Journal of Hospital Infection (2008) 69, 148e155

Available online at www.sciencedirect.com

www.elsevierhealth.com/journals/jhin

Prevalence of anti-legionella antibodies amongItalian hospital workers

P. Borella a,*, A. Bargellini a, I. Marchesi a, S. Rovesti a, G. Stancanelli b,S. Scaltriti a,b, M. Moro b, M.T. Montagna c, D. Tato c, C. Napoli c,M. Triassi d, S. Montegrosso d, F. Pennino d, C.M. Zotti e,S. Ditommaso e, M. Giacomuzzi e

a Department of Public Health Sciences, University of Modena and Reggio Emilia, Italyb S. Raffaele Hospital, Milan, Italyc Department of Internal Medicine and Public Health, University of Bari, Italyd Department of Preventive Medical Sciences, University Federico II of Naples, Italye Department of Public Health and Microbiology, University of Turin, Italy

Received 3 August 2007; accepted 6 March 2008Available online 29 April 2008

KEYWORDSLegionella;Occupationalexposure; Hospitalstaff; Anti-Legionellaantibodies; Riskfactors;Seroprevalence

* Corresponding author. Address: Dip2055474; fax: þ39 059 2055483.

E-mail address: borella.paola@uni

0195-6701/$ - see front matter ª 200doi:10.1016/j.jhin.2008.03.004

Summary This study evaluated the prevalence of anti-legionella anti-bodies in workers at hospitals with a long-term history of legionella con-tamination. The hospitals are located in Milan and Turin, northern Italy,and in Naples and Bari, southern Italy. Antibody prevalence and titres ofhealthcare workers, medical and dental students and blood donors wereassessed. In total 28.5% of subjects were antibody positive, most fre-quently to L. pneumophila serogroups 7e14. Major differences were ob-served in seroprevalence and type of legionella antibody in persons fromdifferent geographic areas. Healthcare workers had a significantly higherfrequency of antibodies compared with blood donors in Milan (35.4 vs15.9%, P< 0.001), whereas in Naples both groups exhibited high antibodyfrequency (48.8 vs 44.0%) and had a higher proportion of antibodies to le-gionella serogroups 1e6. Dental workers had a higher seroprevalence thanoffice staff in Bari, but not in Turin, where daily disinfecting procedureshad been adopted to avoid contamination of dental unit water. No associ-ation was found between the presence of antibodies and the presence ofrisk factors for legionellosis, nor with the occurrence of pneumonia and/or flu-like symptoms. In conclusion, the presence of legionella antibodies

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8 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Legionella antibodies in hospital workers 149

may be associated with occupational exposure in the hospital environ-ment, but there was no evidence of any association with disease.ª 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rightsreserved.

Introduction

Legionella spp. should be considered among the oc-cupational hazards of healthcare workers sincethese bacteria are widespread in healthcare facili-ties.1,2 They have been isolated in the majority ofhospital water distribution systems in Italy, perhapsreflecting the large number of old buildings in thiscountry.3,4 Other potential sources of infection inhealthcare facilities include aerosol-generating de-vices, particularly those used during dental care,since legionella frequently colonises dental unitwaterlines.5e7 Patients are known to be at risk ofacquiring legionellosis in contaminated hospitals,but little is known about the risks to hospital staff.8

The production of anti-Legionella spp. anti-bodies reflects exposure to legionella, andincreased antibody titres can be used retrospec-tively to gauge the infectious risk. Seroprevalencestudies have been undertaken during clusters of Le-gionnaires’ disease,1,9e12 and on people living andworking in contaminated environments.13e15 Re-cent studies have documented a higher prevalenceof Legionella spp. antibodies among people whowork in hospitals, thermal spas and dental practicesbut another study found no increase in antibodyprevalence among dental practitioners.16e19

The aim of this investigation was to explore theprevalence of anti-Legionella spp. antibodies in em-ployees of large hospitals with a history of legionellacontamination located in different Italian regions.Professional and personal characteristics likely toaffect both exposure to Legionella spp. and hostsusceptibility to legionellosis were investigated,and variability in seroprevalence according to localcontamination and geographic area was evaluated.The results of the study may clarify whether ex-posed workers in the health environment are atrisk of acquiring disease, which factors are associ-ated with the presence of antibodies, and whichmeasures can be advocated to avoid infection.

