International Journal of

Environmental Research
and Public Health

Article
Efficacy of Three Commercial Disinfectants in Reducing
Microbial Surfaces’ Contaminations of Pharmaceuticals
Hospital Facilities
Giuseppina Di Martino 1 , Salvatore Pasqua 2 , Bruno Douradinha 1,3 , Francesco Monaco 1 , Chiara Di Bartolo 1,2 ,
Pier Giulio Conaldi 1 and Danilo D’Apolito 1,2, *

1 Unità di Medicina di Laboratorio e Biotecnologie Avanzate, IRCCS-ISMETT (Istituto Mediterraneo per i

Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90127 Palermo, Italy;
gdimartino@ismett.edu (G.D.M.); bdouradinha@fondazionerimed.com (B.D.); fmonaco@ismett.edu (F.M.);
cdibartolo@ismett.edu (C.D.B.); pgconaldi@ismett.edu (P.G.C.)
2 Unità Prodotti Cellulari (GMP), Fondazione Ri.MED c/o IRCCS-ISMETT, Via E. Tricomi 5, 90127 Palermo, Italy;
spasqua@fondazionerimed.com
3 Unità Medicina Rigenerativa ed Immunoterapia, Fondazione Ri.MED c/o IRCCS-ISMETT, Via E. Tricomi 5,
90127 Palermo, Italy
* Correspondence: ddapolito@fondazionerimed.com or danilo.dapolito@gmail.com; Tel.: +39-091-2192472

Abstract: To evaluate and validate the efficacy of disinfectants used in our cleaning procedure, in
order to reduce pharmaceutical hospital surfaces’ contaminations, we tested the action of three
commercial disinfectants on small representative samples of the surfaces present in our hospital
cleanrooms. These samples (or coupons) were contaminated with selected microorganisms for the





 validation of the disinfectants. The coupons were sampled before and after disinfection and the
microbial load was assessed to calculate the Log10 reduction index. Subsequently, we developed and
Citation: Di Martino, G.; Pasqua, S.;
validated a disinfection procedure on real surfaces inside the cleanrooms intentionally contaminated
Douradinha, B.; Monaco, F.; Di
Bartolo, C.; Conaldi, P.G.; D’Apolito,
with microorganisms, using approximately 107 –108 total colony forming units per coupon. Our
D. Efficacy of Three Commercial results showed a bactericidal, fungicidal, and sporicidal efficacy coherent to the acceptance criteria
Disinfectants in Reducing Microbial suggested by United States Pharmacopeia 35 <1072>. The correct implementation of our cleaning
Surfaces’ Contaminations of and disinfection procedure, respecting stipulated concentrations and contact times, led to a reduction
Pharmaceuticals Hospital Facilities. of at least 6 Log10 for all microorganisms used. The proposed disinfection procedure reduced the
Int. J. Environ. Res. Public Health 2021, pharmaceutical hospital surfaces’ contaminations, limited the propagation of microorganisms in
18, 779. https://doi.org/10.3390/ points adjacent to the disinfected area, and ensured high disinfection and safety levels for operators,
ijerph18020779 patients, and treated surfaces.

Received: 13 November 2020

Keywords: efficacy; disinfectant; surface contaminations; disinfection procedure; validation
Accepted: 15 January 2021
Published: 18 January 2021

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in
published maps and institutional affil- Microbial contamination of hospital surfaces is a fundamental aspect which requires
iations. constant monitoring and control, in order to minimize the transmission of microorganisms
from surfaces to patients, to reduce both morbidity and mortality due to nosocomial
infections [1–3].
The emergence of multiple resistant pathogens to several antibiotics has increased the
Copyright: © 2021 by the authors.
need for effective disinfection. This is especially true in cleanrooms where pharmaceuticals
Licensee MDPI, Basel, Switzerland.
or advanced therapy medicinal products (ATMPs) are prepared. The design, validation
This article is an open access article
and implementation of documented and approved disinfection programs are fundamental
distributed under the terms and for the maintenance of critical areas. The disinfection protocols must be effective and safe
conditions of the Creative Commons for staff, patients, and the surfaces to be cleaned [4].
Attribution (CC BY) license (https:// A disinfectant can be defined as a chemical which reduces the number of microor-
creativecommons.org/licenses/by/ ganisms present on a surface. Disinfectants vary in their spectrum of activity, mode of
4.0/). action, and efficacy. The disinfectants currently in use are categorized as non-oxidizing and

