INVESTIGATION OF THE PREVALENCE OF LEGIONELLA SPECIES IN HEATING VENTILATION AIR CONDITIONING (HVAC) SYSTEMS

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1 INVESTIGATION OF THE PREVALENCE OF LEGIONELLA SPECIES IN HEATING VENTILATION AIR CONDITIONING (HVAC) SYSTEMS F Martz 1*, S Parat 2, H Fricker-Hidalgo 3, C Pinel 3 and C Pascual 1 1 Research and Development Center, Elyo, Lyon, France 2 Air & Bio, Grenoble, France 3 Service de parasitologie-mycologie, Centre hospitalier universitaire (CHU), Grenoble, France ABSTRACT The aim of this study was the Legionella risk assessment in the water of HVAC systems equipped with cooling coils in France. Here, that means assess whether or not a HVAC system could actually be a source of Legionella by itself, or if it only remains a potential means for conveying a contamination. The prevalence of amoebae was also performed at the same time. Forty three different HVAC systems were investigated during the summer of All samples were collected from cooling coils condensate tanks. Despite the presence of a great number of other organisms, no Legionella sp was isolated by culture from any water sample. No correlation between Legionella, total bacteria and Gram negative bacteria was found. Out of 43 samples, 27 (63%) were found positive for amoebae with high semiquantitative levels. Different criteria like the climate and the Legionella's ecology account for the absence of Legionella. INDEX TERMS HVAC, Legionella, Free-living amoeba, Exposure assessment, Survey. INTRODUCTION The first outbreaks of legionellosis greatly reported in France at the beginning of 1998 were due to cooling towers. But quickly there has been a mix up in the newspapers articles between cooling towers and the HVAC systems (Cadiergues, 1998). A cooling tower is an evaporative heat transfer device in which atmospheric air cools warm water with direct or indirect contact between the water and the air, by evaporating part of the water. But the atmospheric air used in this kind of device is not the same air which goes through the HVAC system. In some cases however, the air spread from a cooling tower could be closed to the air intake of an HVAC system. And in such cases, the cooling tower could be a potential source of contamination of the HVAC system. Although it is frequently asserted that HVAC systems are a potential source of contamination by Legionella, no investigation of Legionella prevalence in HVAC cooling coils were carried out in Europe prior to this study. In particular, most of Legionella guidelines supply recommandations about HVAC systems (ASHRAE, 2000; HSC, 2000). Moreover, in Europe, no case of legionellosis due to a contamination of the cooling coils condensate tanks have been reported. The parts of the HVAC systems which could constitute a Legionella risk are the cooling coil condensate tank, the humidifier tank and the condensation puddles through the air ducts. The humidifiers were already associated with legionellosis cases. In the first report of a legionnaires'disease outbreak associated with an ultrasonic humidifier in 1989 (Mahoney, Hoge, Farley, et al., 1992), 33 patients were hospitalized with legionnaires'disease in Bogalusa, Louisiana. The case-control study revealed that case-patients were more likely than controls to have been shopping in a grocery store and to have bought produce items located close to an ultrasonic mist machine. The first european * Contact author francoise.martz@elyo.fr 383

