.32		0.2841
Vaccination history, pneumococcus		0.59		0.18, 1.99		0.3961
Vaccination history, influenza		1.46		0.50, 4.29		0.4914
Severity on admission, severe		9.04		3.95, 20.71		<0.0001		0.48		0.10, 2.41		0.3729
Premorbid performance status
0		Ref
1-2		3.12		1.15, 8.47		0.0254
3-4		2.42		0.32, 18.23		0.3903
Viral coinfection, yes		1.49		0.63, 3.52		0.3672		1.11		0.30, 4.16		0.876
Bacterial coinfection, yes		1.65		0.49, 5.56		0.4225
PaCO2, Torr
<35		2.11		0.83, 5.36		0.1151
35-45		Ref
45≤		-		-		-
Lactate, mmol/L
<2		Ref
2≤		1.44		0.49, 4.23		0.5094
Lymphocytes, /mm3
<500		8.68		3.51, 21.48		<0.0001		9.07		1.79, 46.01		0.0078
500≤		Ref
D-dimer, μg/mL
<2		Ref
2≤		3.70		1.52, 9.03		0.0040		4.67		1.16, 18.77		0.0300
KL-6, U/mL
<500		Ref
500≤		4.03		1.71, 9.51		0.0015		1.76		0.43, 7.21		0.4316
Ferritin, ng/mL
<500		Ref
500-1,000		12.74		2.85, 57.01		0.0009		15.65		1.70, 144.31		0.0153
1,000≤		11.65		2.51, 54.03		0.0017		3.67		0.39, 34.77		0.2573
Procalcitonin, ng/mL
<0.5		Ref
0.5≤, <1		6.75		1.99, 22.91		0.0022
1≤		4.95		0.66, 37.23		0.1199
qSOFA, 2≤		12.38		1.61, 94.85		0.0155
Treatment during hospital stay
Antibiotics, yes		4.17		1.64, 10.58		0.0027
Corticosteroids, no		Ref
Corticosteroid use in non-respiratory failure		28.03		6.63, 118.50		<0.0001		15.62		1.99, 122.68		0.009
Corticosteroid use in respiratory failure		16.87		4.42, 64.31		<0.0001		10.66		1.57, 72.18		0.0153
Neuraminidase inhibitors, yes		0.86		0.36, 20.26		0.7279
NSAIDs, yes		0.47		0.06, 3.50		0.4631
Pneumonia subtypes
Primary viral pneumonia		Ref
Mixed viral and bacterial pneumonia		1.65		0.49, 5.56		0.4225

KL-6: Krebs von der Lungen-6, qSOFA: quick Sequential Organ Failure Assessment Score, NSAIDs: nonsteroidal anti-inflammatory drugs

Discussion

The present study showed that most of the SARS-CoV-2 pneumonia was primary viral pneumonia, and while bacterial coinfection was not so common, coinfection with other viruses was common. Considering treatment with antivirals and antibiotics, coinfection with M. pneumoniae and influenza virus were the most important pathogens. Coinfection did not affect severity on admission, the need for HFNC or IMV, and mortality.

There have been reports investigating the frequency of viral infection in pneumonia, but limited studies have focused on the characteristics of viral pneumonia itself. Crotty et al. investigated patients with viral pneumonia, half of whom were immunocompromised patients. Eighty-four of 284 patients had coinfection (9), with half coinfected with bacteria and the rest coinfected with viruses. Another report showed the rates of single virus infection, virus-virus coinfection, and virus-bacterial coinfection to be 22%, 2%, and 3%, respectively (1). These reports suggested that viral pneumonia without bacterial coinfection is common, which is compatible with our results. No patients in the present study had secondary bacterial pneumonia. Patients can easily consult physician soon after noticing their impaired condition in Japan and can receive diagnostic tests for COVID-19. When diagnosed as having COVID-19, they are immediately transported to hospital and isolated. These practices can lead to early hospitalization and may reduce the incidence of secondary bacterial infection on admission.

