4-Year Study in Monitoring the Presence of Legionella in the Campania Region’s Healthcare Facilities

Abstract

Legionella bacterium has the aquatic environment as its natural reservoir. In humans, it can cause a form of interstitial pneumonia called legionellosis which can be transmitted by inhalation of contaminated water aerosols. Legionella infection occurs more frequently in certain more susceptible population groups, including smokers, alcoholics, men, the elderly, as well as people with acquired immunodeficiency syndrome, hematological cancers, and diabetes mellitus. This study aimed to evaluate the effectiveness of the new Italian National Guidelines for the prevention of Legionella colonization in water systems application by analyzing the environmental monitoring data of Legionella carried out in healthcare facilities in the Campania region from 2019 to 2022. The secondary objectives were to estimate the most observed serogroups of L. pneumophila and to analyze the possible link between water temperature and the presence of Legionella, respectively. From our data, it emerged that in 2019, 41.1% of the examined facilities were contaminated by the Legionella genus; in 2020, the contamination percentage was 42.9%; in 2021, it was 54.5%; in 2022, it was 45.5%. Instead, the Legionella positivity rate decreased from 2019 (54.3%) to 2022 (52.4%), suggesting a possible positive influence of more restrictive prevention and control measures. The prevalent species was Legionella pneumophila, particularly serogroup 1; water temperature was the risk factor implicated in Legionella contamination.

1. Introduction

Legionella is an aerobic, Gram-negative, non-spore-forming pathogen [1] that was discovered in 1976 following a pneumonia epidemic that occurred in a hotel in the city of Philadelphia [2]. To date, there are approximately 60 known Legionella species that have been isolated in aquatic environments, but new species are progressively being described. Among all Legionella species, L. pneumophila is often associated with legionellosis pneumonia [3], especially serogroup 1, belonging to this species; it is the main pathogen for humans and causes approximately 70–90% of infections [4]. Serogroups 3 [5,6,7], 9 [7,8], and 6 [7,9] have also been shown to cause infections in humans.

Legionellae are obligate aerobes and grow in a temperature range from 20 to 42 °C [10]. Their survival can also be determined by temperatures between 5.7 °C and 63.0 °C [11]. Their natural reservoir is an aquatic environment, where it can be found in planktonic form or inside amoebae. The ideal environments for Legionella reproduction are humid and warm, such as water pipe systems, air conditioners, and the cooling towers of industrial systems [10].

Legionella-infected amoebae were found in a consortium of microorganisms organized to form a biofilm. It happens that Legionellae, amoebae, and other microorganisms constantly detach themselves from the biofilm due to variations in pressure and flow, passing into the planktonic state [10]. Therefore, the destruction of the biofilm, following environmental changes, can determine a sudden release of Legionella into the surrounding water. If this aerosolized water (through a shower, cooling tower, fountain, hot tub, etc.) is inhaled or aspirated, the bacteria can cause illnesses in a susceptible host [10].

The growth of Legionella can also be promoted by stagnant water in which it reproduces copiously in amoebae, low flow, pH range between 5.5 and 9.2, presence of organic and non-organic compounds and inorganic elements (such as zinc, potassium, and iron), deadlegs in complex water structures, water systems from old plants, low levels of total chlorine, and free residual chlorine [11]. Events such as man-made disasters, natural disasters, floods, water system disruptions, changes in water system disinfection methods, and water main breakages also increase the risk of Legionella’s growth [11].

Healthcare-associated Legionella infections and deaths have been shown to be related to demolition and construction activities, involving phases such as repressurization, excavation, underground utility connections, commissioning at the opening of the building, and water efficiency challenges [12].

Legionella infection occurs more frequently in certain population groups who are more susceptible, including smokers [13], alcoholics [14], males, the elderly [15], as well as ill individuals with diseases like diabetes mellitus [16], acquired immunodeficiency syndrome, and hematological tumors [17].

There are various outcomes of Legionella infection, including Legionnaires’ disease, severe interstitial pneumonia, Pontiac fever, or it can be completely asymptomatic. Pontiac fever, especially, is a flu-like condition and, as such, is often undiagnosed and under-reported [18].

