Relationship Between Airborne Fungi Presence and the Position of the High Efficiency Particulate Air Filter in the Heating,Ventilation,and Air Conditioning System

Abstract

Aim:

Establish the influence of the terminal or nonterminal position of High Efficiency Particulate Air (HEPA) filters in the Heating, Ventilation, and Air Conditioning (HVAC) system on the presence of airborne fungi in controlled environment rooms.

Background:

Fungal infections are an important cause of morbidity and mortality in hospitalized patients.

Methods:

This study was realized from 2010 to 2017, in rooms with terminal and nonterminal HEPA filters, in eight Spanish hospitals. In rooms with terminal HEPA filters, 2,053 and 2,049 samples were recollected, and in rooms with nonterminal HEPA filters, 430 and 428 samples were recollected in the air discharge outlet (Point 1) and in the center of the room (Point 2), respectively. Temperature, relative humidity, air changes per hour, and differential pressure were recollected.

Results:

Multivariable analysis showed higher odds ratio (OR) of airborne fungi presence when HEPA filters were in nonterminal position (OR: 6.78; 95% CI [3.77, 12.20]) in Point 1 and (OR: 4.43; 95% CI [2.65, 7.40]) in Point 2. Other parameters influenced airborne fungi presence, such as temperature (OR: 1.23; 95% CI [1.06, 1.41]) in Point 2 differential pressure (OR: 0.86; 95% CI [0.84, 0.90]) and (OR: 0.88; 95% CI [0.86, 0.91]) in Points 1 and 2, respectively.

Conclusions:

HEPA filter in terminal position of the HVAC system reduces the presence of airborne fungi. To decrease the presence of airborne fungi, adequate maintenance of the environmental and design parameters is necessary in addition to the terminal position of the HEPA filter.

Keywords

air conditioning filters fungi hospital environmental monitoring

Introduction

Healthcare Associated Infections (HAIs) are the subject of study and debate in many countries (Allegranzi et al., 2011; Popovich et al., 2019) as they represent an increase in hospitalization costs for the healthcare system (Alfonso et al., 2007; Gili-Ortiz et al., 2015). Invasive fungi are an important cause of morbidity and mortality in hospitalized patients (Lutz et al., 2003), especially in the immunocompromised population (Suleyman & Alangaden, 2016). Infections with filamentous fungi such as aspergillosis are serious for patients and are increasing worldwide (Caggiano et al., 2014). Air is a vehicle for transmission of fungi infections caused by species such as Aspergillus spp., Mucorales spp., and Scedosporium spp.(Alberti et al., 2001; Sehulster et al., 2003; Suleyman & Alangaden, 2016).

Heating, Ventilation, and Air Conditioning (HVAC) systems can help reduce the presence of microorganisms in the air and on surfaces in hospital rooms through dilution, pressurization, and adequate filtration processes (Belizario et al., 2021; Dai et al., 2021; Li et al., 2007; Lin et al., 2022; Sabuco-Tébar et al., 2020; Stockwell et al., 2019). UNE 100713 (2005) is the Spanish standard of reference for air conditioning in hospitals. This standard establishes three levels of filtration for Class I rooms with very high air cleaning requirements (such as ultraclean operating rooms, conventional operating rooms, sterile material warehouse, and delivery rooms), and it also establishes that the third level of High Efficiency Particulate Air (HEPA) filtration is installed in the air supply unit itself. HEPA filters improve air quality of controlled environment rooms by reducing the entry of microorganisms (Dai et al., 2021; Hahn et al., 2002; Oren et al., 2001); they can remove 99.97% of particles ≥0.3 µm in size, as reference Aspergillus spp. spores are 2.5–3.0 µm in diameter (Sehulster et al., 2003).

In addition to the correct maintenance and validation of controlled environment rooms through periodic environmental biosecurity assessment programs, which are a useful tool to maintain and manage proper hospitals indoor air quality (Pasquarella et al., 2012; Sabuco-Tébar et al., 2022), it is essential to carry out a correct design of the facilities. Despite the recommendations in UNE 100713 (2005), we have found no studies on the influence of the terminal position of the HEPA filter in the HVAC system on the presence of airborne fungi in controlled environment rooms in hospitals. Moreover, many studies have not considered the position of the HEPA filter in the HVAC system, which represents a limitation when comparing research results (Eckmanns et al., 2006). The purpose of this study was to establish the influence of the terminal or nonterminal position of the HEPA filter in the HVAC system on the presence of airborne fungi in controlled environment rooms in hospitals.