Int. J. Environ. Res. Public Health 2021, 18, 779. https://doi.org/10.3390/ijerph18020779 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2021, 18, 779 2 of 11

oxidizing agents. The former includes alcohols, aldehydes, amphoteric compounds, phe-
nolics, and quaternary ammonium compounds, while the latter includes oxygen-releasing
compounds such as peracetic acid and hydrogen peroxide [5,6].
The first step of an efficient disinfection program is the choice of disinfectants that
guarantee bactericidal, fungicidal, and sporicidal actions, as previously demonstrated
by the manufacturer in compliance with United States Pharmacopoeia (USP) 35 Chap-
ter <1072> [7]. For our disinfection procedures, we chose 6% hydrogen peroxide, quater-
nary ammonium, and 70% isopropanol based commercial disinfectants. We developed
an in-house quantitative analytical disinfection method to test their efficacy to eliminate
different microorganisms spread over multiple types of surfaces in cleanrooms. We also
considered the feasibility and the costs associated with sampling. Over the last decades,
protocols for the recovery of microorganisms from surfaces often led to variable results due
to differences between sampling tools, individual operators, low microbial recovery rates,
and low levels of reproducibility [8]. To minimize the experimental variability between the
disinfectant efficacy test done in the microbiological laboratory and the one performed for
validation of the disinfection procedure in the controlled areas, we performed the recovery
of microorganisms as recommended for environmental sampling of flat surfaces. Therefore,
we chose to do the surface coupon testing with minor modifications, i.e., avoiding the
use of swabs and creating a support for the coupons. This allows the recovery of the
microorganisms and the replication of potential contaminations present in the coupons
directly into the contact plates containing the disinfectants’ inactivating agents.
Using our method, we were able to detect all tested suspensions of microorganisms
present on the coupons’ surfaces and to evaluate the activity of the chosen disinfectants,
demonstrating the efficacy of the latter to efficiently eliminate the microorganisms, as
required by USP 35 <1072> [7], which specify that disinfectants must have a bactericidal,
fungicidal, and sporicidal activity, showing a Log10 reduction of three for bacteria and
fungi and of two for spores. To demonstrate the effects induced by several environmental
factors, like temperature, pH, detergent residues, mechanical stress, and surface attachment
in the facility, we validated a disinfection procedure in situ, using the microbial species
required by USP 35 <1072> [7] and three additional potential contaminants of our Good
Manufacturing Practices (GMP) facility [9].
Our results showed that the proposed in-house analytical method was efficient for
confirming the tested disinfectants’ efficacy. Moreover, we observed that all chosen dis-
infectants used for the validation of our cleaning procedure were suitable for surface
disinfection, since they reduced and controlled the spread of contamination in surfaces
representative of hospital sensitive areas.

2. Materials and Methods

2.1. Microbial Species Required by USP 35 <1072> and In-House GMP Facility Strains
American Type Culture Collection strains (ATCC; Table 1) were purchased from Mi-
crobiologics (St. Cloud, MN, USA) and supplied as quantitative microorganism lyophilized
pellets. For each strain, a microbial suspension was prepared according to the manufac-
turer’s instructions to obtain a mother suspension with a concentration of 107 ≤ colony
forming units (CFU)/mL < 108 .
Table 1. Microbial strains.

Reference Strains In-House GMP Facility Strains

Candida albicans ATCC 10231 Kocuria rosea
Aspergillus brasiliensis ATCC 16404 Staphyloccus epidermidis
Staphylococcus aureus ATCC 6538 Micrococcus luteus
Pseudomonas aeruginosa ATCC 15442
Escherichia coli ATCC 8739
Bacillus subtilis ATCC 6633
Bacillus subtilis spore ATCC 6633
Int. J. Environ. Res. Public Health 2021, 18, 779 3 of 11

In-house GMP Facility strains (Table 1), isolated from previous environmental mon-
itoring in our facility, were utilized with not more than five passages from the original
culture [9] and grown in tryptic soy broth (TSB; purchased from BD, Franklin Lakes,
NJ, USA) until they reached the early exponential phase. The culture concentration was
adjusted to 0.5 based on the McFarland scale (around 108 CFU/mL).

2.2. Microorganism Suspension Preparation and Titer Determination

Seven serial decimal dilutions from the mother dilution were prepared in peptone
water (BD). To assess viability, purity, and titer determination, 100 µL of each dilution were
plated on tryptic soy agar (TSA, for bacterial suspensions) and Sabouraud dextrose agar
(SDA, for fungal suspensions) plates containing lecithin and polysorbate 80 (both from BD)
and incubated at 37 ◦ C. The CFUs were counted after 2–3 days (bacteria) and 4–5 days
(yeast and mold). The titer was determined by calculating the weighted arithmetic average
of the CFU number for each dilution.

2.3. Disinfectants
The disinfectants used in this study were all ready-to-use Steri-perox 6% (6% hydrogen
peroxide formulated with 94.0% USP water for injection, purchased from Veltek Associates
Inc., Malvern, PA, USA), Dec-quat 100 (based in quaternary ammoniums alkyl dimethyl
benzyl ammonium chloride and alkyl dimethyl ethyl benzyl ammonium chloride; Veltek
Associates Inc.), and Dec-ahol (based in 70% isopropyl alcohol formulated with 30% USP
water for injection; Veltek Associates Inc.). All 3 products were tested for bactericidal and
fungicidal activity. Steri-perox 6% was also tested for sporicidal activity.