According to data from the National Surveillance System for Legionellosis in Italy as well as globally, the number of individuals affected by legionellosis is progressively growing. Furthermore, although legionellosis is an often-underestimated disease because surveillance systems are not always used adequately, the number of cases has progressively increased over the last thirty years in Italy [19].

Monitoring and preventing the presence of Legionella in the environment is a crucial aspect to avoiding the appearance of legionellosis [4]. Indeed, the problem of Legionella distribution through different water systems has been widely studied over the years, both in domestic and public water distribution systems [20].

In healthcare facilities, the main objective concerns minimizing colonization by Legionella and achieving its total absence in facilities hosting immunocompromised subjects [21]. For this reason, the World Health Organization recommends, as a preventive measure, the establishment of a water safety plan, which is always valid and can be practiced not only during construction or demolition activities as well as guaranteeing the desired quality of water. Among the measures adopted to reduce the colonization of Legionella is chlorine-based disinfection, which is currently the mainly adopted technique in the world, particularly in Italy [22,23].

Since Legionella infection represents an issue related to the public health due to its spread and the resulting costs [24], Italy has developed Guidelines for the prevention and control of legionellosis, drawing inspiration from the guidelines developed by the WHO and in Europe by the EWGLI (European Working Group for Legionella Infections) study group [25].

In Italy, since 2005, there has been a network of regional reference centers responsible for environmental control of legionellosis in the respective regions [25,26]. The reference center in the Campania region, in the south of Italy, is ARPA Campania (ARPAC, “Regional Agency for the Protection of the Environment of Campania”), accredited according to the ISO/IEC 17025:2005 [27] and ISO 11731:2017 [28] standards for the anti-Legionella test. Following confirmed clinical cases and/or control actions prescribed by the regional health authorities, ARPA Campania carries out its activity aimed at identifying and preventing possible outbreaks. ARPA Campania follows the legionnaires’ disease surveillance network of the ECDC (European Center for Disease Prevention and Control), called European Legionnaires’ Disease Surveillance Network (ELDSNet), for the investigation of epidemic clusters associated with travel and acquired in hospitals to highlight the risk factors and interrupt the chain of transmission [28,29].

In this document, the files from the environmental overview carried out in the Campania region by ARPAC in healthcare structures (hospitals, nursing homes, RSAs, and spa facilities) from 2019 to 2022 were analyzed. Currently, few studies have carried out environmental monitoring of Legionella in the aforementioned healthcare facilities in the Campania region. For example, Torre et al. [30] carried out a 5-year monitoring of hospital facilities only. Lombardi et al. [11] also monitored Legionella only in Campania’s hospitals. Therefore, the analysis of the various types of healthcare facilities represents a novel element in this study, compared to the others with the same aim.

Furthermore, since the new Italian national guidelines [21] for the prevention of Legionella colonization in water systems were published in 2015, this study aimed to evaluate the effectiveness of their application through the analysis of environmental monitoring data of the Legionella Finally, the secondary objectives of the study was to estimate the most present L. pneumophila serogroups and analyze the possible link between water temperature and the presence of Legionella.

2. Materials and Methods

2.1. Study Area, Type of Facilities Analyzed, and Collected Samples

To develop this study, a total of 4884 water samples were examined, coming from 117 facilities differently distributed across the regional territory and located in the five provinces of the Campania region (Naples, Salerno, Avellino, Caserta, Benevento). The study lasted 4 years (from 2019 to 2022). The water used for sampling came from cold and hot water systems as well as from thermal plants (thermal water is water that comes from natural underground sources). The hot water had an average temperature of 38 °C and the cold water had an average temperature of 22 °C. In no cases did the water used for sampling come from tanks but always came from water mains. The water supplied to the health facilities comes from the water of the public network, which contain free chlorine as a residual disinfectant in drinking water. The examined facilities were hospitals, nursing homes, healthcare residences, and spas.

