Table 2.

General characteristics of included studies that verified the virucidal effect of CHX against Influenza A, Human coronavirus (HCoV) and Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) strains in this review (n = 18)

Author (year) Country	CHX concentration; how CHX was administered? Time of contact	Control group (concentration); how product(s) was (were) administered? Time of contact	Virus assessed (origin)	Main results
Ansaldi 2004 [33] Italy	1%	Negative control: contaminated cells did not receive disinfectant solutions	Influenza A and HCoV	Infectivity, detected by inoculation of samples in suitable cell culture; genome integrity, detected by nested RRT‐PCR for SARS-CoV and multiplex nested RRT‐PCR
	CHX in direct contact with contaminated cells
			NR	Influenza virus and SARS-CoV RNA are still detectable after 30’ of contact time with 1% CHX
		NR		Last contact time resulting positive by cell culture and PCR for CHX: Influenza (culture): 30’ Influenza (PCR): 30’ SARS-CoV (culture): negative after 30’ SARS-CoV (PCR: 30’ 1% CHX in 2 ml cell culture medium did not significantly damage the cell monolayer
	30”, 1’, 2’, 5’, 15’, and 30’
Bernstein 1990 [31] United States of America	0.12%	Negative control substance: similar to CHX, but without the active ingredient. The substance was in direct contact with contaminated cells	Influenza A	Virus titers in plaque-forming units, PFU/ml. Values are presented, respectively, for CHX and control groups:
			Strain/Bethesda 1/85
	CHX in direct contact with contaminated cells
				CHX demonstrated an effective virucidal effect against Influenza A after 30' of contact
				30’: 93% and 0% 5’: > 98% and 1% 15’: > 98% and 1%
	30″, 5’, and 15’	30″, 5’, and 15’
Davies et al. 2021 [40] United Kingdom	Product 1—CHX 0.2%—formulation contains ethanol; Product 1—CHX 0.2%—alcohol-free formulation	HP (1.5%) PVP-I (0.58%) Essential oils mouthrinse (Listerine total care)	SARS-CoV-2	Log10 reduction of virus titer calculated as log10 TCID50 of control—log10 TCID50 of CHX
			England 2 strain
	CHX in direct contact with contaminated cells	Antiseptic agents in direct contact with contaminated cells		TCF unconcentrated—mean (95% CI)
	1’	1’		Product 1—CHX (0.2%) formulation ethanol: 0.5 (0.1–0.9) Product 2—CHX (0.2%) alcohol-free formulation: 0.2 (-0.2–0.7) HP (1.5%): 0.2 (-0.1–0.5) PVP-I (0.58%): ≥ 4.1 (3.8–4.4) Essential oils mouthrinse (Listerine total care): ≥ 4.1 (3.8–4.4)
Eduardo et al. 2021 [41] Brazil	0.12%	Placebo—distilled water; CPC (0.075%) + zinc (Zn) lactate (0.28%) HP (1.5%)	SARS-CoV-2	RRT‐PCR cycle threshold (Ct). RRT‐PCR assessed before rising, immediately after, 30'' and 60'' after. Mean Ct value for each experimental group was compared to the baseline value. Comparisons among groups were also performed
			Saliva from contaminated patients
	CHX in direct contact with contaminated saliva (mouthwash)
		Antiseptic agents in direct contact with contaminated saliva (mouthwash)		CHX: significantly lower viral load when compared to baseline after 30'' and 60''
	30’			CPC + Zn: significantly lower viral load when compared to baseline and immediately after rising
				HP: significantly lower viral load when compared to baseline and immediately after, 30'', and 60''
				CPC + Zn and HP promoted higher reductions of viral load when compared to CHX
		Placebo: 1’’ CPC + Zn: 30’ HP: 1’’
Elzein et al. 2021 [42] Lebanon	0.2%	Placebo—distilled water PVP-I (1%)	SARS-CoV-2	Saliva samples were collected before and 5 min after rinsing. Subsequently, SARS-CoV-2 RRT‐PCR was performed. The change in cycle threshold (delta Ct) values of salivary SARS-CoV-2 were calculated
	CHX in direct contact with contaminated saliva (mouthwash)	Antiseptic agents in direct contact with contaminated saliva (mouthwash)	Saliva from contaminated patients
				CHX: significantly lower viral load was detected in comparison to baseline. No significant difference was found between the delta Ct of patients using CHX and PVP-I solutions
				PVP-I (1%): significantly lower viral load was detected in comparison to baseline
	30’	30’
Geller 2009 [34] France	0.05%	Negative control: contaminated cells did not receive disinfectant solutions	HCoV	Infected wells were counted and viral titers or 50% cell culture infective dose (TCID50) were estimated:
	CHX in direct contact with contaminated cells
			VR 70—American Type Culture Collection
				CHX showed a moderate anti-HCoV 229E activity but insufficient to be antiseptic Log10 reduction of viral titers: 5’: 0.8 ± 0.7 (10–4 mol/L) and 1.4 ± 0.8 (10–3 mol/L); 15’: 0.5 ± 0.4 (10–4 mol/L) and 2.1 ± 0.4 (10–3 mol/L); 30’: 1.4 ± 1.5 (10–4 mol/L) and 2.4 ± 0.6 (10–3 mol/L); 60’: 2.1 ± 1.2 (10–4 mol/L) and 3.0 ± 0.2 (10–3 mol/L)
	5’, 15’, 30’ and 60’	5’, 15’, 30’ and 60’
Hirose et al. 2021 [47] Japan	0.2% and 1.0%	Negative control: contaminated cells did not receive disinfectant solutions	Influenza A and SARS-CoV-2	The measurement limits of the titers of Influenza A and SARS-CoV-2 were 101 focus-forming units/ml and 100.5 50% tissue culture infectious dose (TCID50)/ml, respectively
	CHX in direct contact with contaminated cells
			NR	Influenza A: CHX presented low virucidal efficacy in both in vitro and on skin models SARS-CoV-2: CHX presented low virucidal efficacy in both in vitro and on skin models The disinfection efficacy of CHX in SARS-CoV-2 was slightly greater than that in Influenza A
		NA
	5’, 15’ and 60’
Huang et al. 2021 [43] United States of America	0.12%	Negative control: contaminated patients did not receive disinfectant solutions. Two negative control groups were involved: (1 − n = 55) and (2 − n = 80)	SARS-CoV-2	After 4 days of CHX administration, the oropharynx was swabbed and tested for the presence of SARS‐CoV‐2 by rRT‐PCR
			Saliva from contaminated patients
	CHX in direct contact with contaminated saliva (1) Only mouthwash (n = 66); (2) Mouthwash and 1.5 mL of CHX spray (n = 93)
				(1) Most individuals in the group that received CHX intervention protocol were considered negative for SARS-CoV-2 in the oropharynx (62.1%; n = 41). In the control group, only 3 (5.5%) were negative for SARS-CoV-2 in the oropharynx (p < 0.01) (2) Among individuals who used CHX rinse, 80 (86.0%) of patients were negative for the presence of SARS-CoV-2, while 5 (6.2%) of the control patients were negative for the virus (p < 0.01)
		NA
	30’ twice a day for 4 days
Imai et al. 2021 [36] Japan	0.1% and 0.5%	Negative control: contaminated cells did not receive disinfectant solutions	HCoV	Viral titers (log10 TCID50/ml) were measured by quantal tests of six wells per dilution
	CHX in direct contact with contaminated cells		VR-1558—American Type Culture Collection
				CHX (0.5%) was ineffective against two coronavirus strains
		NA		The virucidal efficacy of CHX against HCoV through the suspension test [mean log10 reduction (95% CI)] after 15', 30' and 60 were, respectively, 0.60 (0.34), 0.75 (0.35), and 0.75 (0.38)
	15’, 30’, and 60’
Jain et al. 2021 [44] India	0.2% and 0.12%	PVP-I (1%)	SARS-CoV-2	Analysis of the virus inactivation was based on the quantification of viral RNA (Cycle threshold [Ct]) present in the culture supernatant using rRT‐PCR
	CHX in direct contact with contaminated cells	Antiseptic agents in direct contact with contaminated cells	NR
				CHX and PVP-I were able to inactivate SARS-CoV-2. 0.2% CHX performed better than the other substances
				30’; 60’ = relative Ct change log10 reduction (standard deviation) CHX (0.2%): 12.5 (0.5); 13 (0) CHX (0.12%): 10.5 (0.5); 11 (1.0) PVP-I: 9.5 (0.5); 11 (2)
		30’ and 60’
	30’ and 60’
Kawana 1997 [32] Japan	0.05%, 0.1%, 0.5%	Povidone-iodine (solution: 0.05%, 0.1%, 0.5%, 1% and 5%; gargle: 0.07%, 0.1%, 0.5%, 1%; cream 0.05%)	Influenza A	Reduction in virus titer after treatment:
			A/Kitakyushu/159/93	CHX: effective virucidal effect for Influenza A PVP-I solution and PVP-I gargle: virucidal effect clearly effective for Influenza A PVP-I cream: not performed
	CHX in direct contact with contaminated cells
		NR
	NR
Komine et al. 2021 [45] Japan	0.12%	Negative control: contaminated cells did not receive disinfectant solutions CPC toothpaste (0.05%) CPC mouthwash (0.05%): CPC spray (0.3%): CPC mouthwash (0.075%): CPC mouthwash (0.04%)	SARS-CoV-2	Log10 reduction of virus titer (PFU/ml)
	CHX in direct contact with contaminated cells	Antiseptic agents in direct contact with contaminated cells	JPN/TY/WK-521 strain—National Institute of Infectious Disease	CHX (0.12%) did not show a sufficient inactivation effect against SARS-CoV-2; inactivation effectiveness = 42.5%