2.4. Preparation of Coupons

The surfaces present in the pharmaceutical cleanrooms were the following: glass, AISI
304 steel, PVC, melamine, and compact polycarbonate. For each material type, coupons
were prepared with dimensions of 6 × 6 cm. Each square was glued using a 3M adhesive
over the lid of a 50 mL tube. The coupons and tubes were sterilized in a tyvec bag (Johnson
& Johnson, New Brunswick, NJ, USA) in a STERRAD sterilizer, using hydrogen peroxide
gas plasma for 54 min at 45 ◦ C. The sterilization efficiency was evaluated by the color
change of the indicator strips present in the tyvec bags and the biological indicators “Sterrad
Cyclesure” (Johnson & Johnson), and incubated for 24 h at 37 ± 2 ◦ C.

2.5. Analytical Method and Experimental Protocol to Evaluate Disinfectants’ Efficacy

The first scope of our study was to evaluate the efficiency of the 3 types of disinfectants
mentioned above by measuring their action against the microorganisms suggested by USP
35 <1072> [7] spread on the coupons. The experimental protocol involved testing the action
of each disinfectant on 6 × 6 cm coupons contaminated with titrated microbial suspensions.
The experimental plan (Figure 1) involved three phases to be performed in parallel using
the same microbial suspensions.
The recovery of microorganisms was performed using TSA or SDA plates containing
lecithin and polysorbate 80 (BD) for bacteria/spores and fungi, respectively. The use of
lecithin and polysorbate 80 prevented the sanitizer’s carryover inhibition [10]. The first
phase regards the evaluation of the viability, purity, and titer of microbial suspensions.
One hundred microliters of each microbial suspension were plated in triplicate, and for
each microorganism, the titer of the mother suspension was determined (N). In the second
phase, the number of microorganisms that survived the action of the disinfectant on the
coupon surface (Na ) was determined by applying, in triplicate, one hundred microliters of
each microbial suspension on the coupons with a spreader. After drying, the disinfectant
was applied without mechanical removal (considered the worst case scenario) and allowed
to act for 10 min, as suggested by the manufacturer. After drying, sampling of the coupon
surface was done by direct replication on plates, to asses Na . In the third phase, the number
of vital microorganisms dispensed on the coupon surface (Nv ) was quantified by spreading,
Int. J. Environ. Res. Public Health 2021, 18, 779 4 of 11

in triplicate, one hundred microliters of each microbial suspension on the coupons. After
drying, sampling of the coupon surface was performed by direct replication on plates.

Figure 1. Scheme of the experimental plan used to evaluate disinfectants’ efficacy. In phase 1, 100 µL of each microbial
suspension was plated in triplicate, and for each microorganism, the titer of the mother suspension was determined (N).
In phase 2, 100 µL of each microbial suspension was applied, in triplicate, on the coupon surface with a spreader. After
drying, the disinfectant was applied without mechanical removal (worst case scenario). After 10 min of action, sampling of
the coupon surface was performed by direct replication on plates to determine the number of vital microorganisms that
survived the action of the disinfectant (Na ). In phase 3, 100 µL of each microbial suspension was applied, in triplicate, on
the coupon surface with a spreader. After drying, sampling of the coupon surface was performed by direct replication on
plates to determine the number of vital microorganisms dispensed on the coupon surface (Nv ).

2.6. Log10 Reduction Index and Acceptance Criteria

The efficacy of the disinfection action was determined by calculating the Log10 reduc-
tion index. Log10 reduction = Log10 (Nv(Msusp) /Na(Msusp) ).
The formulas applied are the following:
- Na(Msusp) = Na × DF × V
- Nv(Msusp) = Nv × DF × V
in which DF is the dilution factor of the first dilution, taken into consideration for
the calculation of the weighted average; and V is the suspension volume inoculated on a
plate (100 µL). Subsequently the Log10 reduction index was calculated as stated above. The
acceptance criteria, in accordance with USP 35 <1072> [7] and also declared by the disin-
fectants’ manufacturer, were a Log10 reduction ≥3, in the case of bactericidal/fungicidal
activity; and a Log10 reduction ≥ 2, in the case of sporicidal activity.

2.7. Microorganisms Identification

For each step of the experiments, the purity of each microorganism was determined by
microscope observation and gram staining, and species identification was performed using
Vitek MS (Biomerieux, Marcy-l’Étoile, France), according to manufacturer’s instructions.
Int. J. Environ. Res. Public Health 2021, 18, 779 5 of 11

2.8. Robustness
An analytic method is considered robust when it is not influenced by small but inten-
tional variations in method parameters or usual conditions, e.g., different days, materials,
or users, and gives an indication of its reliability during normal use. To evaluate the
robustness of our disinfection procedure, the experiments described below were performed
3 times in 3 different days by 3 different operators.