2.2. Sample Collection

Sampling was carried out following Italian national guidelines [21]. For each sample, 1 L of water was collected in sterile polyethylene bottles. The content of the bottles was added with 0.01% sodium thiosulphate useful for neutralizing the effects of chlorine. The collection was carried out without removing the filters from the taps and without carrying out flaming and washing. The structure’s water consumption was simply simulated. Each sample was uniquely identified and noted on a spreadsheet. The transport to the ARPA Campania laboratories was performed while maintaining an adequate temperature and protected from light, as well as separating the hot water samples from the cold ones. The transportation did not affect the samples integrity, so all the samples were used.

2.3. Microbiological Analysis and Identification

Research into environmental sources of contagion for Legionella spp. carried out by the ARPA Campania Laboratory began following the reporting of individual cases or clusters of cases in the same site by the National Institute of Public Health. The laboratory is accredited according to the UNI CEI EN ISO/IEC 17025 standard [27] for the isolation, quantification, and typing of Legionella spp. in environmental samples using the cultural method, according to the UNI EN ISO 11731:2017 method [28], while sample collection follows to the UNI EN ISO 19458:2006 standard [31].

Specifically, microbiological analysis was performed within 2 h following sample collection. The selection of Legionella was carried out using Petri plates containing GVPC agar medium. The plates were incubated for 7–10 days at 36 °C. Confirmation of the presence of Legionella spp. was obtained using BCYE agar medium supplemented with L-cysteine and BCYE agar without L-cysteine. Differential sowing in unsupplemented soil is very useful for further confirmation. The plates were incubated for 2–5 days at 36 °C.

Legionella pneumophila DNA, recovered from the environmental sample under examination through an extraction phase, is amplified using a real-time amplification system. The amplified product is determined using a probe with a fluorescent dye reporter specific for Legionella pneumophila.

Species identification is performed on an antigenic basis with serological tests and latex agglutination tests. This is based on the immune response (antigen–antibody type). It is performed using sensitized latex particles, i.e., by coating latex spheres, which has the purpose of making the agglutination phenomenon clearly visible, with specific antigens of the pathogen. Particle agglutination is considered a positive result, confirming that the microbial species in question (Legionella) is the same as the one for which the test was performed.

2.4. Data Analysis

The data of the sample’s analysis were entered into a Microsoft Excel 2016 file. The analysis of quantitative data (Legionella serotype/matrix type) was carried out using a two-tailed c² test, while quantitative data (bacterial count) was carried out using the t-student test. Results were considered statistically significant with p values < 0.05. All statistical analysis were also performed on Microsoft Excel 2016.

3. Results

Colonization by Legionella: Levels of Contamination and Species Distribution

Data deriving from Legionella monitoring from 2019 to 2022 were considered.

In total, 4884 samples were analyzed, all of which were taken from the cold water, hot water, and thermal water circuits (when spas are present). In general, there were less positive test results observed in cold water than those in hot water and in thermal water (

Table 1. Number of positive samples taken from hot water, cold water, and thermal water, as well as the average bacterial count for each year of investigation.

Year	Sample Source	Number of Positive Samples Broken Down by Sample Source	Percent Positivity by Sample Source Type	Bacteria Count (CFU/L) (Average Value)	Minimum and Maximum Bacterial Count Value (CFU/L)
2019 2020 2021 2022	Cold Water	127 24 74 85	11.8%	65,866.43 5700.00 1037.50 1980.00	3 × 10–10.2 × 103
2019 2020 2021 2022	Hot Water	838 179 437 656	79.9%	104,553.92 21,257.69 4904.08 12,568.67	102–1.5 × 106
2019 2020 2021 2022	Thermal Water	76 13 30 99	8.3%	9816.67 135,000.00 925.00 2938.46	3 × 102–13.5 × 104

).

In 2019, 41 structures were monitored and differently distributed across the regional territory: Naples (n. 29), Salerno (n. 7), Avellino (n. 2), Caserta (n. 1), Benevento (n. 2). The Legionella genus contaminated 17 (41.4%) of all structures evaluated. The distribution of the positivity rate by type of facility over the years is shown in Figure 1.

A total of 1918 water samples were analyzed. Of the 1918 samples, 1041 (54.3%) were positive for L. pneumophila (Figure 2).

