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Air sampling methods to evaluate microbial

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Journal of Hospital Infection 80 (2012) 128e132

Available online at www.sciencedirect.com

Journal of Hospital Infection

journal homepage: www.elsevierhealth.com/journals/jhin

Air sampling methods to evaluate microbial

contamination in operating theatres: results of
a comparative study in an orthopaedics department
C. Napoli a, *, S. Tafuri a, L. Montenegro b, M. Cassano b, A. Notarnicola b,
S. Lattarulo b, M.T. Montagna a, B. Moretti b
a
b

Department of Biomedical Sciences and Human Oncology, Section of Hygiene, University of Bari Aldo Moro, Bari, Italy
Department of Clinical Methodology and Surgical Techniques, University of Bari Aldo Moro, Bari, Italy

A R T I C L E

I N F O

Article history:
Received 6 May 2011
Accepted 11 October 2011
by J.A. Child
Available online 3 December
2011
Keywords:
Air quality
Environmental microbial
monitoring
Operating theatre
Surgical site infection
Orthopaedics

S U M M A R Y

Aim: To evaluate the level of microbial contamination of air in operating theatres using
active [i.e. surface air system (SAS)] and passive [i.e. index of microbial air contamination
(IMA) and nitrocellulose membranes positioned near the wound] sampling systems.
Methods: Sampling was performed between January 2010 and January 2011 in the operating theatre of the orthopaedics department in a university hospital in Southern Italy.
Findings: During surgery, the mean bacterial loads recorded were 2232.9 colony-forming
units (cfu)/m2/h with the IMA method, 123.2 cfu/m3 with the SAS method and
2768.2 cfu/m2/h with the nitrocellulose membranes. Correlation was found between the
results of the three methods. Staphylococcus aureus was detected in 12 of 60 operations
(20%) with the membranes, five (8.3%) operations with the SAS method, and three
operations (5%) with the IMA method.
Conclusion: Use of nitrocellulose membranes placed near a wound is a valid method for
measuring the microbial contamination of air. This method was more sensitive than the
IMA method and was not subject to any calibration bias, unlike active air monitoring
systems.
2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Air biocontamination and related health effects are an
emerging public health problem. Airborne bacteria, fungi and
viruses can cause infection in diverse living or working environments. This is particularly relevant in medical facilities
where there are susceptible patients and tissues are exposed to
the air during surgery. As such, there is a need for various
systems to minimize the introduction, generation and
* Corresponding author. Address: Department of Biomedical Sciences
and Human Oncology, Section of Hygiene, University of Bari Aldo Moro,
Piazza G. Cesare, 11, 70124 Bari, Italy. Tel./fax: 39 080 5478472.
E-mail address: c.napoli@igiene.uniba.it (C. Napoli).

retention of particles in these environments.1,2 In this context,

microbiological monitoring of air quality is useful in order to
determine the potential exposure of individuals at risk.
The control of air biocontamination was first deemed to be
necessary in order to reduce the risk of deep wound infections
in prodedures such as hip arthroplasties.3,4 It is generally
accepted that bacterial contamination of the air in operating
theatres, predominantly caused by contaminated skin scales
shed from the surgical team, is the main factor causing surgical
site infection after clean operations.1,5,6
Whilst the procedures for microbiological assessment of
other environmental matrices are established in European Law
(e.g. methods are specified for the identification of microbes
from water samples), there are no established regulations for

0195-6701/$ e see front matter 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jhin.2011.10.011

