Co-infection in COVID-19, a cohort study.

Dear editor,

Co-infection in COVID-19 patients may inflence the outcome of the disease and needs more attention and investigations. In this journal, Lansbury and colleges reported a meta-analysis of co-infections in COVID-19 patients. In this study, we investigated a COVID-19 cohort in Shanghai, China. We screened viruses include Human parainfluenza virus 1, Human parainfluenza virus 2, Human parainfluenza virus 3, Human parainfluenza virus4, Influenza A virus, Influenza B virus, Human rhinovirus, Human metapneumovirus, Human respiratory syncytial virus, Human Bocavirus, Human adenovirus, Human Coronavirus 229E, Human Coronavirus NL63, Human Coronavirus HKU1, Human Coronavirus OC43; bacteria include Pseudomonas aeruginosa, Moraxella catarrhalis, Mycobacterium tuberculosis, Legionella pneumophila, Group A Streptococcus, Haemophilus influenza, Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumonia, Klebsiella.peneumoniae, Escherichia coli and Mycoplasma pneumonia, Chlamydia pneumonia by a taqman-based real time PCR methods.

Eighty-nine patients were enrolled with disease outcomes include mild, moderate, severe and critical (Chinese clinical guidance for COVID-19 pneumonia diagnosis and treatment (7th edition) published by China National Health Commission on March 4, 2020. http://www.gov.cn/zhengce/zhengceku/2020–03/04/content_5486705.htm). Nine patients showed severe or critical symptoms. As reported, age is a risk factor for severe symptoms (Table 1 ). , Nucleic acids from the throat swab samples of the COVID-19 patients were used as template for Real-time PCR. Real-time PCR was performed by using One Step PrimeScript RT-PCR kit (Takara) in a 25µl reaction mixture as following: 2.5µl of nucleic acid was added in the mixture of 12.5µl of 2 × one step RT-PCR buffer, 0.5µl of EX Taq HS, 0.5μl of RT Enzyme, 1.6µl of primer mix (10µM each), 0.4µl of probe (10µM) and H2O up to 25µl. The R-PCR program was run with incubation at 42 °C for 10 min, followed by 40 cycles of a program (95 °C, 10 s; 95 °C 10 s, 60 °C, 1 min). The primers and probes for Influenza A virus were as following: sense (5′-CTT CTA ACC GAG GTC GAA ACG TA-3′), antisense: (5′-GGT GAC AGG ATT GGT CTT GTC TTT A-3′) and probe: (5′-FAM-TCA GGC CCC CTC AAA GCC GAG −3′-BHQ1); For E. coli were as flowing: sense (5′-GGA TAT CGT CTG GGA CTT CCG-3′), antisense (5′-GCG GAG CCA GAC CGA ATT T-3′) and probe (5′-FAM-GTG AAA TCG ATC AGT GCT TCA GGC CA −3′-BHQ1). Screening for other pathogens were performed by using real-time PCR detection kits from BioGerm (Shanghai, China) as following: human parainfluenza virus 1/human parainfluenza virus 3(PIV1/PIV3) (BioGerm, SJ-HX-215–2), human parainfluenza virus 2/human parainfluenza virus 4 (PIV2/PIV4)(BioGerm, SJ-HX-216–2), Influenza B virus (FLUB)(BioGerm, SJ-LG-006–2), human rhinovirus/human metapneumovirus/Human respiratory syncytial virus (HRV/HMPV/RSV)(BioGerm, SJ-HX-303–2), human Bocavirus/human adenovirus (HBOV/RADV)(BioGerm, SJ-HX-207–2), human Coronavirus 229E/human Coronavirus NL63 (BioGerm, SJ-HX-204–2), human Coronavirus HKU1/ human Coronavirus OC43 (BioGerm, SJ-HX-205–2), Pseudomonas aeruginosa (PA)(BioGerm, SJ-HX-039–2), Moraxella catarrhalis (MC)(BioGerm, SJ-HX-027–2), Mycobacterium tuberculosis (MTB)(BioGerm, SJ-HX-040–2), Mycoplasma pneumonia/Chlamydia pneumonia/Legionella pneumophila (MP/CP/LP)(BioGerm, SJ-HX-302–2), Group A Streptococcus/Haemophilus influenzae/Staphylococcus aureus (GA/HI/SA)(BioGerm, SJ-HX-319–2), Acinetobacter baumannii/Streptococcus pneumoniae/Klebsiella peneumoniae (AB/SP/KP)(BioGerm, SJ-HX-309–2).