870 positive samples were found in hot water circuits, 76 in thermal water circuits, and the remaining in cold water circuits (Table 1). Serological typing of the 1041 L. pneumophila isolates revealed that 600 (57.6%) belonged to Legionella pneumophila serogroup 1, 197 (18.9%) to serogroup 10, 108 (10.4%) to serogroup 8, 79 (7.6%) to serogroup 6, and 57 (5.5%) to serogroups 2–14, (Figure 3).

Out of the total 1918 samples, 1041 positive samples composed 259 (29.9%) samples containing a bacterial load between 10 and 10³ CFU/L, 547 (52.5%) samples containing a bacterial load between 10³ and 10⁴ CFU/L, and 235 (22.6%) samples containing a bacterial load between 10⁴ and 10⁵ CFU/L (

Table 2. Sampling data (monitored facilities, positive and negative samples, bacterial counts) classified by year.

Year	Facilities Analyzed	Samples Collected	Positive Samples	Bacteria Count (CFU/L)
2019	41	1918	1041	In 259 samples, between 10 and 103 In 547 samples, between 103 and 104 In 235 samples, between 104 and 105
2020	21	424	216	In 183 samples, between 102 and 103 In 33 samples, between 104 and 105
2021	21	938	541	In 519 samples, between 102 and 103 In 22 samples, between 103 and 104
2022	33	1604	840	In 757 samples, between 10 and 103 In 83 samples, between 104 and 105

).

782 (75.1%) counts exceeded the public health limit (1000 CFU/L), requiring legally mandated disinfection measures. The larger bacterial load was greater than 1.5 × 10⁶ CFU/L. L. pneumophila was isolated in 17/41 (39.6%) facilities, specifically in 1041/1928 water samples (

Table 3. Number of facilities where positive samples were found and highest bacterial counts classified by year.

Year	Highest Counts (CFU/L)	N° of Facilities with Positive Samples/n° of Hotel Examined
2019	1.5 × 106	17/41
2020	4 × 105	9/21
2021	8.5 × 104	12/21
2022	6 × 105	15/33

).

In 2020, 21 facilities were monitored. The structures were differently arranged across the regional territory: Naples (n. 10), Salerno (n. 6), Avellino (n. 1), Caserta (n. 1), Benevento (n. 3). The Legionella genus contaminated 9 (42.9%) of all structures evaluated. A total of 424 water samples were analyzed and 216 (50.9%) water samples were positive for L. pneumophila (Figure 2).

Of the 216 positive samples, 179 samples came from hot water circuits, 13 from thermal water circuits, and the rest from cold water circuits (Table 1). Serological typing revealed that 193 (89.4%) samples belonged to Legionella pneumophila serogroup 1, 4 (1.8%) to serogroup 8, and 19 (8.8%) to serogroup 12 (Figure 3).

The 216 positive samples consisted of 183 (84.7%) samples containing a bacterial load between 10² and 10³ CFU/L and 33 (15.3%) samples containing a bacterial load between 10⁴ and 10⁵ CFU/L (Table 2).

Therefore, 91 (42.1%) counts exceeded the public health limit (1000 CFU/L), requiring legally mandated disinfection measures. The larger bacterial load observed was over 4 × 10⁵ CFU/L. L. pneumophila was isolated in 9/21 (42.9%) facilities, from 216/424 water samples (Table 3).

In 2021, 21 structures were monitored. They were differently arranged across the regional territory: Naples (n. 12), Salerno (n. 5), Avellino (n. 3), Benevento (n. 1). The Legionella genus contaminated 12 (54.5%) of all structures evaluated. A total of 938 water samples were analyzed. Of the 938 samples, 541 (57.7%) were positive for L. pneumophila (Figure 2).

There were 437 samples from hot water circuits, 30 from thermal water circuits, and the rest from cold water circuits (Table 1). Serological typing of the 541 isolates revealed that 336 (62.1%) belonged to Legionella pneumophila serogroup 1, 55 (10.2%) to serogroups 2–14, 31 (5.7%) to serogroup 3, 37 (6.8%) to serogroup 6, 35 (6.5%) to serogroup 8, and 33 (6.1%) to serogroup 11 (Figure 3).

The 541 positive samples consisted of 519 (95.9%) samples containing a bacterial load between 10² and 10³ CFU/L and 22 (4.1%) samples containing a bacterial load between 10³ and 10⁴ CFU/L (Table 2).