Methods

Participating units

This study was conducted as part of a wider surveyof legionella infection that was aimed at better

defining environmental contamination in Italianhospitals, providing data on the prevalence ofcommunity and nosocomial infections and evalu-ating the risk to personnel working in contami-nated hospitals.20 Four research groups recruitedsubjects in their respective university hospitals.Two were located in northern Italy (Turin and Mi-lan) and two in southern Italy (Naples and Bari).The participating units were selected on the basisof a history of legionella contamination.

Hospital dental workers (N¼ 61) were recruitedin Turin, where Legionella pneumophila serogroup1 and L. non-pneumophila species have been foundsporadically in the water of drill units subjected todaily disinfection procedures, and in Bari (N¼ 44),where drill units were not subjected to regular dis-infection and were frequently contaminated byL. non-pneumophila species and L. pneumophilaserogroup 1.21,22 In both cases, office staff fromthe same hospital (N¼ 70 and 44 respectively)were selected as controls to evaluate specificand/or additive exposure. In addition, dental stu-dents (N¼ 58) were included in Turin. Water distri-bution systems in both hospitals have been foundhighly contaminated by Legionella spp. and hospi-tal-acquired infection has occurred.

In Milan and Naples, ward personnel (N¼ 65 and41 respectively) were selected as subjects whileblood donors (N¼ 132 and 75 respectively) servedas controls. In addition, a group of office staff(N¼ 15) was studied in Milan, and a group of med-ical students was studied in Naples (N¼ 59), ac-cording to local co-operation. The water suppliesin the hospitals in Milan and Naples hospitalshave both been shown to be heavily colonisedwith legionella, and in both cases this has beenassociated with hospital-acquired infection.23,24

In Milan L. pneumophila serogroups 2e14 weremost prevalent (serogroup 1 was rarely detected),whereas in Naples L. pneumophila serogroups 3and 1, and L. non-pneumophila species weremainly detected.

Guidelines for healthcare facilities have beenavailable in Italy since 2000, making recommen-dations for the appropriate management oflegionellosis, the assessment of environmentalcontamination in at-risk wards, and the implemen-tation of preventive measures on the basis oflegionella concentration.

150 P. Borella et al.

Recruited subjects

Different groups were selected in each city asdescribed in the previous section. A total of 340hospital workers (131 males, 209 females) withvarious occupations (physician, dentist, nurse,healthcare assistant, cleaner, and clerk) wereexamined. They were classified into three groups:persons working in dentistry wards (dental staff,N¼ 105), those working in medical/surgical wards(ward staff, N¼ 106) and those employed in ad-ministrative and/or technical services (officestaff, N¼ 129). Students and doctors attendingspecialisation courses in medicine and/or den-tistry were also included (N¼ 117). As controlsin Milan and Naples, 207 subjects were randomlyselected among blood donors and paired forage. Participation in the study was voluntarywith written informed consent, and the study re-ceived the formal approval of the local EthicalCommittees.

Hospital staff (dental workers, ward staff andoffice staff) and blood donors did not differ inmean age (39.4� 10.1 vs 39.1� 10.0 years), butthere were significantly fewer men among hospitalstaff (38.5% vs 64.7%, P< 0.001). Smoking habitswere similar (27.8% vs 25.1%), whereas signifi-cantly fewer hospital workers than blood donorsdeclared a daily alcohol intake (48.1 vs 65.2%,P< 0.005). The students were similar to hospitalstaff in sex composition and alcohol intake, butfewer of them smoked (23.1%). This group wasyounger (27.4� 6.0 years, P< 0.001) and hada shorter length of service (3.6� 1.9 vs12.6� 10.2 years, P< 0.001).

A detailed questionnaire was used to collectinformation on demographic characteristics (sex,age, civil state, education), personal habits(smoking, alcohol consumption, type of drinkingwater), present and past diseases, personal hy-giene (bathtub and/or shower, jacuzzi, saunause), sporting activities and related water expo-sure, type of job, length of service, exposure towater at work (use of shower and/or sink, use ofaerosol-producing devices, pressured water jets,etc.), housing (flat or house, year of construc-tion/refurbishment, water supply), water-heatingsystems and related characteristics (central orindependent, electric or gas heater, existence ofa tank, service frequency, existence of a softeningsystem, etc.). Particular attention was paid torecording episodes of pneumonia during the pre-vious five years (time of occurrence, antibioticsand hospital admissions) and flu-like symptoms inthe previous year.