2.9. Cleaning and Disinfection Standard Operating Procedure

Subsequently, the study included the design of the standard operating procedure
(SOP), the theoretical and practical training of the personnel, and the final validation of
the SOP. The aspects taken into consideration to write the SOP were the materials to be
used, the method of application of disinfectants, and their rotation, to avoid the onset of
potential resistance. The experimental protocol includes intentional contamination of the
surfaces present in controlled areas, with approximately 107 –108 CFU (worst case scenario:
heavy contamination of surfaces) of reference microorganisms and in-house GMP facility
strains (Table 1). For our study, we also used Kocuria rosea, Staphylococcus epidermidis, and
Micrococcus luteus, which are typical of cleanrooms’ microbiota [11]. The surfaces were
subjected to sampling to evaluate the efficacy, reproducibility, and robustness of our cleaning
and disinfection procedure. Accordingly, we decided to perform a weekly rotation program
of Steri-perox 6% and Dec-quat 100. Furthermore, for all steel or glass surfaces and for all
those on which the pharmaceutical product is prepared, an extra final step of disinfection
with Dec-ahol was done, in order to remove any traces of the first disinfectant, thus ensuring
a higher level of disinfection and greater safety for patients and surfaces. The disinfectants
were applied on the surface using polyester sterile wipes. The steps to follow were:
(1) Saturation of the wipes by immersion in the first disinfectant solution
(2) Squeezing the wipes to remove excess liquid;
(3) Passing the wipe over the surface, proceeding from top to bottom and from left to
right, overlapping the passes and making sure that there were no areas where the
wipe has not been passed;
(4) Contact time of action of disinfectant was 10 min;
(5) For the surfaces described above, repetition of the first 4 points with the Dec-ahol.

2.10. Validation of Cleaning and Disinfection Procedure

At the beginning of validation procedure, the rooms were initially cleaned and sub-
jected to sterilization using vapor phase hydrogen peroxide (VPHP) by a specialized
company (Belsar Srl, Tradate, Italy) thoroughly, to eliminate any potential contaminations.
The coupons that were contaminated were representative of the surfaces. For each type
of material, we selected 3 areas of 4 × 4 cm. These dimensions allowed us to directly
sample the entire surface with the Replicate Organism Detection and Counting (RODAC)
contact plates (BD), specifically for environmental monitoring. Each of these areas were
contaminated with approximately 107 –108 CFU of the microorganisms detailed in Table 1.
The procedure was performed blindly to all surfaces, e.g., one operator contaminated
specific surface points, while another would clean the surfaces homogeneously without
knowing which areas had been contaminated. Subsequently, the trained personnel did
the environmental sampling using TSA or SDA RODAC contact plates, containing lecithin
and polysorbate 80. In addition, to confirm that spreading of the microorganisms to the
areas adjacent to those contaminated did not occur, we also sampled the surrounding
area [12]. The number of total CFU that survived the cleaning procedure was determined
by calculating the arithmetic average of the values obtained in the 3 plates in the same
experiment. As before, the validation procedure was repeated 3 times by 3 different opera-
tors on three different days to confirm repeatability and robustness of the assay [13]. The
results obtained for the 3 experiments are expressed as the Log10 reduction of the average
value of the 9 experiments performed, for each microorganism. After validation, the rooms
were subjected to sterilization using VPHP.
Int. J. Environ. Res. Public Health 2021, 18, 779 6 of 11

3. Results
3.1. Microorganisms Identification and Purity
For each step of the experiments, a representative colony of the microorganisms recov-
ered from the sampled surfaces was subjected to identification by Vitek MS to confirm its
identity, purity, and correspondence to the original suspension inoculated on the coupons.
All samples were confirmed as the ones applied to the surface, and no cross-contaminations
were found (data not shown).

3.2. Disinfectants’ Efficacy and Log10 Reduction Index

Log10 reduction values were obtained by evaluating the disinfectants efficiency over the
reduction of microorganisms’ viability on each tested surface (Table 2). Aspergillus brasiliensis
was consistently the most resistant to both quaternary ammonium compound and iso-
propyl alcohol. Our results were consistent with the acceptance criteria required by
USP 35 <1072> [7].

Table 2. Log10 reduction index measured by disinfectants’ efficacy test method. The tests were performed using a contact
time of 10 min. Each test was done in triplicate on three different days. The Log10 reduction index was calculated. The
acceptance criteria, in accordance with USP 35 <1072>, were a Log10 reduction ≥ 3, in the case of bactericidal/fungicidal
activity; and a Log10 reduction ≥ 2, in the case of sporicidal activity.