C. Napoli et al. / Journal of Hospital Infection 80 (2012) 128e132

Methods
This study was undertaken between January 2010 and
January 2011 in the operating theatre of the Department of
Orthopaedics and Traumatology of the University Hospital
Policlinico Consorziale, Bari, Italy. Microbial sampling was
undertaken during 60 total hip arthroplasties, all performed in
the same operating theatre (dimensions 8.7 m  4.3 m, height
3.14 m, total volume 117.467 m3). The operating theatre is
equipped with a turbulent ventilation system of four low-level
inlets with air forced through 0.3-mm H14 99.995 highefficiency particulate air filters, and three high-level outlets;
this provides an air change rate of 19.3 air changes/h and
a pressure differential of 7 Pa.
Microbial air contamination was evaluated through active
sampling (to measure the concentration of micro-organisms in
the air) and passive sampling (to measure the rate at which
viable particles settle on surfaces). Total viable count and the
presence of Staphylococcus aureus were recorded by active
and passive sampling using tryptone soya agar (TSA) and
maltose salt agar (MSA), respectively, with plates incubated at
a mean of 36 [standard deviation (SD) 1]  C for 48 h. The Health
Protection Agencys standard method was used for the

identification of S. aureus.20 The presence of fungi was evaluated by both active and passive sampling using plates containing Sabouraud chloramphenicol dextrose agar (SDA). The
plates were incubated for seven days at a mean of 27 (SD 1) C,
and filamentous fungi were identified in accordance with
standard methods on the basis of their macroscopic and
microscopic morphological features.21
Active sampling was performed using a surface air system
(SAS) (International PBI, Milan, Italy), with a flow rate of 180 L/
min and a suction volume of 500 L. The sampler was placed in
the operating theatre approximately 1 m above the floor and
approximately 1 m from the operating table. The number of
cfus was adjusted using the conversion table provided by the
manufacturer, and the value was expressed in cfu/m3. Passive
sampling was performed to determine the index of microbial
air contamination (IMA).22 This corresponds to the number of
cfus counted on a Petri dish with a diameter of 9 cm placed
according to the 1/1/1 scheme (i.e. for 1 h, 1 m above the
floor, 1 m away from walls or any major obstacles), and the
results were expressed in cfu/m2/h.
Sampling near the wound was performed using three nitrocellulose membranes with a diameter of 47 mm (Millipore,
Billerica, MA, USA), placed horizontally near the incision.
Adhesive incise drape film (Steri-DrapeTM; 3M Health Care,
St Paul, MN, USA) was applied to the skin before incision. This
provides a sterile operative surface all the way to the wound
edge, and a barrier to bacteria on the skin for the duration of
the operation. The nitrocellulose membranes were positioned
on the lower adhesive border of the film; this was folded
forwards so the membranes were held in place below the
wound and not beside it. In this position, they did not interfere
with the surgical procedure. Figure 1 illustrates the experimental set-up.
The membranes were exposed for 1 h from the start of the
operation. Each membrane was subsequently transferred to
a specific nutrient pad (i.e. one to TSA, one to MSA and one to
SDA), and incubated as described previously for IMA and SAS
procedures.18,19 All laboratory tests were performed at the
Operative Unit Hygiene Laboratory (quality certified according to ISO 9001:2008) at the University Hospital Policlinico
Consorziale, Bari, Italy. In order to evaluate a possible correlation between the number of people in the operating theatre
and the level of microbial contamination, the number of
people present was recorded for each surgical intervention.

Operating team

Anaesthetist

air monitoring other than the international norm ISO 14698.

This states that different sampling methods exist and different
types of devices are commercially available, each with limitations, thus leaving the choice of system open.7,8 Microbiological content of the air can be monitored by two principal
methods: active and passive monitoring of air flows.8 In active
monitoring, a microbiological air sampler physically draws
a known volume of air through or over a particle collection
device, which can be liquid or solid, and the number of microorganisms present is given in colony-forming units (cfu)/m3 of
air. This system is applicable when the concentration of microorganisms is not very high, such as in an operating theatre.2,9,10
Passive monitoring uses settle plates (i.e standard Petri
dishes containing culture media) that are exposed to the air for
a given time to collect biological particles; these sediment
out and are subsequently incubated. Results are expressed in
cfu/plate/time or cfu/m2/h.11
Several studies have compared the two sampling methods
with discordant results; some studies have found significant
correlation between the methods,11e14 while other studies
have found no correlation.15,16 In particular, Friberg et al.
proposed an equation that permits the transformation of the
number of cfus settling on a plate over 1 h (cfu/plate/h) into
air contamination units (cfu/m3).11,17
One of the most important questions that still needs to be
resolved is whether the data from these monitoring systems are
actually relevant to what is happening on the operating table.
If one of the principal causes of air biocontamination is the
surgical team, the results from the general room monitoring
systems may underestimate the real risk of exposure to the
patient on the surgical table, which may be surrounded by air
with a higher level of micro-organisms.
To evaluate this hypothesis, the present study investigated
and compared the levels of air contamination measured with
active and passive systems, and also evaluated the level of air
contamination near the wound using nitrocellulose
membranes.18,19