Table 1

Patients in this study.

Age	Patients (Total number)	Patients (Severe or critical symptom)
15–60	66	1
61–75	20	5
>75	3	3

As shown in the Table 2 , we detected co-infections in 18 patients. We detected co-infection with Kp (Klebsiella.peneumoniae) in 6 patients, co-infection with EC (E. coli) in 5 patients, co-infection with Mcat (Moraxella catarrhalis) in 4 patients, co-infection with Hi (Haemophilus influenzae) in 4 patients, co-infection with Ab (Acinetobacter baumannii) in 2 patients, co-infection with Sa (Staphylococcus aureus) in 2 patients, co-infection with PA (Pseudomonas aeruginosa) in 1 patient, and co-infection with GAS (Group A Streptococcus) in 1 patients. Notably, 6 patients got coinfection with more than two bacteria. One patient with a moderate to severe disease and one patient with severe disease got co-infection with Mcat (Moraxella catarrhalis). One patient with a critical disease got co-infection with Ab (Acinetobacter baumannii). We didn't detect co-infection with any virus in these patients.

Table 2

Co-infection in the COVID-19 patients.

Patients	Pathogen	Age	Ct Value	Disease Severity
277#	PA, Pseudomonas aeruginosa	15	35.47	Mild
726#	Mcat, Moraxella catarrhalis	76	35.14	Moderate to Severe
144#	Kp, Klebsiella.peneumoniae	32	32.87	Mild
973#	Hi, Haemophilus influenzae	57	30.52	Moderate
994#	Mcat, Moraxella catarrhalis	35	24.16	Mild
	GAS, Group A Streptococcus		32.46
903#	Hi, Haemophilus influenza	21	26.55	Mild
	Kp, Klebsiella.peneumoniae		35.82
830#	Ab, Acinetobacter baumannii	65	30.17	Critical
14#	Mcat, Moraxella catarrhalis	37	34.49	Mild
	Hi, Haemophilus influenza		34.01
	Kp, Klebsiella.peneumoniae		26.19
743#	Hi, Haemophilus influenza	36	35.13	Mild
	Ab, Acinetobacter baumannii		32.11
952#	Sa, Staphylococcus aureus	33	31.94	Moderate
	EC, E. coli		35.77
976#	Kp, Klebsiella.peneumoniae	32	35.42	Mild
75#	EC, E. coli	45	26.40	Mild
1002#	Sa, Staphylococcus aureus	19	34.68	Moderate
225#	Mcat, Moraxella catarrhalis	67	28.62	Severe
63#	Kp, Klebsiella.peneumoniae	34	32.02	Mild
	EC, E. coli		27.38
67#	EC, E. coli	27	23.01	Mild
74#	EC, E. coli	66	28.43	Mild
327#	Kp, Klebsiella.peneumoniae	64	33.97	Mild

M. catarrhalis typically infect adults with a weakened immune system. Elderly COVID-19 patients have impaired Cytotoxic CD8+ T Cell Responses, which may make them highly risk for infection. A. baumannii is a common pathogen in Intensive Care Units (ICU) and evolves rapidly to be resistant to many antibiotics and should be seriously considered for critical COVID-19 patients.

In summary, we carried out an extensive pathogen screening in a COVID-19 cohort. We didn't detect co-infection of SARS-CoV-2 with other viruses. Co-infection with bacteria was detected in 18 of the 89 patients. M. catarrhalis was detected in two patients with severe symptoms and A. baumannii was detected in one patient with critical symptoms. These bacterial co-infections should be taken care in managing the COVID-19 patients.

Ethics statements

This study was approved by the ethics committee of Shanghai public health clinical center under the study number YJ-2020-S077-02, and the procedures were carried out in accordance with approved guidelines. Informed consent was obtained from the subjects.

Authors’ contributions

YZ conceived the manuscript; SW and JX performed experiments; ZX, LY collected samples; YZ all authors performed litera- ture search; YZ wrote the first draft of the manuscript; all authors reviewed it. SW and JX contributed equally.

Declaration of Competing Interest

The authors declare no conflicts of interest.