Serological study

Serum anti-Legionella spp. antibodies were de-tected by indirect immunofluorescence (IFA,RIDA� FLUORlegionella IgG, R-Biopharm AG, Darm-stadt, Germany). This method distinguishes IgGantibodies against L. pneumophila serogroups 1e6,L. pneumophila serogroups 7e14, andL. non-pneumophila (L. bozemanii, dumoffii, gor-manii, jordanis, longbeachae and micdadei). Lab-oratory workers in the different cities weretrained to ensure standardisation of test perfor-mance. A cut-off for positivity of 1:128 was se-lected to reduce the risk of false-positive resultsalthough cross-reaction with other micro-organismscannot be completely ruled out.25

Statistical analysis

Statistical analyses were conducted by SPSS/pc(SPSS Inc., Chicago, IL, USA). Pearson’s Chi-squared test was used to evaluate the relationshipbetween anti-Legionella spp. antibodies (positive/negative, titres and species) in the examinedgroups and the investigated variables, and odds ra-tios (ORs) and [95% confidence intervals (CI)] werecalculated on bivariate variables to assess the risksassociated with the presence of antibody. Condi-tional logistic regression was used to evaluate in-dependent predictors of seropositivity.

Results

Table I summarises the anti-Legionella spp. anti-body frequency, type and titres in the groups ex-amined. In total, 189 out of 664 subjects (28.5%)had detectable antibodies. Seven of these had de-tectable titres to both L. pneumophila serogroups7e14 and serogroups 1e6, while 19 had detectabletitres to both L. pneumophila serogroups 7e14 andto L. non-pneumophila species. Since multiple an-tibodies were found in both hospital staff andblood donors (5.0 vs 3.4%, respectively), the totalnumber of antibodies was used for statistical anal-ysis. A slightly higher positivity was observedamong hospital staff, but direct comparison be-tween groups was not performed due to differ-ences in selection by the participating units. Inall groups the most frequently detected antibodieswere directed against L. pneumophila serogroups7e14 and the most frequent titre was 1:256, buttitre distribution differed between groups.

Table II presents the results separately. In Turinno difference was observed between dental and

Table I Legionella spp. antibodies in the examined groups

Hospital staff Students Blood donors Total

Positive subjects No. (%) 106/340 (31.2) 29/117 (24.8) 54/207 (26.1) 189/664 (28.5)Antibody type

LP 1e6No. (% of total) 9/340 (2.6) 4/117 (3.4) 7/207 (3.4) 20/664 (3.0)(% of positive) (8.5) (13.8) (13.0) (10.6)

LP 7e14No. (% of total) 74/340 (21.8) 17/117 (14.5) 34/207 (16.4) 125/664 (18.8)(% of positive) (69.8) (58.6) (63.0) (66.1)

LSNo. (% of total) 40/340 (11.8) 8/117 (6.8) 20/207 (9.7) 68/664 (10.2)(% of positive) (37.7) (27.6) (37.0) (36.0)

Total antibodiesNo. (% of total) 123/340 (36.2) 29/117 (24.8) 61/207 (29.5) 213/664 (32.1)

Antibody titrea

No. (% of positive)1:128 24/106 (22.6) 17/29 (58.6) 20/54 (37.0) 61/189 (32.3)1:256 54/106 (50.9) 7/29 (24.1) 12/54 (22.2) 73/189 (38.6)1:512 28/106 (26.4) 5/29 (17.2) 22/54 (40.7) 55/189 (29.1)

LP 1e6, L. pneumophila serogroups 1e6; LP 7e14, L. pneumophila serogroups 7e14; LS, other Legionella spp.a When multiple antibody types were detected, only the highest titre was selected.