129

Wound

Figure 1. Experimental set-up. S, sedimentation plates; A,

surface air system; M, nitrocellulose membranes.

130

C. Napoli et al. / Journal of Hospital Infection 80 (2012) 128e132

The results from the three sampling methods were loaded

into a database created with File Maker software (Santa Clara,
CA, USA), and data elaboration was performed using Epi-Info
Version 6.00 (Centers for Disease Control and Prevention,
Atlanta, GA, USA). Students t-test for independent samples
was used to compare continuous variables. Categorical variables were expressed as percentages, and c2 test was used to
compare percentages. Pearsons correlation was used to
measure the relationships between the results obtained using
different sampling methods. Linear regression was used to
analyse the relationship between the number of people
present in the operating theatre and the bacterial load for each
method. A P-value <0.05 was regarded as significant.

Results
The mean bacterial load for the IMA samplings was 2232.9
(SD 859.7) cfu/m2/h (range 786e4246), which was significantly
lower than the results obtained with the nitrocellulose
membranes [mean 2768.2 (SD 1325.4) cfu/m2/h, range
1153e6344; t 2.62, P 0.0049]. A mean value of 123.2 (SD
58.7) cfu/m3 (range 40e288) was measured with the SAS
sampler.
A significant correlation was found between the IMA values
and the SAS values (r2 0.73, P < 0.0001, Figure 2), and both of
these were significantly correlated with the results from the
nitrocellulose membranes (IMA: r2 0.73, P < 0.001, Figure 3;
SAS: r2 0.67, P < 0.05, Figure 4).
S. aureus was detected in 12 of 60 operations with the
membranes [20%; 95% confidence interval (CI) 6.7e33.3], five
operations with the SAS method (8.3%; 95% CI 0.87e17.54)
and three operations with the IMA method (5%; 95%
CI 2.26e12.26). The number of samples that tested positive
for S. aureus was significantly higher for the membranes
compared with the other two methods (c2 7.53, P 0.02).
Using the membrane results as the reference, the IMA
method to detect the presence of S. aureus had sensitivity of
25% (95% CI 1e100%), specificity of 100%, and positive and
negative predictive values of 100% and 84%, respectively (95%
CI 75e94). The SAS method had sensitivity of 42% (95% CI
14e70), specificity of 100%, and positive and negative predictive values of 100% and 87%, respectively (95% CI 78e96).

Figure 2. Correlation between the bacterial load detected with

Petri dishes (index of microbial air contamination) and the surface
air system during 60 hip arthroplasties. cfu, colony-forming units.

Figure 3. Correlation between the bacterial load detected with

Petri dishes (index of microbial air contamination) and nitrocellulose membranes positioned near the wound during 60 hip
arthroplasties. cfu, colony-forming units.