6 Hydrogen
Microorganisms
Peroxide
Log10 ± St. Dev.
Surface
B. subtilis S. aureus Spores B. subtilis P. aeruginosa E. coli C. albicans A. brasiliensis
Glass 4.46 ± 0.01 4.52 ± 0.01 2.64 ± 0.08 4.59 ± 0.01 4.64 ± 0.01 4.47 ± 0.01 4.11 ± 0.01
AISI 307 Steel 4.49 ± 0.01 4.41 ± 0.01 2.66 ± 0.06 4.51 ± 0.01 4.58 ± 0.01 4.50 ± 0.01 4.09 ± 0.03
PVC 4.50 ± 0.01 4.44 ± 0.01 2.62 ± 0.07 4.66 ± 0.01 4.58 ± 0.01 4.50 ± 0.01 4.07 ± 0.03
Melamine 4.49 ± 0.01 4.44 ± 0.01 2.68 ± 0.05 4.58 ± 0.01 4.63 ± 0.01 4.49 ± 0.01 4.20 ± 0.02
Compact
4.47 ± 0.01 4.44 ± 0.01 2.64 ± 0.04 4.63 ± 0.01 4.59 ± 0.01 4.45 ± 0.01 4.18 ± 0.02
Policarbonate
Quaternary
Microorganisms
ammonium
Log10 ± St. Dev.
Surface
B. subtilis S. aureus Spores B. subtilis P. aeruginosa E. coli C. albicans A. brasiliensis
Glass 4.24 ± 0.02 4.38 ± 0.02 - 4.50 ± 0.01 4.50 ± 0.01 4.24 ± 0.02 3.87 ± 0.03
AISI 307 Steel 4.28 ± 0.02 4.36 ± 0.02 - 4.49 ± 0.00 4.48 ± 0.01 4.36 ± 0.02 3.90 ± 0.01
PVC 4.30 ± 0.02 4.41 ± 0.01 - 4.58 ± 0.01 4.49 ± 0.01 4.28 ± 0.02 3.97 ± 0.02
Melamine 4.38 ± 0.02 4.40 ± 0.02 - 4.46 ± 0.01 4.49 ± 0.01 4.34 ± 0.02 3.91 ± 0.04
Compact
4.23 ± 0.02 4.42 ± 0.00 - 4.60 ± 0.01 4.47 ± 0.01 4.36 ± 0.02 3.99 ± 0.04
Policarbonate
70 Isopropanol Microorganisms
Log10 ± St. Dev.
Surface
B. subtilis S. aureus Spores B. subtilis P. aeruginosa E. coli C. albicans A. brasiliensis
Glass 4.18 ± 0.02 4.26 ± 0.01 - 4.35 ± 0.02 4.31 ± 0.02 4.20 ± 0.02 3.84 ± 0.05
AISI 307 Steel 4.24 ± 0.02 4.33 ± 0.02 - 4.37 ± 0.02 4.36 ± 0.02 4.19 ± 0.02 3.82 ± 0.03
PVC 4.25 ± 0.01 4.30 ± 0.02 - 4.52 ± 0.01 4.35 ± 0.02 4.20 ± 0.01 3.92 ± 0.04
Melamine 4.36 ± 0.02 4.25 ± 0.02 - 4.40 ± 0.00 4.38 ± 0.02 4.25 ± 0.01 3.84 ± 0.05
Compact
4.19 ± 0.03 4.39 ± 0.01 - 4.49 ± 0.01 4.45 ± 0.01 4.21 ± 0.02 3.83 ± 0.05
Policarbonate
Sporicidal activity was determined only for 6% Hydrogen Peroxide.

3.3. Validation of Cleaning and Disinfection Procedure

Our cleaning and disinfection procedure was performed as described above. Environ-
mental sampling results, expressed as Log10 reduction indices, are summarized in Table 3.
Int. J. Environ. Res. Public Health 2021, 18, 779 7 of 11

These results showed that our disinfectant application procedure is able to routinely
eliminate the tested microorganisms on the materials present in the production area. Fur-
thermore, the use of Steri-perox 6% and Dec-ahol, or Dec-quat 100 and Dec-ahol, on the
critical surfaces where the pharmaceutical drugs are produced almost completely removed
the microorganisms present on the surfaces, including the three in-house GMP facility
strains, thus demonstrating the efficiency of our disinfection program. Additionally, the
sampling of points near to those of the validation showed the complete absence of mi-
croorganisms, confirming that the disinfectant procedure does not lead to microorganisms’
diffusion to the adjacent areas.

3.4. Robustness
During the validation period, the rooms were cleaned once daily. We obtained similar
and robust results for all tested conditions (Table 4), thus demonstrating that the proposed
cleaning procedure eliminates almost all surface contaminations without showing differences
that might have been induced by either different operators or different execution days.
The robustness was also confirmed by the trend analysis of environmental monitoring
data obtained over three years, which showed that our procedure mantains environmental
contamination below the limits set by GMP guidelines, even in extreme situations such as
extraordinary maintenance or restructuring, with no signs of corrosion on the treated surfaces.