Legionella antibodies in hospital workers 151

office staff, whereas in Bari dental staff were sig-nificantly more likely than office staff to be anti-body positive (OR: 4.67; 95% CI: 1.39e15.61;P< 0.01), mainly due to a higher frequency of L.non-pneumophila antibodies (3.75; 1.10e12.74;P< 0.005). In Milan ward staff were significantlymore likely than blood donors to be antibody pos-itive (2.89; 1.45e5.77; P< 0.002), particularly forL. non-pneumophila antibodies (4.62; 1.51e14.14; P< 0.01). Naples had the highest seropreva-lence in both hospital staff and blood donors anda higher frequency of antibodies against L. pneu-mophila serogroups 1e6. Blood donors in Napleswere significantly more likely to be antibody posi-tive than those in Milan (44.0 vs 15.9%; c2¼ 19.57;P< 0.001), although age, job and gender were sim-ilar. Among blood donors in Naples, antibody prev-alence was higher in those who consumed alcoholcompared with non-consumers (53.3 vs 30.0%;P< 0.05), those living in flats compared with thoseliving in houses (51.8 vs 21.2%; P< 0.02), thosedrinking tap water compared with those drinkingmineral water (73.3 vs 36.7%; P< 0.05) and thosetaking baths instead of showers (83.3 vs 35.7%;P< 0.05). Students had significantly fewer anti-bodies, probably related to their lower age andshorter length of service.

Table II also presents the distribution ofantibody types together with data on local water

contamination. L. pneumophila serogroups 7e14prevailed in all groups of subjects living in northernItaly, even though in Turin hospital contaminationhas mainly involved serogroup 1 and L. non-pneumophila species. In Milan serogroups 2e14were frequently isolated in the environment bothwithin and without the hospital, perhaps explain-ing the high percentage of antibodies toL. pneumophila serogroups 7e14, but not the higherprevalence of antibodies to L. non-pneumophilaspecies in ward staff. Similarly, in Bari antibodiesto L. non-pneumophila species prevailed in bothdental and office staff, even though legionellaewere frequently detected in the water of dentalchair equipment but rarely in the hospital watersystem. A higher proportion of antibodies toL. pneumophila serogroups 1e6 was confirmed inNaples, in accordance with the environmentalisolates.

Among hospital staff, subjects older than 50years were more likely to be seropositive (43.4 vs28.9%, P< 0.05). In addition, seroprevalence in-creased with length of service: from 19.4% in thoseworking less than 5 years to 41.1% in those workingmore than 20 years (c2¼ 13.6, P< 0.01). Whenage, length of service and cities were introducedas independent variables into a multivariate re-gression model, length of service remained inde-pendently associated with an increased risk of

Table II Distribution of anti-Legionella spp. antibodies according to group and location

Subjects positiveNo. (%)

No. of antibodiesdetected

Antibody distribution [no. (% total) (% positive)] Local water contamination

LP 1e6 LP 7e14 LS

Turin (North)Dental staff (N¼ 61) 18 (29.5) 18 0 17 (27.9) (94.4) 1 (1.6) (5.6) Dental unit: sporadically

LP 1 (25%), LS (75%)Dental students (N¼ 58) 14 (24.1) 14 3 (5.2) (21.4) 9 (15.5) (64.3) 2 (3.4) (14.3)

Office staff (N¼ 70) 23 (32.9) 26 1 (1.4) (3.8) 20 (28.6) (77.0) 5 (7.1) (19.2) Hospital: LP 1 (50%),LP 3 (3.3%), LS (46.6%)

Bari (South) ** *Dental staff (N¼ 44) 14 (31.8) 17 0 5 (11.1) (29.4) 12 (23.3) (70.6) Dental unit: LS (67%),

LP 1 (21%), LP 2e14 (12%)Office staff (N¼ 44) 4 (9.1) 6 0 2 (4.5) (33.3) 4 (9.1) (66.7) Hospital: LP 2e14 (65%),

LP 1 (30%), LS (5%)

Milan (North) ** *Ward staff (N¼ 65) 23 (35.4) 30 2 (3.1) (6.7) 18 (27.7) (60.0) 10 (15.4) (33.3) Hospital: LP 2e14 (98.8%),

sporadically LP 1Office staff (N¼ 15) 4 (26.7) 5 0 4 (26.7) (80.0) 1 (6.7) (20.0)Blood donors (N¼ 132) 21 (15.9) 24 0 19 (14.4) (79.2) 5 (3.8) (20.8) Home/hotel: LP 2e14