Materials and Methods

This longitudinal study retrospectively analyzed data collected from 2010 to 2017 in eight hospitals in the provinces of Alicante and Murcia in the southeast of Spain. Samples were collected during a periodic environmental biosecurity assessment program for the validation of controlled environment rooms (ultraclean operating rooms, conventional operating rooms, sterile material warehouse, delivery rooms, radiology interventionist, and intensive care).

Environmental parameters (temperature, relative humidity, fungal colony-forming units (CFUs) present in the air), and design parameters (air changes per hour, differential pressure) were recollected. All the equipment was used in current calibration status. All sampling was carried out by appointment by two trained researchers with experience in the field and independent of the hospitals analyzed.

The samples were collected between 1 and 5 hr after room cleaning. The rooms were with all equipment installed and operating but with no staff present during sampling (ISO 14698-1, 2003a), except the person in charge of collecting samples. The HVAC system had 100% external air intake, turbulent air flow, and positive pressure. The rooms analyzed had three filtration levels, the first-level F5 (filtration efficiency 40%–60%), the second-level F9 (filtration efficiency 80%–95%), and the third-level HEPA filters, class H13 (filtration efficiency > 99.95%), following the recommendations of UNE 100713 (2005). The filters of the HVAC system were replaced following the manufacturer’s recommendations.

Air Microbiological Analysis

To analyze the presence of airborne fungi, two control points were identified: the first 0.5 m (1.64 ft) below the air discharge outlet (Point 1), and the second in the center of the room, approximately 1.2 m (3.94 ft) from the ground (Point 2). In rooms equipped with HEPA filters in terminal position, 2,053 and 2,043 samples were collected in Points 1 and 2, respectively. In rooms with HEPA filters in nonterminal position, 430 and 428 samples were collected in Points 1, and 2, respectively.

The air samples were taken following the recommendations of European and international standards ISO 14698-1 (2003a) and ISO 14698-2 (2003b). A redundant sampling was performed at both Point 1 and Point 2. The samplings were static, volumetric, by impact, in a semi-solid medium, using a microbial impactor air sampler (MAS-100®; Merck, Darmstadt, Germany) with a suction flow of 1.66 l/s (0.059 ft3/s) and with a volume per sample of 1 m³ (35.31 ft3) of air, over a period of 10 min. The impactor was disinfected before each sampling.

To identify fungi, sabouraud dextrose chloramphenicol plates were used, being incubated first at 37 ± 1 °C (98.6 °F) for 2 days, and then kept at room temperature (22 °C to 25 °C [71.6 °F to 77 °F]) for the next 3 days. To prevent contamination, each plate was sealed after sampling. An external laboratory analyzed the air samples. Opportunistic fungi (Aspergillus spp., Rhizopus spp., Mucor spp., and Scedosporium spp.) were analyzed quantitatively, and nonopportunistic fungi were analyzed qualitatively. The results were expressed in colony-forming units per m³ (CFU/m³). The CFU count figure was corrected using Feller’s statistical correction table for the MAS-100® sampler provided by the manufacturer.

Temperature and Relative Humidity

Temperature and relative humidity parameters were determined following the recommendations of UNE 171340 (2020), using multifunction equipment (Testo 400®; 0563 4001, Lenzkirch, Germany; and Testo 480®; 0560 0480, Lenzkirch, Germany) with an integrated probe for temperature (Testo 400®; 0636 9741, Lenzkirch, Germany); and for relative humidity (Testo 480®; 0635 1543, Lenzkirch, Germany). Setting the probe in the center of the room 1–1.2 m (3.28–3.94 ft) from the ground, away from heat/cold focal points, 30 measurements per minute were taken, with an interval between them of 2s. The results were expressed in (°C) for temperature and (%) for relative humidity.