2 legionnaires'disease due to an humidifier was reported in 1999 (CDR, 2000). Molecular typing showed that isolates of Legionella pneumophila serogroup 1 (sg 1) from two patients who died from legionnaires'disease were indistinguishable from isolates from a food display humidifier in the dining room of a hotel visited by the patients. These two cases show that some kinds of humidifiers may behave as a vehicle for Legionella transmission. As far as cooling coils condensate tanks are concerned, no investigation or contamination has been published. The main purposes of this study were : (1) to provide data on the prevalence of Legionella sp. in the water of the cooling coils condensate tanks from a randomly-selected sample of HVAC systems in South-East France, and (2) to investigate factors (eg water temperature, stagnant water quantity ) that could be associated with such contamination. METHODS A total of 43 different HVAC systems in South-East France were investigated during summer Out of the 43 systems, 22 used only outdoor air and 21 outdoor air and a part of recirculated air. All samples were collected directly from cooling coils condensate tanks and never from the drainage valve. The number of analysis realised was respectively : 54 for Legionella, 43 for free-living amoebae (29 water samples and 14 biofilm swabs), 16 for total bacteria and 16 for Gram negative bacteria. For Legionella, the number of samples is higher than the number of systems studied because some samples were taken several times on the same system but in different conditions during the summer : at the beginning of the cooling time, at the end of the cooling time, several times on the whole cooling time in order to have a good assessment of the prevalence of Legionella during the whole cooling time. Physical and chemical parameters were also recorded at the same time for each sample. Only sterile bottles were used throughout the study, made of plastic for Legionella and made of glass for amoeba. The water was sampled from the tank with sterile glass syringe for amoeba and sterile plastic syringe for Legionella. A new sterile plastic syringe was used for each isolate. The glass syringe was cleaned after each sample with deionised water. The samples were taken during the day, handed to a conveyor at the end of the day, stored in a cold store at +4 C for Legionella and +22 C for amoeba and delivered to the laboratory the next morning before noon. Analytical methods: The Legionella were sought using a culture method in accordance with the French standard (AFNOR, 1993) required by the French regulations. The medium used is the GVPC medium (the GVPC medium is a Buffered Charcoal Yeast Extract -α (BCYE-α) medium supplemented with cysteine, ferric pyrophosphate, glycine and filter-sterilized antibiotics). The sample is divided in different parts, some for direct incubation, some for concentration by filtration, centrifugation and acid treatment and thermal treatment. All the plates are incubated at 37 C +-1 C for 10 days and examined at 3, 5 and 10 days. Colonies with a morphology similar to that of Legionellaceae are subcultured on different BCYE mediums and on blood agar plates; those that fail to grow on the blood agar plates but grow on BCYE with cysteine are identified as Legionella. On these colonies, the identification of Legionella pneumophila sg1 is confirmed by immunofluorescence test. There is no standardized method for the analysis of amoeba. A direct examination of samples was done after centrifugation. The sampled water was concentrated by filtration through a 1.2 µm pore-size filter. The membrane was directly incubated onto a non-nutritional agar covered with Escherichia coli. The biofilm swabs were directly incubated on the same medium. The plates were incubated at 27 C and examined at 48 hours, 5, 10 and 20 days. When the plates were negative, they were examined for one month. The positive results were given with semiquantitative estimations (+ = rare, ++ =,few, +++ = many) and genus identification. The semi-quantitative estimations were performed by evaluating the progression of amoebae growth, which means by correlating the number of amoebae and the incubation time. Direct microscopical investigations and various coloration tests were performed on positive samples. 384

3 RESULTS Despite the presence of a great number of other organisms in the collected water, no Legionella sp was isolated by culture from any of the 54 water samples analysed for Legionella. The results of Legionella were always less than 50 CFU/l (Colony Forming Unit per liter) what means "no detectable" (50 CFU/l is the limit of detection for the French Legionella culture method). As regards the total bacteria, 9 samples out of 16 were negative (0 to 49 CFU/ml) and 5 out of the 7 positive samples contained more than CFU per ml. As regards the Gram negative bacteria, 10 samples out of 16 were negative and 3 out of the 6 positive samples contained more than CFU per ml. The total bacteria found in the cooling coils condensates appeared to be mainly Gram negative bacteria. Samples number Total bacteria Gram - bacteria < 50 < 10^3 10^3 < < 10^4 > 10^4 CFU per ml Figure 1. Total bacteria and Gram negative estimations on the 16 water samples collected in 16 HVAC systems. In 4 samples out of 16, Legionella, total bacteria, Gram negative bacteria and amoeba were not detected. In 4 samples out of 16, Legionella, total bacteria and Gram negative bacteria were not detected but amoebae were found positive : the only genus always found was Acanthamoeba sp. On the whole, no correlation between Legionella, total bacteria and Gram negative bacteria was found. The number of total bacteria could not be used as an indicator of Legionella prevalence. However detecting high levels of total bacteria indicates a non hygienic environment and a potential poor indoor air quality. Out of 43 samples, 27 (63%) samples were found positive for amoebae with high semiquantitative evaluations. Out of 27 positive samples, 14 samples had at least one amoeba genus equal to +++, 5 equal to ++ and 8 equal to +. The main genera found were Acanthamoeba sp (22/27, 81%), Hartmanella sp (11/27, 41%), Naegleria sp (9/27, 33%) and Valkampfia sp (1/27). Fourteen out of 27 positive samples included at least two genera at the same time. The associated genera were : Acanthamoeba sp + Hartmanella sp (7/14, 50%), Acanthamoeba sp + Naegleria sp (3/14, 22%), Hartmanella sp + Naegleria sp (1/14, 7%), Acanthamoeba sp + Valkampfia sp (1/14, 7%), Acanthamoeba sp + Hartmanella sp + Naegleria sp (2/14, 14%). The proportion of amoeba genera was not the same between the systems using only outdoor air and the ones using outdoor air and a part of recirculated air. The systems using a part of recirculated air look favour the presence of Acanthamoeba sp. (figures 2 & 3). 385