4. Discussion
Nowadays, effective cleaning and disinfection procedures are required due to an
increase in the number of microorganisms’ strains which display resistance to multiple
antibiotics. Such procedures are important for cleanrooms designed to handle products
which will be in contact with patients, especially those under an immunosuppression
regimen. Contamination of medical environments with a range of pathogens may lead to
undesirable healthcare-associated infections. Several pathogens can survive on surfaces for
extended periods and could be disseminated in the hospital via healthcare workers who
have direct contact with infected patients and contaminated environmental surfaces [14].
The choice of sanitizing agents is fundamental and must take into consideration their abil-
ity to destroy microorganisms, user safety, application method, instrument compatibility,
minimization of the induction of bacterial resistance, and their ability to avoid corrosion of
materials subjected to disinfection and reduce associated costs [1,6,9,14]. The disinfectants
on the market are generally alcohols, chlorine, aldehydes, peroxygenes, and quaternary am-
monium compounds [6], and each of them have advantages and disadvantages. Alcohol is
cheap and highly inflammable, with a rapid bactericidal effect without bacteriostatic action
and relevant toxicity issues, but it is not sporicidal and has a poor efficacy to inactivate some
viruses. Chlorine-based disinfectants are widely used in hospital disinfection procedures
and show a vast bactericidal spectrum. However, they may cause corrosion of hospital
equipment and of metal materials, leading to additional costs for their maintenance and
eventual replacement. Moreover, such disinfectants can be easily inactivated by organic
matter, cause irritation and burning in several organs, such as skin, eyes, and mucous
membranes [6,15]. Hydrogen peroxide is a disinfectant with a good sporicidal action, and
due to its rapid action, it is desirable from an environmental point of view. In recent years,
the development of accelerated hydrogen peroxide (AHP) has made it possible to achieve
higher compatibility with materials, increasing its use for disinfection procedures. An
excellent compound with fast action at low concentrations towards all microorganisms
and spores is peracetic acid. It is not affected by the presence of organic substances and
does not leave residual matter, but it is unstable and has a corrosive effect on metals [16,17].
Together with chlorine-based compounds, quaternary ammonium-based disinfectants are
the most commonly used in hospital disinfection procedures. They have a broad spectrum
of action towards many microorganisms and sporostatic activity, but exhibit low efficacy
against gram-negative bacteria and non-enveloped viruses [15].
Int. J. Environ. Res. Public Health 2021, 18, 779 8 of 11

Table 3. Log10 reduction index of surface contaminations determined after the disinfection procedure. Prior to the disinfection procedure, surfaces were contaminated with 107 –108 colony
forming units (CFU)/mL. For each disinfectant, the contact time was 10 min.

6% Hydrogen
Microorganisms
peroxide IPA 70%
Log10 ± St. Dev.
Surface
B. subtilis S. aureus Spores B. subtilis P. aeruginosa E. coli C. albicans A. brasiliensis K. rosea S. epidermidis M. luteus
Glass 7.40 ± 0.16 7.40 ± 0.22 6.74 ± 0.09 7.40 ± 0.13 7.40 ± 0.16 7.40 ± 0.16 7.70 ± 0.26 7.22 ± 0.11 7.22 ± 0.12 7.40 ± 0.15
AISI 307 Steel 7.22 ± 0.19 7.70 ± 0.30 6.66 ± 0.08 7.40 ± 0.20 7.70 ± 0.32 7.70 ± 0.34 7.40 ± 0.13 7.40 ± 0.10 7.40 ± 0.15 7.22 ± 0.16
PVC 7.40 ± 0.27 7.40 ± 0.23 6.70 ± 0.08 7.70 ± 0.30 7.70 ± 0.23 7.70 ± 0.30 7.70 ± 0.30 7.22 ± 0.09 7.40 ± 0.18 7.40 ± 0.20
Melamine 7.22 ± 0.21 7.40 ± 0.20 6.66 ± 0.07 7.40 ± 0.26 7.70 ± 0.28 7.70 ± 0.20 7.70 ± 0.26 7.40 ± 0.10 7.22 ± 0.12 7.22 ± 0.16
Compact Policarbonate 7.70 ± 0.30 7.22 ± 0.21 6.66 ± 0.07 7.70 ± 0.23 7.70 ± 0.28 7.70 ± 0.30 7.40 ± 0.15 7.22 ± 0.12 7.10 ± 0.11 7.40 ± 0.15
Quaternary
Microorganisms
ammonium IPA 70%
Log10 ± St. Dev.
Surface
B. subtilis S. aureus Spores B. subtilis P. aeruginosa E. coli C. albicans A. brasiliensis K. rosea S. epidermidis M. luteus
Glass 6.80 ± 0.13 6.74 ± 0.14 6.52 ± 0.10 6.70 ± 0.16 6.70 ± 0.09 6.74 ± 0.11 6.74 ± 0.11 6.70 ± 0.10 6.70 ± 0.11 6.66 ± 0.08
AISI 307 Steel 6.66 ± 0.09 6.74 ± 0.12 6.66 ± 0.09 7.22 ± 0.16 6.74 ± 0.10 6.80 ± 0.16 6.74 ± 0.11 6.70 ± 0.09 6.66 ± 0.07 6.70 ± 0.09
PVC 6.80 ± 0.14 6.80 ± 0.16 6.49 ± 0.07 6.74 ± 0.07 6.80 ± 0.12 6.92 ± 0.17 6.80 ± 0.12 6.74 ± 0.12 6.74 ± 0.09 6.70 ± 0.08
Melamine 6.74 ± 0.12 6.80 ± 0.14 6.62 ± 0.11 6.74 ± 0.09 6.74 ± 0.11 6.74 ± 0.14 6.74 ± 0.09 6.74 ± 0.10 6.74 ± 0.08 6.70 ± 0.07
Compact Policarbonate 6.74 ± 0.09 6.74 ± 0.15 6.49 ± 0.11 6.74 ± 0.10 6.74 ± 0.11 6.80 ± 0.13 6.80 ± 0.09 6.70 ± 0.13 6.66 ± 0.07 6.70 ± 0.10
Int. J. Environ. Res. Public Health 2021, 18, 779 9 of 11