Differential Pressure

Differential pressure was determined following the recommendations of UNE 171340 (2020), using equipment (Testo 400®; 0563 4001, Lenzkirch, Germany; and Testo480®; 0560 0480, Lenzkirch, Germany) with a differential pressure probe (Testo 400®; 0638 1347, Lenzkirch, Germany; and Testo 480®; 0560 0480, Lenzkirch, Germany). Differential pressure was measured in all doors of the analyzed rooms, placing the negative terminal in the annexed room through a chink in the door and the positive terminal inside the sampled room. The results were expressed in (Pa).

Air Changes per Hour (ACH)

ACH was determined following the recommendations of UNE 171340 (2020) and was calculated by applying the formula (R = Q/V) where “R” is the ACH, “Q” is the exterior air flow rate in m³/h, and “V” is the volume in m³ of the sampled rooms. Flow rates were calculated using the formula (Q = V × S × K × 3,600), where “Q” is the air flow rate in m³/h, “V” is average speed in m/s, “S” is the total area of the air passage in the grids in m², and “K” is the correction factor determined by the characteristics of the grilles or diffusers provided by the manufacturer and multiplied by 3,600s per hour.

To obtain the average extraction and discharge speed, a multifunction equipment was used (Testo 400®; 0563 4001, Lenzkirch, Germany; and 480®; 0560 0480, Lenzkirch, Germany) with a hot ball thermal probe for low-speed range (Testo 400®; 0635 1049, Lenzkirch, Germany; and Testo 480®; 0635 1543, Lenzkirch, Germany). Following the recommendations UNE-EN ISO 14644-3 (2006), the air speed at the surfaces of grilles was measured, placing the probe in six different positions approximately 30 mm (1.18 inch) from all the extraction and discharge outlets of the room for a period of 1 min.

Statistical Analyses

CFUs of fungi (CFU/m³) and the environmental and design parameters were summarized using the median and interquartile range (P₂₅–P₇₅) and 95th, mean, and standard deviation (SD) and comparisons in Absence/Presence of airborne fungi groups were performed by the Mann–Whitney test. We used the chi-squared test to assess the association between categorical variables represented by frequencies and percentages. Multivariable logistic regression was performed to assess association between Absence/Presence of airborne fungi and HEPA filter position in the HVAC system and environmental and design parameters at Points 1 and 2. Adjusted and unadjusted odds ratio (OR) were reported. Statistical analyses were performed with the statistical package IBM SPSS (IBM Corporation, Armonk, NY). Analyses were two sided, considering p < .05 as statistically significant.

Results

Table 1 shows the descriptive statistics of the environmental and design parameters of the HVAC system. Our results showed significant differences among rooms with absence and presence of fungi for all environmental and design parameters, except for relative humidity. What called our attention was that median values of ACH were highest in rooms with presence of fungi, which goes against scientific evidence.

Table 1.

Descriptive Statistics of the Environmental and Design Parameters of the Heating, Ventilation, and Air Conditioning System, Classified Into Presence or Absence of Airborne Fungi in Points 1 and 2.

Fungi		Point 1 ^a				Point 2 ^b
Fungi		T (°C)	RH (%)	ACH	DP (Pa)	T (°C)	RH (%)	ACH	DP (Pa)
Absence	N	2,043	2,043	1,233	1,164	1,940	1,940	1,155	1,096
	Mean	21.9	53	21	12.1	21.8	53	20	12.5
	SD	1.7	13	17	9.7	1.6	13	17	9.7
	P₂₅	20.8	45	13	6.4	20.8	44.5	13	7.0
	Median	21.9	53	17	12.8	21.8	52	17	13.1
	P₇₅	23.0	61	22	16.7	23.0	61.7	22	16.8
Presence	N	212	212	80	75	309	309	137	125
	Mean	22.9	52	34	1.5	23.0	51.6	34.9	2.2
	SD	1.6	11	29	6.6	1.7	11.5	30.7	7.5
	P₂₅	22.2	44	9	1.0	22.2	44.1	10.1	0.8
	Median	23.1	51	24	0.8	23.3	51.6	21.3	1.0
	P₇₅	24.1	60	51	3.8	24.2	59.3	55.2	4.60
	p Value ^c	.000	.348	.000	.000	.000	.122	.000	.000

Note. T = temperature; RH = relative humidity; DP = differential pressure; ACH = air changes per hour; SD = standard deviation.