4 80% 70% 60% 50% 40% 30% 20% 10% 0% Acanthamoeba Naegleria Valkampfia Hartmanella negative Figure 2. Proportion of amoeba genera in the systems with a part of recirculated air 60% 50% 40% 30% 20% 10% 0% Acanthamoeba Naegleria Valkampfia Hartmanella negative Figure 3. Proportion of amoeba genera in the systems with only outdoor air DISCUSSION In France, the cooling coil condensate tanks don't seem to be a potentially hazardous component towards contamination by free-living Legionella. The short cooling time linked to a low humidity rate and low air temperatures implies little stagnant water. Indeed, the months of June and July were quite cold : from the 06/26/00 to 09/27/00, the mean air temperature was 24.4 C and the mean humidity rate 51%. That is because the HVAC systems were put on only at the beginning of July. Moreover, the mean temperature of all the water samples that means of the stagnant water was 16.4 C which is not the adequate temperature to allow Legionella's growth. Nevertheless, the study showed an important presence of free-living amoebae. But, amoebae and particularly Acanthamoeba, Naegleria and Hartmanella are well known to be potential hosts for Legionella (Harb and Kwaik, 2000). Furthermore, the growth of Legionella in amoebae is supposed to enhance the virulence and thus the efficient inoculum rate could be lower (Cirillo, Cirillo, Yan, et al., 1999; Neumeister, Reiff, Faigle, et al., 2000). That's why one of the study's follow-up issues will be to check the detection of Legionella inside amoebae or to check if the number of Legionella is lower than 50 CFU/l as soon as appropriate routine detection methods are available. 386

5 The second explanation for the absence of Legionella could be the presence of viable but nonculturable Legionella. The only detection method in that case would be a molecular biology method like PCR (Polymerase Chain Reaction). But one may question the meaning of the viable but non-culturable state, regarding the human infection risk, and in particular about pathogenicity, virulence and contamination risk of such a state? CONCLUSION AND IMPLICATIONS As people spend most of their time at work, it was essential to evaluate as a priority the risk level due to air conditioning in office buildings regarding Legionella. The results can be extrapolated to other buildings, for the technical features of most French office buildings HVAC systems are similar to those investigated, as are the environmental and climatic conditions. The follow-up issues are to carry out similar investigations on the one hand in different meteorological conditions such as in a tropical climate (with high temperature and high hygrometry) which produces continuous condensates and could stand for a higher risk than in France, and on the other hand with new analysis methods, in order to improve the Legionella detection sensitivity. ACKNOWLEDGEMENTS We thank all the study participants for their co-operation, in particular Stéphanie Arnaud from the French laboratory ALPA in Montmélian and R. Grillot from the CHU of Grenoble. This work was supported by the Suez Innovation Found, France. 387

6 REFERENCES AFNOR AFNOR Standard T90-431, Recherche et dénombrement des Legionella et Legionella pneumophila, Paris : Association Française de Normalisation. ASHRAE ASHRAE Guideline , Minimizing the Risk of Legionellosis Associated with Building Water Systems, Atlanta : American Society of Heating, Refrigerating, and Air-Conditioning Engineers, Inc. Cadiergues R Erreurs et vérités sur la légionellose. Chauffage, Ventilation, Conditionnement d'air. Vol. 11, pp CDR Legionella from guests of Welsh hotel indistinguishable from humidifier isolates. CDR weekly. Vol.10, No. 16. Cirillo JD, Cirillo SLG, Yan L, et al Intracellular Growth in Acanthamoeba castellanii Affects Monocyte Entry Mechanisms and Enhances Virulence of Legionella pneumophila. Infection and Immunity, Vol. 67, No. 9, pp Harb OS, and Kwaik YA Interaction of Legionella pneumophila with Protozoa Provides Lessons. ASM News, Vol. 66, No. 10, pp HSC L8 Legionnaires' disease, The control of legionella bacteria in water systems, Health & Safety Commission. Mahoney FJ, Hoge CW, Farley TA, et al Communitywide Outbreak of Legionnaires' Disease Associated with a Grocery Store Mist Machine. The Journal of Infectious Diseases. Vol. 165, pp Neumeister B, Reiff G, Faigle M, et al Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes. Applied and Environmental Microbiology, Vol. 66, No. 3, pp