The mean number of people in the operating theatre during

the 60 operations was 6.4 (SD 1.44, range 4e10). Linear
regression revealed a relationship between the number of
people present and the total viable count with all three
methods: IMA (r2 0.23, P < 0.0001), SAS (r2 0.28; P < 0.001)
and nitrocellulose membranes (r2 0.12, P < 0.001). For the
IMA method, the mean number of people in the operating
theatre did not differ significantly between operations when
S. aureus was detected [7.7 (SD 1.5)] compared with operations
when S. aureus was not detected [6.3 (SD 1.4); t 1.62,
P>0.05]. However, the difference was significant for the SAS
method, with a mean of 7.8 (SD 1.3) people in the operating
thatre when S. aureus was detected compared with 6.2
(SD 1.4) people when S. aureus was not detected (t 2.03,
P 0.04). Similarly, the mean number of people in the operating theatre when S. aureus was detected on the nitrocellulose membranes was 7.3 (SD 1.3), compared with 6.1 (SD 1.4)
people when S. aureus was not detected (t 2.48, P 0.01).
Fungi were detected during two separate surgical operations: Aspergillus spp. was detected with the IMA method, and

Figure 4. Correlation between the bacterial load detected with

the surface air system and nitrocellulose membranes positioned
near the wound during 60 hip arthroplasties. cfu, colony-forming
units.

C. Napoli et al. / Journal of Hospital Infection 80 (2012) 128e132

Alternaria spp.
membranes.

was

detected

with

the

nitrocellulose

Discussion
Infections can be debilitating complications of primary hip
arthroplasty, and have been cited as the most common cause of
implantation failure.23 Many contributing factors have been
implicated such as patient factors, surgical technique and
postoperative factors,24 but the environment, progress of the
operation, clothes worn, etc. are also important. In this
context, the microbiological quality of the air in operating
theatres is a significant parameter to control surgical infection,
and sampling may be undertaken to assess both the quality of
air and the ability of the air systems to dilute or exclude
contamination.25 The present study compared active and
passive collection methods, and the results showed correlation
between the two. Both methods can be used to measure air
contamination, but if air sampling is performed during surgery
to monitor the risk of microbial wound contamination, passive
methods should be used to predict the likely contamination
rate at the surgical site rather than volumetric sampling, as this
allows direct measurement of the number of micro-organisms
settling on surfaces.8,26,27 Active measurement also provides
results on the concentration of particles in the air.8
In addition to traditional sampling methods, this study also
used nitrocellulose membranes in order to evaluate microbial
contamination near the wound. The use of nitrocellulose
membranes is a passive technique, but comparison of the
microbial sedimentation on the settle plates (IMA), which were
near the operating table but outside the active operating area,
and the microbial sedimentation on the membranes, which were
actually placed on the operating table, revealed higher microbial loads on the membranes, confirming that the surgical team is
a source of contamination. Contamination levels of the air in
operating theatres are normally measured near the table but not
near the wound, thus underestimating bacterial numbers.
The membrane method was also useful for the detection of
S. aureus, which, together with streptococci, causes 65e85% of
all prosthetic joint infections.28e30 The membranes detected
S. aureus during 12 of 60 operations, compared with five
operations for the SAS method and three operations for the IMA
method. The membranes revealed that S. aureus was more
likely when more people were present, which was not the case
with the IMA method.
This study only looked specifically for S. aureus, but other
staphylococcal species are considered to be opportunistic
pathogens, and are increasingly recognized as causes of clinically significant nosocomial infections.31
The efficiency of nitrocellulose membrane sampling may be
influenced by the electrostatic potential of these membranes,
as they have been found to have a general negative electric
charge at neutral pH.32 However, the authors believe that the
differences in air contamination measured by the IMA and SAS
methods compared with the membrane method were more
a result of their positioning than the efficiency of the method
used. When using passive sampling methods, Friberg et al. reported higher counts on settle plates near the wound than in the
periphery of the operating theatre.17 A possible explanation for
this is that heavier bacteria-carrying particles (i.e. those more
likely to settle) tend to settle close to their main origin (i.e. the