Therefore, 252 (46.6%) counts exceeded the limit of public health (1000 CFU/L), requiring legally mandated disinfection measures. The larger bacterial load observed were over 8,5 × 10⁴ CFU/L. L. pneumophila was isolated in 12/21 (57.1%) facilities from 541/938 water samples (Table 3).

In 2022, 33 structures were monitored. The facilities were differently arranged across the regional territory: Naples (n. 24), Salerno (n. 5), Caserta (4). The Legionella genus contaminated 15 (45,5%) of all structures evaluated. A total of 1604 water samples were analyzed. Of the 1604 samples, 840 (52.4%) were positive for L. pneumophila (Figure 2).

656 positive samples came from hot water circuits, 99 from thermal water circuits, and the rest from cold water circuits (Table 1). Serological typing revealed that 443 (52.7%) samples belonged to Legionella pneumophila serogroup 1, 123 (14.6%) to serogroup 3, 47 (5.6%) to serogroup 6, 66 (7.9%) to serogroup 10, 14 (1.7%) to serogroup 11, 128 (15.2%) to serogroup 12, and 4 (0.5%) to serogroup 13 (Figure 3).

The 840 positive samples consisted of 757 (90.1%) samples containing a bacterial load between 10 and 10³ CFU/L as well as 83 (9.9%) samples containing a bacterial load between 10⁴ and 10⁵ CFU/L (Table 2).

Therefore, 569 (67.7%) counts exceeded the limit of public health (1000 CFU/L), requiring legally mandated disinfection measures. The larger bacterial load observed were over 6 × 10⁵ CFU/L. L. pneumophila was isolated from 840/1604 water samples derived from 15/33 facilities (45.5%) (Table 3).

4. Discussion

The data collected from the samples in various healthcare facilities of Campania from 2019 to 2022 were analyzed. Furthermore, in these 4 years, the COVID-19 pandemic occurred, a non-negligible factor.

The facilities have been chosen on the basis of previously experienced cases of Legionella disease, thereby introducing a bias.

Furthermore, the conclusions of our study probably have been underrated due to the reduced number of positive individuals found during the pandemic period and the resulting low number of controlled structures. Members of the Legionella genus contaminated 45.7% of all facilities evaluated in this study. These data are comparable to what emerged from our previous study which investigated the presence of Legionella in the accommodation facilities of the Campania region [19]. We observed a positivity rate of 55%. The Legionella positivity rate decreased from 2019 (54.3%) to 2022 (52.4%).

In 2020 and 2021, the COVID-19 pandemic decreased attention to environmental Legionella prevention. Indeed, analysis of 2021 data highlighted a positivity rate of 57.7%.

Instead, the 2020 data show that the positivity rate has decreased (50.9%). This deflection is caused by the increase in hospitalizations during the pandemic period which led to the need of implementing hygiene rules (for example, hand washing) [32], that probably favored the flow of water and prevented water stagnation. Indeed, it is known that water stagnation is responsible for the disappearance and poor stability of the residual disinfectant and the microbial growth of Legionella [33]. In addition to the implementation of hygiene standards, the publication of the Italian National Guidelines in 2015 had probably a positive impact as well. In 2022, a differential diagnosis protocol for cases of pneumonia was adopted by hospitals. It requires that the rapid test for the detection of Legionella urinary antigen and the test for the detection of SARS-CoV-2 (COVID-19) must be carried out. The Regional Health Department and ARPAC have highlighted that the activation of this diagnostic protocol has caused a significant increase in diagnoses of legionellosis in Campania. Despite this, however, the positivity rate in 2022 (52.4%) appears to have decreased compared to the year 2019, suggesting a possible positive influence of more restrictive preventive and control measures. The sanitization interventions of the workplaces foreseen by the guidelines for the control and prevention of the SARS-CoV-2/COVID-19 virus, issued by the Ministry of Labor and the Ministry of Health [34], have somehow had a positive impact on the positivity rate of the samples taken from the facilities included in this study.