(73%), LP 1 (17%)a

Naples (South) * *Ward staff (N¼ 41) 20 (48.8) 21 6 (14.6) (28.6) 8 (19.5) (38.1) 7 (17.1) (33.3) Hospital: LP 3 (70%),

LS (17%), LP 1 (13%)Ward students (N¼ 59) 15 (25.4) 15 1 (1.7) (6.7) 8 (13.6) (53.3) 6 (10.2) (40.0)Blood donors (N¼ 75) 33 (44.0) 37 7 (9.3) (18.9) 15 (20.0) (40.5) 15 (20.0) (40.5) Home/hotel: LP 1 (94%),

LP 2e14 (6%)a

LP 1e6, L. pneumophila serogroups 1e6; LP 7e14, L. pneumophila serogroups 7e14; LS, other Legionella spp.*P< 0.05; **P< 0.01 (Chi-squared test).

a References 4 and 26.

152P.

Bore

llaet

al.

Legionella antibodies in hospital workers 153

seropositivity (OR: 1.04; 95% CI: 1.01e1.06;P< 0.002).

The presence of antibodies was not associatedwith known personal or professional risk factors forlegionellosis, except that antibodies to L. pneu-mophila serogroups 1e6 were slightly more com-mon in hospital workers using aerosol-generatingdevices: 5/75 (6.7%) vs 7/265 (2.6%) (P¼ 0.55).No association was found between seroprevalenceand the occurrence of flu-like symptoms and/orpneumonia, except for a higher risk of pneumoniain subjects positive for antibodies to L. pneumo-phila serogroups 1e6 compared with negativesubjects: 2/16 (12.5%) vs 6/459 (1.3%) (OR:10.79; 95% CI: 2.00e58.24; P< 0.001). The twosubjects were: (i) a 48-year-old woman reportingpneumonia two years previously, living in north-ern Italy, a housekeeper, heavy smoker and alco-hol consumer with hypertension; (ii) a 60-year-oldman who had had pneumonia five years previ-ously, working as a health assistant in Naples,ex-smoker, alcohol consumer, regularly takinganalgesic drugs.

Discussion

This study has defined an endemic level of Legion-ella spp. exposure in different Italian regions withspecial reference to hospital staff, and demon-strates geographic variations in both the frequencyand type of antibodies to legionella in line with thevariety of species and serogroups distributednationwide.3,4

Although much emphasis has been placed onthe role of cell-mediated immunity in legionellainfection, antibodies acting in concert withpolymorphonuclear leucocytes may play a rolein overcoming infection.26 Hence it is not surpris-ing that antibody prevalence was so high ina country where legionella contamination iswidespread.3,4,27 Indeed, the rate might havebeen underestimated by our cut-off of 1:128for positivity.

Irrespective of their roles, personnel working incontaminated Italian hospitals were more likely tobe seropositive than controls, although differencesemerged depending on the local environmentalcontamination. Legionella spp. antibody titres>1:256 have also been found in a significant pro-portion of hospital staff in Poland, and their speci-ficity reflected the distribution of environmentalisolates.17 Seroprevalence was associated withlength of service, whereas no association couldbe demonstrated with risk factors for disease orwith the occurrence of pneumonia or flu-like

symptoms. This suggests that the antibodiesmeasured represent the frequency of exposure tothe bacterium rather than to disease. Recent sur-veys of other legionella-contaminated working en-vironments documented increased antibody levelsamong personnel, but no cases of legionellosiswere recorded.12e14

As regards dental care services, our data suggestthat the risk of legionella infection due to watercontamination of dental equipment can be pre-vented by adopting rigorous disinfection proced-ures. Antibodies to L. non-pneumophila specieswere significantly more common among dentalstaff in Bari where L. non-pneumophila havebeen frequently isolated from dental units. By con-trast, in Turin no difference could be observed be-tween office and dental staff, probably becausedaily disinfection has been implemented. In a sim-ilar study carried out in London and NorthernIreland, the prevalence of L. pneumophila anti-bodies in dentists was low and did not exceedthe levels seen in blood donors, and environmentalcontamination was consistently very low.19

It is instructive to examine the different distri-bution and types of antibodies in the areasstudied. Healthcare personnel in Milan were sig-nificantly more at risk than blood donors, but thiswas not the case in Naples where both groups hadhigh seroprevalence (48 and 44%, respectively).One explanation is that persons living in Naplesreceive more exposure to legionella in the com-munity, perhaps because of the greater use of airconditioners, although this is speculation. How-ever, our data suggest that lifestyle factors such asalcohol consumption, more frequent exposure totap water and housing characteristics are riskfactors for increased exposure.