^a 0.5 m (1.64 ft) below air discharge outlet. ^b1.2 m (3.94 ft) from the ground in the center of the room. ^cMann–Whitney test.

Most samples with absence of fungi were found in rooms with HEPA filter in terminal position: 1,868 (88.5%) in Point 1, and 1,782 (88.9%) in Point 2. We found significant differences between the terminal and nonterminal position of the HEPA filter and the presence of airborne fungi in all sampling points (Table 2). In this study, the samples were recollected during a periodic environmental biosafety assessment program for validation of controlled environment rooms, and most samples showed absence of fungi, N = 2,111 (2331) in Point 1 and N = 2,005 (2,326) in Point 2.

Table 2.

Relationship Between the Percentage (%) of Samples With and Without Fungi, and the Terminal or Nonterminal Position of the HEPA Filter in the HVAC System.

Fungi	HEPA Filter	Point 1 ^a		Point 2 ^b
Fungi	HEPA Filter	N	%	N	%
Absence	Terminal position	1,868	88.5	1,782	88.9
Absence	Nonterminal position	243	11.5	223	11.1
Presence	Terminal position	84	38.2	156	48.6
Presence	Nonterminal position	136	61.8	165	51.4
p Value ^*		.000		.000

Note. HEPA = high efficiency particulate air; HVAC = heating, ventilation, and air conditioning.

^a 0.5 m (1.64 ft) below air discharge outlet. ^b 1.2 m (3.94 ft) from the ground in the center of the room. ^* Chi-squared test.

In rooms with a terminal HEPA filter, more than 90% of samples showed absence of airborne fungi, whereas in rooms with a nonterminal HEPA filter only 64% and 57% of samples showed absence of airborne fungi in Points 1 and 2, respectively. When airborne fungi were present, the percentages of samples with presence of fungi were higher in Point 2 than in Point 1 in both positions of the HEPA filter (Figure 1).

Figure 1.

Percentage (%) samples with absence and presence of fungi in Points 1 and 2, in rooms with the terminal and nonterminal position of the High Efficiency Particulate Air filter in the Heating, Ventilation, and Air Conditioning system. Point 1 is 0.5 m (1.64 ft) below the air discharge outlet, and Point 2 is 1.2 m (3.94 ft) from the ground in the center of the room.

In addition to finding lower percentages of fungi presence, the number of CFU/m³ of opportunistic fungi was very low in rooms with HEPA filter in terminal position in both sampling points. In this study, the majority of CFUs were from nonopportunistic fungi. Among the opportunistic fungi, Aspergillus spp. was the most frequently isolated fungus in both points. There were significant differences in the CFU/m³ of Aspergillus spp. and nonopportunistic fungi in rooms with terminal and nonterminal HEPA filters. The other types of opportunistic fungi (Rhizopus spp., Mucor spp., and Scedosporium spp) were not significant but cannot be assessed due to the absence or low number of CFU/m³ found. (Table 3).

Table 3.

Relation Between CFUs of Nonopportunistic and Opportunistic (Aspergillus spp., Mucor spp., Scedosporium spp., and Rhizopus spp.) Fungi and the HEPA Filter Position in the Heating, Ventilation, and Air Conditioning System in Points 1 and 2.