Several studies investigated coinfection of SARS-CoV-2. One study showed 23 (19.8%) of 116 patients with COVID-19 had coinfection; rhinovirus and enterovirus were the most common viruses, followed by RSV and common cold coronavirus (10). Another study showed that 18 of 89 patients (20.2%) with COVID-19 showed coinfection, all of which were due to bacteria (11). A multicenter study in the U.S. showed 1,690 of 12,075 (14.0%) patients had coinfection, and the number of pathogens coinfecting with SARS-CoV-2 ranged from 1 to 6 (12). Frequent pathogens included Staphylococcus aureus, human herpes virus-4, M. catarrhalis, Klebsiella pneumonia, hMPV, and adenovirus (12). Another multicenter study of 5,700 COVID-19 patients showed the common coinfecting pathogens to be enterovirus, rhinovirus, of which the common cold coronavirus was the most common, followed by RSV, HPIV, C. pneumoniae, hMPV, influenza virus, and M. pneumoniae (13). Other studies also showed that coinfection with viruses, including RSV, hMPV, HPIV, and common cold coronavirus (14,15), was common.

Previous studies suggested that coinfection is usually connected with the need for a higher level of care, increased length of stay, and development of acute respiratory distress syndrome (16). Because of the serious damage to the immune system caused by the coinfection (17), the condition of patients who are positive for both SARS-CoV-2 and other viruses may be more serious, and their treatment can be more complicated and require a longer treatment cycle (18). However, in the present study, coinfection did not affect severity on admission, the need for HFNC or IMV, and mortality, the results of which were compatible with those of a previous report (19). Another previous study showed mixed viral and bacterial pneumonia to be an independent factor for mortality (20) from influenza-associated pneumonia, and an additional report showed higher mortality from viral pneumonia when coinfected by bacteria, e.g., Streptococcus pneumoniae (21,22). In one study that investigated patients with cystic fibrosis, coinfection of other pathogens in addition to SARS-CoV-2 led to intensive care, antibiotics use, and an increased mortality rate (23). In the present study, the pneumococcal coinfections were minor, and underlying diseases of bronchiectasis and pulmonary non-tuberculous mycobacteriosis, both of which are risk factors of mixed viral and bacterial infection (23), were infrequent. These factors may have affected our results that mixed bacterial coinfection was minor and bacterial coinfection did not affect either severity or mortality. In other words, in COVID-19 patients without such underlying diseases, bacterial coinfection is uncommon, which indicates that the use of routine broad-spectrum antibiotics is not recommended. Prediction models to distinguish bacterial coinfection from primary viral pneumonia are desirable to judge the need for antibiotics therapy. The most frequent bacterial pathogens coinfecting in the present study were M. pneumoniae followed by S. pneumoniae and Legionella spp., and thus, macrolides or quinolones may be recommended in regions with a low rate of infection with macrolide-resistant S. pneumoniae for the time being. Future prospective studies are needed to clarify recommendations for routine antibiotics use in COVID-19.

Although the significance of viral coinfection is unknown, the mechanisms of coinfection include virus-induced airway damage, reduced mucociliary clearance, and damage to the immune system (24), which indicates a role of coinfection as a gatekeeper of SARS-CoV-2. Because our study could not clarify this matter, the significance of viral coinfection should be investigated in future studies. Another important issue is the efficacy of antivirals on coinfection. A few studies showed that early use of neuraminidase inhibitors decreased intensive care unit admission and mortality in patients with influenza-associated pneumonia (25). Options for the treatment of viruses other than influenza virus are extremely limited, and the efficacy of antivirals against these viruses coinfecting with COVID-19 remains unknown but should be elucidated in future studies.

Our study has several limitations. First, because this is a non-randomized observational study, the level of confidence was reduced. Second, clinical tests to detect causative microorganisms were not used in all patients. For example, sputum culture was performed in only 62 (20.8%) of 298 patients because of the low frequency at which patients expectorate sputum. This may result in underestimation of the coinfection rate. Third, this study was carried out in a single institution, and the results may not be applicable to other settings. Finally, some viral infections may have been missed in this study because only a limited number of viruses were screened in the assay.

In conclusion, the present study showed that coinfection was frequent in CAP with COVID-19, especially by other viruses, and primary viral pneumonia was dominant. The rate of bacterial coinfection was less than 10%. Coinfection, both of viral and bacterial origin, did not appear to affect severe respiratory conditions or mortality.

The authors state that they have no Conflict of Interest (COI).

Financial Support

This study was partially supported by a grant from Saitama Cardiovascular and Respiratory Center (16ES, 17ES, 18ES, 19ES, 20ES).