Table 4. Surface contamination determined after disinfection procedure. The tests were performed under practical
conditions. For each disinfectant, we used a contact time of 10 min. Each test was done by three operators in triplicate on
three surfaces. Each operator worked on different days, for a total of nine replicates. Results are presented as the arithmetic
mean of the CFU obtained in the nine samplings ± standard deviation.

6% Hydrogen
peroxide + Microorganisms a
IPA 70%
B. S. Spores B. P. aerugi- C. A. S. epider-
Surface E. coli K. rosea M. luteus
subtilis aureus subtilis nosa albicans brasiliensis midis
Glass 2 ± 0.9 2 ± 1.3 9 ± 2.2 2 ± 0.7 2 ± 0.9 2 ± 0.9 1 ± 0.8 3 ± 0.9 3±1 2 ± 0.8
AISI 307 Steel 3 ± 1.6 1±1 11 ± 2.1 2 ± 1.2 1 ± 1.1 1 ± 1.2 2 ± 0.7 2 ± 0.5 2 ± 0.8 3 ± 1.3
PVC 2 ± 1.7 2 ± 1.4 10 ± 1.9 1±1 1 ± 0.7 1±1 1±1 3 ± 0.7 2.4 ± 1 2 ± 1.2
Melamine 3 ± 1.9 2 ± 1.2 11 ± 1.9 2 ± 1.6 1 ± 0.9 1 ± 0.6 1 ± 0.8 2 ± 0.5 2.7 ± 1 3 ± 1.3
Compact
1±1 3 ± 1.9 11 ± 2 1 ± 0.7 1 ± 0.9 1±1 2 ± 0.8 3±1 4 ± 1.2 2 ± 0.8
Policarbonate
Quaternary
ammonium + Microorganisms a
IPA 70%
B. S. Spores B. P. aerugi- C. A. S. epider-
Surface E. coli K. rosea M. luteus
subtilis aureus subtilis nosa albicans brasiliensis midis
10 ± 10 ±
Glass 8 ± 2.7 9 ± 3.4 15 ± 4 10 ± 4.5 9 ± 2.5 9 ± 2.6 10 ± 2.8 11 ± 2.2
2.2 2.6
11 ± 10 ±
AISI 307 Steel 9 ± 2.9 11 ± 2.5 3 ± 1.3 9 ± 2.3 8 ± 3.5 9 ± 2.6 11 ± 2 10 ± 2.3
2.5 2.3
PVC 8 ± 3.1 8 ± 3.6 16 ± 3 9 ± 1.6 8 ± 2.6 6 ± 2.8 8 ± 2.5 9±3 9 ± 2.2 10 ± 2
Melamine 9±3 8±3 12 ± 3.5 9 ± 2.1 9 ± 2.6 9 ± 3.4 9 ± 2.2 9 ± 2.3 9 ± 1.8 10 ± 1.7
Compact 10 ±
9 ± 2.1 9 ± 3.7 16 ± 4.5 9 ± 2.4 8 ± 2.5 8 ± 2.9 8 ± 1.8 11 ± 2 10 ± 2.7
Policarbonate 3.5
a CFU mean ± standard deviation.