		Point 1 ^a					Point 2 ^b
		Fungi (CFU/m³)					Fungi (CFU/m³)
HEPA Filter		Aspergillus	Mucor	Rhizopus	Scedosporium	NO	Aspergillus	Mucor	Rhizopus	Scedosporium	NO
Terminal position	N	2,053	2,053	2,053	2,053	2,053	2,049	2,049	2,049	2,049	2,049
	Mean	0.02	0.00	0.00	0.00	0.18	0.03	0.00	0.00	0.00	0.39
	SD	0.39	0.03	0.00	0.00	1.50	0.56	0.02	0.02	0.00	2.76
	P₂₅	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	Median	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	P₇₅	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	P₉₅	0.00	0.00	0.00	0.00	0.50	0.00	0.00	0.00	0.00	1.00
	P₉₉	0.50	0.00	0.00	0.00	4.23	0.50	0.00	0.00	0.00	9.00
Nonterminal position	N	430	430	430	430	430	428	428	428	428	428
	Mean	0.27	0.00	0.00	0.00	2.16	0.23	0.00	0.00	0.00	3.50
	SD	1.27	0.02	0.00	0.00	12.79	0.87	0.02	0.02	0.02	14.21
	P₂₅	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	Median	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	P₇₅	0.00	0.00	0.00	0.00	1.00	0.00	0.00	0.00	0.00	1.50
	P₉₅	1.00	0.00	0.00	0.00	6.95	1.00	0.00	0.00	0.00	13.28
	P₉₉	8.22	0.00	0.00	0.00	19.69	6.00	0.00	0.00	0.00	70.97
	p Value ^*	.000	.464	1.000	1.000	.000	.000	.462	.221	.029	.000

Note. HEPA = high efficiency particulate air; CFU = colony-forming unit; NO = nonopportunistic fungi; SD = standard deviation; P₂₅ = 25th quantile; P₇₅ = 75th quantile; P₉₀ = 90th percentile; P₉₅ = 95th percentile; P₉₉ = 99th percentile.

^a 0.5 m (1.64 ft) below air discharge outlet. ^b 1.2 m (3.94 ft) from the ground in the center of the room. ^* Mann–Whitney test.

The unadjusted results showed an increased probability of the presence of airborne fungi when HEPA filters were in nonterminal position (OR: 12.44; 95% CI [9.19, 16.85]) in Point 1, and (OR: 8.45; 95% CI [6.52, 10.95]) in Point 2. The multivariate analysis maintained the same results although the probability decreased (OR: 6.78; 95% CI [3.77, 12.20]) in Point 1 and (OR: 4.43; 95% CI [2.65, 7.40]) in Point 2.

We found a higher probability of airborne fungi when the temperature was higher (OR: 1.23; 95% CI [1.06, 1.41]). Moreover, the probability of airborne fungi increased both in Point 1 (OR: 0.86; 95% CI [0.84, 0.90]) and Point 2 (OR: 0.88; 95% CI [0.86, 0.91]) when the differential pressure decreased (Table 4). Although relative humidity plays a fundamental role in the presence of airborne fungi, in this research, it was outside the multivariate model because no significant differences were found with the absence or presence of fungi (Table 1).

Table 4.

Multivariable Analysis—Association Between Presence of Airborne Fungi and HEPA Filter Position in the HVAC, and Environmental and Design Parameters in Points 1 and 2.

Parameters	Point 1 ^a				Point 2 ^b
	Unadjusted		Adjusted		Unadjusted		Adjusted
	OR	95% CI	OR	95% CI	OR	95% CI	OR	95% CI
T (°C)	1.48	[1.35, 1.63]	0.93	[0.80, 1.09]	1.06	[1.48, 1.78]	1.23	[1.06, 1.41]
DP (Pa)	0.87	[0.84, 0.89]	0.86	[0.84, 0.90]	0.87	(0.85, 0.89]	0.88	(0.86, 0.91)
ACH	1.02	[1.01, 1.03]	1.05	[1.01, 1.02]	1.03	[1.02, 1.04]	1.02	(1.02, 1.03]
RH (%)	–	–	–	–	–	–	–	–
HEPA filter ^c	12.44	[9.19, 16.85]	6.78	[3.77, 12.20]	8.45	[6.521, 10.95]	4.43	[2.65, 7.40]

Note. CFU = colony-forming unit; T = temperature; DP = differential pressure; ACH = air changes per hour; RH = relative humidity; HEPA = high efficiency particulate air; OR = odds ratio; CI = confidence interval.

^a 0.5 m (1.64 ft) below air discharge outlet. ^b 1.2 m (3.94 ft) from the ground in the center of the room. ^c Nonterminal as reference.