The most prevalent species was Legionella pneumophila and serogroup 1 showed the highest percentage of positivity. This is perfectly comparable with what was found in clinical diagnosis. Actually, L. pneumophila serogroup 1 is the most frequently isolated species and the one associated with the majority of human cases in Europe and North America [35]. However, the presence of other serogroups (3, 2–14, 6, 8, 10, 11, 12, 13) was found in 39.3% of samples (1037 samples). This result suggests that targeting the clinical diagnosis on serogroups other than serogroup 1 is also crucial. Currently, the sensitivity and specificity of diagnostic methods for L. pneumophila serogroup 1 are quite high while they are lower for other L. pneumophila serogroups or for other Legionella species. Since the results of serological typing in our study showed a clear prevalence of serogroup 1, they support a more precise identification and, therefore, timely preventive interventions.

Regarding bacterial counts, 34.9% of positive samples have a count that exceeded the limits set by public health and the highest count (1.5 × 10⁶ CFU/L) was recorded in 2019.

Data analysis showed that the risk factor that influenced contamination was the temperature of the circulating water. The highest average contamination levels were found in hot water systems. Furthermore, the higher percentage of positive samples was found in the samples deriving from the hot water circuit (79.9%) compared to those deriving from the cold circuit (11.8%) and the thermal circuit (8.3%). This result is consistent with what was observed in our previous study [19] and in a work by Stojek et al. [36], which showed a greater frequency of isolation of Legionella in hot water samples.

The percentage of positivity in the thermal water was 8.3%; therefore, a lower positivity value was recorded than that observed for the samples deriving from the cold ester circuit (10.5%). Another study had already investigated the correlation between the maximum temperature of the water at the point of use and the concentration of L. pneumophila. In that case, a significant reduction of concentrations of the bacterium was observed at temperatures ≥ 55 °C [37]. In fact, a study by Falkinham JO 3rd also suggests that to reduce exposure to Legionella, it could be useful to raise the temperature of the hot water supplied by the water circuits [38].

This study provided a partial picture of the presence of Legionella in healthcare facilities. The monitoring work is constantly evolving thanks to the presence of the regional reference center for legionellosis at the provincial department of ARPAC in Salerno. The aim of the environmental surveillance work carried out by ARPAC is the identification of the “critical points” of water and air conditioning systems and is the result of the effective cooperation between the Environment Agency and the Healthcare System, which relieves pressure on the Healthcare System. Furthermore, in 2015, the National Guidelines for the prevention and control of Legionellosis were published, in which the types of interventions to be carried out in healthcare facilities were clearly indicated based on the positivity rate, the concentration of Legionella (expressed in CFU/L), and the presence or absence of clinical cases [21]. Regarding healthcare facilities, specifically in departments that host immunocompromised and very high-risk patients, as well as in the water used for tub births, procedures must be carried out to ensure the non-detection of Legionella in blood treatment systems, air, and water. Furthermore, according to Italian guidelines, points sampled and tested positive must be decontaminated and resampled later (after one, three, and six months).

In our study, post-remediation sampling was excluded from the analysis. Since this is a bias, we are also working to include post-remediation checks in monitoring practices. Despite the attention paid to the environmental surveillance of Legionella, even today, legionellosis seems to be underestimated due to two factors: there is an objective difficulty in cultivating the bacterium [39] and the surveillance systems are not used adequately in all countries.

5. Conclusions

Legionnaires’ disease still presents a public health problem, but the general population is not provided with sufficient information about the risks associated with this pathology. Furthermore, the aging of the population makes it more likely that the impact of legionellosis on the health of the most vulnerable groups will increase. The results of this work could induce greater interest in the problem linked to the presence of Legionella, both on the part of healthcare workers and users of these facilities; the latter very often are not at all aware of the possibility of contracting a Legionella infection. It would, therefore, also be desirable to provide information campaigns about this problem to educate the population and those who frequent healthcare facilities. Furthermore, in addition to ascertaining cases of Legionella and the presence of the bacterium in water systems, attention should be paid to the preventive and counteractive measures to be adopted to resolve this problem. In the future, it would also be interesting to analyze the post-remediation samples to appreciate the effectiveness of the preventive measures implemented once the presence of Legionella has been verified.

The data at our disposal suggest that further exploration of this area of research would be useful to ensure public health.