Antibodies to L. pneumophila serogroups 1e6were found in only 20 samples, i.e. 9.3% of totalantibodies and 3% of sera tested, even thoughthese serogroups are those most frequently in-volved in human disease.28 Interestingly, 14/20were detected in Naples, in both hospital workers(28.6% of positive sera) and blood donors (18.8% ofpositive sera). A previous study conducted in Na-ples on healthy persons confirmed this trend: usinga cut-off of 1:256, antibodies to serogroups 1e6were found in 23% of samples.29 This is in linewith environmental contamination in Naples,where 83% of hospital isolates were L. pneumo-phila serogroups 1 and 3, and 94% of domestic/hotel isolates were L. pneumophila serogroup1.4,24,27 By comparison, antibodies to L. pneumo-phila serogroups 7e14 and to L. non-pneumophilaspecies prevailed in Milan, and the frequency ofpositive sera was much lower. This partly reflects

154 P. Borella et al.

the environmental data for Milan, where L. pneu-mophila serogroups 2e14 are the predominant iso-lates. The reported incidence of Legionnaires’disease is much higher in Milan than in Naples.30

The reasons for this might be numerous, and in-clude differences in detecting and/or reportingcases, or different age composition of the two pop-ulations, but we also suggest that persistent andmore frequent contact with L. pneumophila sero-groups 1e6 from infancy can induce protectionagainst the disease later in life.

Excluding Naples, antibodies to serogroups 1e6were found in only six subjects out of 489 (1.2%),four of whom were from Turin hospital: threedental students and one office worker. In thishospital contamination by L. pneumophila se-rogroups 1 and 3 and non-pneumophila speciesprevailed, and numerous hospital-acquired caseshave been detected in spite of costly environmen-tal control measures.21 A previous study on blooddonors in Turin revealed that 0.3% had antibodiesto serogroup 1 (cut-off: 1:16),31 suggesting spor-adic exposure in the community. Under these con-ditions a marginal risk of developing disease mayexist for hospital staff, particularly those withrisk factors. In our study, persons positive for anti-bodies to serogroups 1e6 showed a significantlyhigher prevalence of pneumonia, not specificallyrelated to their hospital employment but more totypical risk factors for legionellosis (smoking, alco-hol and chronic disease).

In conclusion, hospital personnel may be athigher risk of developing anti-Legionella spp. anti-bodies as a result of exposure in the workplace. Wedid not demonstrate an association between sero-positivity and disease, however, probably becauseworkers are generally in good health which helpsto prevent the progression of infection to disease.

The public health implications of such wide-spread exposure to legionella in Italian hospitalsalso deserve attention. The facilities examinedhave a long history of hospital-acquired legion-ellosis, and have adopted preventive strategies toreduce water contamination, but eradication isdifficult particularly in old and large buildings.21e24

For instance in Turin the incidence of healthcare-associated legionellosis has not decreased despiteextensive disinfection procedures; we confirmedthe importance of daily disinfection of dentalunit water to avoid exposure in dental staff.21

Furthermore, generally only high-risk wards aresubjected to control measures according to thenational guidelines, such that little information isavailable for lower-risk environments.32 Strategiesfor preventing legionellosis in healthcare workersshould include attention to education on the

adverse effects of lifestyle factors such as smokingand alcohol consumption, and the use of protec-tive devices in at-risk persons in case of exposureto potentially contaminated aerosols.

Acknowledgements

We thank A. Collins for editing the English text.

Conflict of interest statementNone declared.

Funding sourcesThis work was supported by a grant from theItalian Ministry of University Research Projectsof National Interest, years 2003 and 2005.

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