Discussion

Recommendations differ for thresholds of CFU/m³ of airborne fungi of controlled environment rooms depending on the country (Charkowska, 2008). In Spain, UNE 171340 (2020) establishes as acceptable 0 CFU/m³ of opportunistic fungi (Aspergillus spp., Rhizopus spp., Mucor spp., and Scedosporium spp.) in the air of all controlled environment rooms. In this study, the most frequently isolated fungi were nonopportunistic, but when opportunistic fungi were isolated, Aspergillus spp. was the most frequent (Sabuco-Tébar et al., 2022). This is in line with Caggiano et al. (2014) who found that Aspergillus spp. was the most frequently isolated fungus both in the air and on surfaces in a hospital in southern Italy. Alastruey-Izquierdo et al. (2013) carried out a study on antifungal resistance in 29 Spanish hospitals and indicated that Aspergillus spp. was the genus most frequently isolated in patients with a positive culture of filamentous fungi in respiratory samples, blood cultures, or biopsies, followed in lower frequency by Scedosporium spp. and Mucor spp.

UNE 171340 (2020) establishes the center of the room (Point 2) as the only point for taking air samples for validation of controlled environment rooms. Moreover, this standard contemplates taking additional samples at the air discharge outlet (Point 1) in controlled environment rooms with three levels of filtration that do not have a terminal HEPA filter position in the HVAC system. In this study, we observed that in Point 1, when the filter was in the terminal position, we obtained 96% of samples without fungi. When the filter was not in the terminal position, this percentage of samples without fungi decreased to 64%. Hence, based on our results, we corroborate that the sampling points suggested in the Spanish standard are correct to perform room validation, provided that correct maintenance of the facilities is carried out.

HVAC systems can help control airborne fungi transmission by influencing dilution, exposure time, and airflow patterns. The high-efficiency filtration in HVAC systems reduces airborne infectious particles and prevents the spread of fungal spores (Dai et al., 2021; Hahn et al., 2002; Oren et al., 2001). The samples were recollected in a periodic environmental biosecurity assessment program for validation of controlled environment rooms; these programs are a useful tool that contribute to improving and maintaining indoor environmental conditions (Sabuco-Tébar et al., 2022). A limitation of retrospective studies is that they do not use measuring instruments designed to observe the event (Hernández-Avila et al., 2000). However, the instruments used in this study were in a current state of calibration and are those recommended by Spanish standards for the validation of hospital controlled environment rooms (UNE 171340, 2020). Our results show the median of CFU/m³ of opportunistic and nonopportunistic airborne fungi was 0.00 (0.00–0.00) in all rooms. When we found airborne fungi, the amount of CFU/m³ was higher in rooms with a nonterminal position HEPA filter in both sampling points. An HVAC system equipped with nonterminal HEPA filters may experience fungal contamination downstream of the filter and introduce unfiltered contaminated air into the room. Lutz et al. (2003) carried out a study of an outbreak of Aspergillus spp. infection in which they described the contamination of the insulating material in the ducts downstream of the final filters and emphasized the importance of terminal filtration. This is in line with other published studies such as Crimi et al. (2006), who concluded that the placement of absolute HEPA filters directly in the discharge outlet of the emission openings is important in order to obtain a total filtering prior to air emission.

The high-efficiency filtration in HVAC systems reduces airborne infectious particles and prevents the spread of fungal spores (Dai et al., 2021; Hahn et al., 2002; Oren et al., 2001).

placement of absolute HEPA filters directly in the discharge outlet of the emission openings is important in order to obtain a total filtering prior to air emission

Despite the scientific evidence and standards, and although the influence of the HEPA filter on air cleaning has been studied (Dai et al., 2021; Hahn et al., 2002; Oren et al., 2001), we have found no studies that link the influence of filter position in HVAC systems with the presence of airborne fungi. Eckmanns et al. (2006) conducted a systematic review to compare the effectiveness of HEPA filters and non-HEPA filters in reducing mortality and the rates of fungal infection in patients. The authors could not draw a definitive conclusion from the available data, and they proposed as one of the limitations that the reviewed research did not reflect the position of the HEPA filter. In our study, the odds of presence of fungi for nonterminal HEPA filter position were 12.44 and 8.45 times that for terminal HEPA filter position in Points 1 and 2, respectively. The odds were reduced to 6.78 and 4.43 times when the position of the HEPA filter is related with other environmental and design parameters.