	30’	Negative control: 20’, 30’ and 3’’ Toothpaste: 3’’ CPC: 20’		CPC toothpaste [0.05% (1/4 slurry: 0.0125)]: 99.94% CPC mouthwash (0.05%): 99.994% CPC spray (0.3%): > 99.96%; CPC mouthwash (0.075%): > 99.995% CPC mouthwash (0.04%): > 99.996%
Meister 2020 [37] Germany	(1) Product B—0.2% (2) Product D—0.2%	Hydrogen peroxide (1.5%) Polyvidone-iodine (1%) Essential oils	SARS-CoV-2	Viral titers were determined upon titration on Vero cells: virucidal activities could be observed with log reduction factors ranging between 0.3 and 1.78
			UKEssen strain
				Although CHX demonstrated mild virucidal activities, it did not significantly reduce the viral infectivity of the three strains of SARS-CoV-2
	CHX in direct contact with contaminated cells	Antiseptic agents in direct contact with contaminated cells
				Active compound: strain 1/strain 2/strain 3 (1) CHX 0.2%: 1.00/0.78/1.17 (2) CHX 0.2%: 0.50/0.56/0.50
	30”	30”
				Hydrogen peroxide: 0.78/0.68/0.73
				Polyvidone-iodine: ≥ 3.11/ ≥ 2.78/ ≥ 2.61
				Essential oils: ≥ 3.11/ ≥ 2.78/ ≥ 2.61
Okunishi 2009 [30] Japan	0.5% and 0.1%	Negative control: contaminated cells did not receive disinfectant solutions	Influenza A	Log10 reduction of virus titer calculated as log10 TCID50 of control—log10 TCID50 of CHX
	CHX in direct contact with contaminated cells		NR	CHX showed virucidal efficacy against Influenza A 0.5% and 0.1% CHX failed to show virucidal efficacy against Influenza A at any exposure time lower than 5 min
				CHX decreased only 45.1% of Influenza A titers after 10 min of exposure
	15”, 30” and 60”
Sattar 1989 [35] Canada	0.008% and 0.08%	Povidone-iodine (10—1% iodine)	HCoV	The criterion of efficacy (PFU/disk) for the disinfectants was ≥ 3 log10 reduction in the number of infectious virus units (reduction of virus titer by > 99.9%):
	CHX applied in the surface test (stainless steel disks)	Quaternary ammonium (Dimethyl benzyl ammonium chloride)—50% C14, 40% C12, 10% C16—0.04%) applied in the surface test (stainless steel disks)	NR
				CHX was ineffective in reducing viral replication CHX: no Povidone-iodine: yes Quaternary ammonium: no
	1’
		1’
Seneviratne 2020 [39] China	0.02%	Povidone-iodine (0.5%) Cetylpyridinium chloride (0.075%)	SARS-CoV-2	RRT‐PCR cycle threshold (Ct)
	CHX in direct contact with contaminated saliva		UKEssen strain
				The relative change in the doubling of the cycle limit values in patients in the mouthwash and water group demonstrated no significant difference in viral reduction in the CHX group than the others (p > 0.05)
		Antiseptic agents in direct contact with contaminated saliva
	5’, 120’, and 240’	5’, 120’, and 240’
Steinhauer et al. 2021 [46] Germany	0.1% and 0.2%	Negative control: contaminated cells did not receive disinfectant solutions	SARS-CoV-2	Viral titers were determined upon limited end-point titration on Vero E6 cells. Tissue culture infectious dose 50% (TCID50/ml) was calculated—Log10 reduction of virus titer
	CHX in direct contact with contaminated cells
			NR
				Both formulations based on CHX were found to have limited efficacy against SARS-CoV-2
		NA		CHX 0.1%: reduced the virus titer even at a prolonged contact time of 10 min by < 1 log10
	CHX 0.1%: 5’’ and 10’’ CHX 0.2%: 1’’ and 5’’
				CHX 0.2%: reduced SARS-CoV-2 within a contact time of 1 min as well as at a prolonged contact time of 5 min when tested by < 1 log10
Xu 2020 [38] United States of America	0.12%	HP (1.5%) PVP-I (1%) Essential oils mouthrinse	SARS-CoV-2	Plaque assays determined virus titers
	CHX in direct contact with contaminated saliva
	20’
		Antiseptic agents in direct contact with contaminated cells	USA_WA1/2020	Viruses that came into contact with CHX (50%) completely lost their infectivity. Treatment with 5% essential oils or CHX had a moderate antiviral effect; Hydrogen peroxide and povidone-iodine had greater inhibitory effects on viruses than CHX or essential oils. In general, the effect of CHX on the viruses after removal of the mouth rinse during the infection, 5% (v/v) CHX had only a moderate effect, reducing infection by 35–55%
		20’

NA not applicable, NR not reported, PVP-I povidone-iodine, HP hydrogen peroxide, CPC cetylpyridinium chloride