131

surgical team).17 Moreover, from a practical point of view, to

assess microbial contamination close to the wound, membranes
are much easier to position than settle plates.
This study shows that it is important to note that the limits
of sampling systems are not exclusively influenced by their
sensitivity and specificity (active or passive), but also by
external factors such as the point of collection. Standardized
sampling protocols should be developed for use within
a hospital, for different clinical settings and for comparisons
between different institutions and countries.
The two passive systems detected two different strains of
fungi, but the SAS system did not detect any fungi. This is in
contrast to a study by Verhoeff et al., which found that active
sampling was better for the detection of fungi.33 Furthermore,
in a recent study by Asefa et al. comparing the SAS super 180 air
sampler and settle plates, the SAS sampler detected more
fungal species and a higher mean cfu/plate compared with the
settle plates. Qualitatively, both methods detected similar
predominant fungal genera and species.34
In conclusion, this study found that an air sampling system
near the surgical wound may be the best way to identify the
risk of exposure to pathogens, and to develop infection
measures for operations with a high risk of contamination (e.g.
arthroplasty). Cooperation of the surgical team is necessary,
and during this study, various team members were unhappy
with the presence of the sampling equipment, particularly the
membranes, which had to be positioned in the sterile operating
field before the first incision. Therefore, personnel need to be
trained in sampling techniques, but it is also necessary to make
staff aware of the importance of environmental surveillance
programmes in order to plan corrective action in the case of
any anomalies, thus allowing the avoidance of infective
complications.
Conflict of interest statement
None declared.
Funding sources
University of Bari financial support for scientific research.

References
1. Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR. Guideline for prevention of surgical site infection, 1999. Hospital
Infection Control Practices Advisory Committee. Infect Control
Hosp Epidemiol 1999;20:250e278.
2. Center for Disease Control and Prevention. Guidelines for environmental infection control in health-care facilities. Atlanta, GA:
CDC; 2003.
3. Charnley J. Post operative infection after total hip replacement
with special reference to air contamination in the operating room.
Clin Orthop 1972;87:167e187.
4. Lidwell OM. Air, antibiotics and sepsis in replacement joints.
J Hosp Infect 1988;11:18e40.
5. Gosden PE, MacGowan AP, Bannister GC. Importance of air quality
and related factors in the prevention of infection in orthopaedic
implant surgery. J Hosp Infect 1998;39:173e180.
6. Whyte W, Hodgson R, Tinkler J. The importance of airborne
bacterial contamination of wounds. J Hosp Infect 1982;3:123e135.
7. Council Directive 98/83/EC of 3 November 1998 on the quality of
water intended for human consumption. Official Journal of the
European Community 1998;L330:32e54.

132

C. Napoli et al. / Journal of Hospital Infection 80 (2012) 128e132

8. ISO 14698-1. Cleanrooms and associated controlled environments e

biocontamination control. Part 1: General principles and methods.
Milan: UNI; 2003.
9. Pasquarella C, Albertini R, Dallaglio P, Saccani E,
Sansebastiano GE, Signorelli C. Air microbial sampling: the state of
the art. Ig Sanita Pubbl 2008;64:79e120.
10. Blomquist G. Sampling of biological particles. Analyst 1994;119:
53e56.
11. Friberg B, Friberg S, Burman LG. Inconsistent correlation between
aerobic bacterial surface and air counts in operating rooms with ultra
clean laminar air flows: proposal of a new bacteriological standard
for surface contamination. J Hosp Infect 1999;42:287e293.
12. Orpianesi C, Cresci A, La Rosa F, Saltalamacchia G, Tarsi R.
Evaluation of microbial contamination in a hospital environment.
Comparison between the surface air system and the traditional
method. Nuovi Ann Ig Microbiol 1983;34:171e185.
13. Perdelli F, Sartini M, Orlando M, Secchi V, Cristina ML. Relationship between settling microbial load and suspended microbial load
in operating rooms. Ann Ig 2000;12:373e380.
14. Whyte W. Sterility assurance and models for assessing airborne
bacterial contamination. J Parenter Sci Technol 1986;40:188e197.
15. Petti S, Iannazzo S, Tarsitani G. Comparison between different
methods to monitor the microbial level of indoor air contamination in the dental office. Ann Ig 2003;15:725e733.
16. Sayer WJ, MacKnight NM, Wilson HW. Hospital airborne bacteria as
estimated by the Andersen sampler versus the gravity settling
culture plate. Am J Clin Pathol 1972;58:558e566.
17. Friberg B, Friberg S, Burman LG. Correlation between surface and
air counts of particles carrying aerobic bacteria in operating rooms
with turbulent ventilation: an experimental study. J Hosp Infect
1999;42:61e68.
18. Pasquarella C, Pitzurra O, Herren T, Poletti L, Savino A. Lack of
influence of body exhaust gowns on aerobic bacterial surface
counts in a mixed ventilation operating theatre. A study of 62 hip
arthroplasties. J Hosp Infect 2003;54:2e9.
19. Pasquarella C, Sansebastiano GE, Ferretti S, et al. A mobile
laminar airflow unit to reduce air bacterial contamination at
surgical area in a conventionally ventilated operating theatre.
J Hosp Infect 2007;66:313e319.
20. Health Protection Agency. Identification of Streptococcus species,
Enterococcus species and morphologically similar organisms.