In addition to correct maintenance by the validation of the correct positioning (UNE 171340, 2020) and the periodic change of the HEPA filters according to the manufacturer, HEPA filters need to be placed in terminal position in the HVAC system to decrease airborne fungi presence. Moreover, this will mean savings in the room validation procedure by only having to take microbiological samples at Point 2 (UNE 171340, 2020). Although the filters were changed according to the manufacturer’s recommendations, we do not have the HEPA filter change dates nor the results of the leak tests.

In addition to the correct maintenance of the HVAC system to reduce the presence of microorganisms in the air, the quality of the HVAC duct systems is important. In Spain, the Regulation of Thermal Installations (RITE) approved by Royal Decree (RD) 178 (2021) establishes the energy efficiency and safety requirements that must be met by thermal installations in buildings designed in line with well-being and hygiene criteria, during their design and sizing, execution, maintenance and use, as well as determining the procedures for accrediting compliance. As of the publication of the latest version of the RITE, UNE-EN 16798-3 (2018) becomes the mandatory standard on energy efficiency requirements for ventilation and air conditioning systems in nonresidential buildings. A limitation of this study is that we do not know the airtightness class of the ductwork installed after the HEPA filter, which may influence the number of microorganisms in the room. Further research should study in depth the influence of the terminal position of HEPA filters on the presence of airborne microorganisms.

Temperature and relative humidity have a significant influence on the presence of microorganisms in the air and on surfaces (Eames et al., 2009; Fu Shaw et al., 2018; Sabuco-Tébar et al., 2020; Shajahan et al., 2019). Humidity, nutrients, and temperature are the most important factors that influence the growth of airborne fungi (Haleem Khan & Mohan Karuppayil, 2012). UNE 171340 (2020) establishes the thresholds of relative humidity between 40% and 60% for all controlled environment rooms. This is in line with the review by Mousavi et al. (2019) which concludes that relative humidity between 40% and 60% is the most unfavorable condition for the survival of microorganisms. However, in this study, relative humidity was outside the multivariable model, because we obtained no significant differences in relative humidity (between rooms) and the absence and presence of fungi.

UNE 171340 (2020) determines temperature thresholds between 20 °C and 26 °C (68 °F to 78.8 °F) for all controlled environment rooms. In our study, in rooms with a presence of airborne fungi, the median temperature was higher, 23.1 °C (22.2 °C to 24.1 °C)—73.58 °F [71.96 °F to 75.38 °F]—and 23.3 °C (22.2 °C to 24.2 °C)—73.94 °F [71.96 °F to 75.56°F]—in Points 1 and 2, respectively, than in rooms with an absence of airborne fungi, which had a median temperature of 21.9 °C (20.8 °C to 23.0 °C)—71.42 °F [69.44 °F to 73.4 °F]—in both points. In a review by Tang (2009) on the effect of environmental parameters on the survival of airborne infectious agents, the results confirmed a positive correlation between spore levels of airborne fungi and higher temperatures. Moreover, the optimum temperature for fungi sporulation is 25 °C to 30 °C (77 °F to 86 °F), and indoor air temperature can be an important factor influencing bioaerosols (Cabral, 2010). The multivariate analysis showed a 2.3 time increase in the odds of the presence of fungi in Point 2 for one-unit temperature increase, this is in line with what was published by other authors (Cabral, 2010; Tang, 2009). The temperature of the rooms was only measured in Point 2. This is a study limitation because we cannot say whether the temperature at Point 1 was exactly the same as at Point 2. Further studies are needed on the influence of environmental parameters on the growth and survival of airborne fungi.