Disinfectants can be applied with or without mechanical action. The latter, for example,
spray disinfection, is an easy method but causes relatively high atmospheric concentrations
of some of the disinfectant components. The former option ensures removal of the vast
majority of organic debris or dirt present that could hinder the disinfecting action. The
following methods are based in mechanical application: “Spray and Wipe”, “Dip and
Wipe”, and “Soak and Wipe”. The “Spray and Wipe” method requires the disinfectant
to be sprayed directly onto the surface. However, there are several drawbacks, such as
possible over-spraying and the possibility of the disinfectant being inhaled by workers
and patients [18]. The “Dip and Wipe” method requires a dry wipe to be immersed in a
disinfectant solution for 5–10 s, squeezed to remove excess disinfectant, and used to evenly
distribute the liquid over the surface. It is important that the wipe is soaked in an adequate
manner to prevent it from losing its antimicrobial activity and becoming a potential vehicle
for the transmission of pathogens itself [19]. The “Soak and Wipe” method is similar to
“Dip and Wipe” and requires that the wipe remain immersed in a disinfectant solution
from 10 min to 8 h. The wipe is then used as described above. This method allows a contact
time of the wipe with the disinfectant to ensure sufficient wetting of the fabric before
use. However, it was reported that longer soaking led to a decrease in the antimicrobial
activity of the disinfectant [20]. A particular “Soak and Wipe” method uses ready-to-use
disinfecting wipes distributed by the manufacturer in sealed packs. They are timesaving
and ensure convenient implementation and practical and reliable performance. However,
their long shelf-life could increase the probability of loss of antimicrobial activity due to
the degradation of the disinfectant or interactions between the wipe material and active
ingredients, with a risk of increasing healthcare-associated infections [21].
Int. J. Environ. Res. Public Health 2021, 18, 779 10 of 11

In order to define a cleaning and disinfection procedure capable of keeping the micro-
bial surface contaminations under the limits set by the respective guidelines that was safe
for operators, patients, and equipment, and was also economically sustainable, we chose
to use three ready-to-use commercial disinfectants to be applied with the “Soak and Wipe”
method to limit potential harmful aerosols, reduction of preparation time, and distribute
the disinfectants evenly on all surfaces.
The efficacy of our method showed that for all three selected disinfectants, when
applied for 10 min on all tested surfaces, a Log10 reduction index was obtained that was
consistent with the acceptance criteria expressed in USP 35 <1072> and in agreement
with the technical specifications from the manufacturer. These results demonstrated the
bactericidal and fungicidal efficacy of the three commercial disinfectants chosen for the
disinfection of clean rooms. Moreover, Steri-perox 6% also showed sporicidal activity.
Furthermore, our protocol based on direct sampling, i.e., without the help of swabs,
allowed robust and reproducible results and was more cost-effective in respect to the
procedures that used alternative recovery methods [1,9]. These results were useful for
estimating the validity of the procedural disinfection parameters and for developing the
SOP for the disinfection of pharmaceutical surfaces.
The next aim of the work was to validate the disinfection procedure when applied
under practical conditions. Accordingly, we initially wrote a disinfection procedure with
the goal of maximizing disinfection performance and minimizing the presence or spread
of both microbial contaminations and resistance to the disinfectants. We have chosen to
rotate the disinfectants weekly and to apply them by the “Soak and dry” method. Our
disinfection protocol involved the sequential use of two different disinfectants, in particular,
Steri-perox 6% or Dec-quat 100, followed by Dec-ahol. The validation of the procedure,
through in situ studies done on surfaces contaminated with 107 –108 CFU (worst case
scenario) of the 10 microorganisms detailed in Table 1, have demonstrated strong reduction
in the surfaces’ contamination, with at least 6 Log10 reduction for all bacteria, fungi, and
spores used (Table 3) and no propagation of microorganisms to the points adjacent to the
disinfected area.
The results derived from our in situ study validated our disinfection procedure, con-
firming that this practical approach is able to reduce the microbial surface contaminations.
Furthermore, the continuous environmental monitoring of the surfaces over a period of
three years has shown that our procedure is safe for the operators who perform the cleaning
and keeps the environmental contamination below the stipulated limits. Moreover, the
routine inspection of the surfaces showed no signs of corrosion, confirming that the three
commercial disinfectants used in our cleaning procedures are safer than other disinfectants,
i.e., chlorine based solutions [22]. In addition, these disinfectants are not based on chlorine
or phenol, making them less dangerous to personnel, surfaces, and to the environment [18].

5. Conclusions
In conclusion, our validation protocol is an example of an efficient and reliable method
which evaluates the efficacy and safety of disinfectants in the materials commonly present
in cleanrooms. Together with our disinfection procedure, it can be easily applied in different
hospital settings, limiting the propagation of microorganisms and maintaining high levels
of safety for operators, patients, and integrity of surfaces.

Author Contributions: Conceptualization, G.D.M., S.P., B.D., C.D.B., P.G.C., and D.D.; methodology,
G.D.M., S.P., F.M., C.D.B., and D.D.; software, D.D.; validation, G.D.M., S.P., B.D., C.D.B., and D.D.;
formal analysis, B.D. and D.D.; investigation, G.D.M., S.P., F.M., P.G.C., and D.D.; resources, F.M. and
D.D.; data curation, G.D.M. and D.D.; writing—original draft preparation, D.D.; writing—review
and editing, B.D. and D.D.; visualization, D.D.; supervision, D.D.; project administration, P.G.C.
and D.D.; funding acquisition, P.G.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Int. J. Environ. Res. Public Health 2021, 18, 779 11 of 11

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available on request to corresponding author.
Acknowledgments: We gratefully acknowledge Salvatrice Lo Giudice for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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