21.
22.
23.

24.

25.
26.

27.
28.

29.

30.
31.
32.

33.

34.

National Standard Method BSOP ID 4 Issue 2.1. London: Standard

Unit, Evaluations and Standards Laboratory; 2007.
De Hoog GS, Guarro J, Gene
J, Figueras MJ. Atlas of clinical fungi.
3rd ed. Utrecht: Centraalbureau voor Schimmelcultures; 2009.
Pasquarella C, Pitzurra O, Savino A. The index of microbial air
contamination. J Hosp Infect 2000;46:241e256.
Fish DN, Hoffman HM, Danziger LH. Antibiotic-impregnated
cement use in U.S. hospitals. Am J Hosp Pharm 1992;49:
2469e2474.
Jackson WO, Schmalzried TP. Limited role of direct exchange
arthroplasty in the treatment of infected total hip replacements.
Clin Orthop 2000;381:101e105.
Hoffman P, Humphreys H. Air sampling: settle plates or slit
samplers? J Hosp Infect 2001;49:299e300.
Kundsin RB. The microbiologists role in evaluating the hygienic
environment. In: Kudsin RB, editor. Architectural design and
indoor microbial pollution. New York: Oxford University Press;
1988. p. 103e122.
Whyte W. In support of settle plates. PDA J Pharm Scien Technol
1996;50:201e204.
Salgado CD, Dash S, Cantey JR, Marculescu CE. Higher risk of
failure of methicillin-resistant Staphylococcus aureus prosthetic
joint infections. Clin Orthop Relat Res 2007;461:48e53.
Tattevin P, Cremieux AC, Pottier P, Huten D, Carbon C. Prosthetic
joint infection: when can prosthesis salvage be considered? Clin
Infect Dis 1999;29:292e295.
Zimmerli W, Ochsner PE. Management of infection associated with
prosthetic joints. Infection 2003;31:99e108.
Rogers KL, Fey PD, Rupp ME. Coagulase-negative staphylococcal
infections. Infect Dis Clin North Am 2009;23:73e98.
Pristoupil TI, Kramlova
M, Strbkova
J. On the mechanism of
adsorption of proteins to nitrocellulose in membrane chromatography. J Chromatogr 1969;42:367e375.
Verhoeff AP, van Wijnen JH, Boleij JS, Brunekreef B, van ReenenHoekstra ES, Samson RA. Enumeration and identification of
airborne viable mould propagules in houses. A field comparison of
selected techniques. Allergy 1990;45:275e284.
Asefa DT, Langsrud S, Gjerde RO, et al. The performance of SASsuper-180 air sampler and settle plates for assessing viable fungal
particles in the air of dry-cured meat production facility. Food
Control 2009;20:997e1001.