Although the influence of the position of the HEPA filter on the presence of fungi in the air has been demonstrated, other factors also influence the level of containment of the rooms (ACH and differential pressure). These parameters contribute to the maintenance of environmental biosafety by preventing the entry of microorganisms from adjacent spaces, by renewing the air, and by maintaining the cleanliness of the air and surfaces (Sabuco-Tébar et al., 2020; Sehulster et al., 2003). UNE 171340 (2020) establishes 15 ACH for very high risk, high risk, and medium risk rooms (type of the rooms analyzed in this research); 10 ACH for low risk; and five ACH for minimal risk rooms. This research was realized during a periodic environmental biosafety assessment program for validation of controlled environment rooms. These programs are an effective tool to improve air cleanliness (Faure et al., 2002; Pasquarella et al., 2012; Sabuco-Tébar et al., 2022), but the collection of samples by appointment represents a limitation for this study. In our study, the results show a greater number of ACH in the rooms with presence of fungi in the air with values of the median and the 75th percentile of ACH above the limits established in the Spanish standard. The results of the multivariable analysis were practically 1 (null value) in both points, which does not allow us to draw conclusions. Based on our experience, we think that it may be influenced by not maintaining constant environmental parameters by the analyzed centers due to the limitation of collecting samples by appointment. According to what is recommended in the standards and in the scientific literature, we expected to find less presence of fungi in the air at more ACH (UNE 171340, 2020; Tung et al., 2009; Sehulster et al., 2003).

Many different suggestions exist with regard to the level of pressure difference necessary to avoid contaminants migration between adjoining rooms. UNE 171340 (2020) establishes a differential pressure difference of 20 Pa (0.0029 psi) in maximum and high risk rooms, 15 Pa (0.00217 psi) in medium risk rooms (the type of rooms analyzed in this research), 10 Pa (0.00145 psi) in low risk rooms, and 6 Pa (0.00087 psi) in minimum risk rooms. Positive differential pressure prevents the entry of microorganisms from adjacent areas into controlled environment rooms (Sehulster et al., 2003). The samples with absence of fungi in Points 1 and 2 have higher values of differential pressure in the rooms. Our results show that higher differential pressure results in less presence of fungi at both sampling points. This relationship is practically maintained with the same odds when the multivariate model was performed. Although there is evidence of the relationship between differential pressure and ACH (Tung et al., 2009; Sehulster et al., 2003), this was not studied in depth in this study due to the inconclusive results of ACH. Based on our findings, we consider it interesting to carry out more studies on the ACH interaction, differential pressure, and position of the HEPA filter with the aim of achieving clean air.

Conclusion

This study was conducted within a periodic environmental biosafety assessment program; these programs contribute substantially to maintaining the air free of fungi. Placement of the HEPA filter in the terminal position of the HVAC system reduces the presence of airborne fungi. To achieve correct biosafety in controlled environmental rooms in addition to the terminal position of the HEPA filter, adequate maintenance of the environmental and design parameters is necessary.

Placement of the HEPA filter in the terminal position of the HVAC system reduces the presence of airborne fungi.

Implications for Practice

Placement of the HEPA filter in the terminal position of the HVAC system reduces the presence of airborne fungi.

Maintaining environmental and design parameters within the values recommended in the standards contributes to achieving cleaner air.

It is necessary to further investigate the position of the HEPA filter in the HVAC system in order to compare results and generate scientific evidence.

Footnotes

Authors’ Note

Ethical approvals were obtained from CEIC, Morales Meseguer, University Clinical Hospital, Murcia, Spain (C.P.-C.I. EST: 39/17).

Authors’ Contribution

Emiliana A. Sabuco Tébar, Julián J. Arense Gonzalo, and F. Javier Campayo-Rojas contributed to the design, conceptualization, methodology, data curation, and investigation. Emiliana A. Sabuco Tébar wrote the original draft and worked on visualization and on funding acquisition. Julián J. Arense Gonzalo performed formal analysis. F. Javier Campayo-Rojas provided the resources and directed the project. All authors discussed the results and contributed to the validation and the final version of the manuscript, writing-review, and editing.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Fundación para la Investigación Sanitaria de la Región de Murcia, n^º. de expediente FFIS17/CE/01/04, Murcia. Spain.

ORCID iDs

Emiliana A. Sabuco-Tébar, RN, MPH, PhD Candidate

Julián J. Arense-Gonzalo, PhD

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