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Legionella is the causative agent of Legionnaires’ disease. The bacteria are widespread in nature and man-made water systems. In Denmark, approximately 120 cases are diagnosed each year, and the disease can be fatal. Legionella pneumophila is the species responsible for approximately 95% of cases. The transmission pathway is through inhalation of contaminated water droplets mainly from technical systems such as hot water systems. Overall, the aim of this PhD thesis was to improve the background knowledge to accomplish risk assessment regarding Legionella in water systems. Based on a literature review and the results from the PhD work the following subjects were addressed: a) prevalence of Legionella in habitations, b) validation of the use of qPCR in risk assessment in hot water systems, c) clarifying risk factors mainly associated with Legionella in habitations, and d) discussion of interventions which could be used to overcome or prevent a Legionella colonisation in water. The standard method to quantify Legionella in water samples is culturing, but since it has long response time (7-14 days) faster methods are needed to evaluate if Legionella is present and to quantify the numbers to assess the risk. Quantitative real-time polymerase chain reaction (qPCR) is an alternative and /or a supplement and two qPCR assays targeting Legionella species and Legionella pneumophila were implemented and validated. Limit of detection for Legionella species was found to be 833 GU/L and for Legionella pneumophila 5000 GU/L. Limits of quantification of the assays were 3333 GU/L for the Legionella species assay, and 8333 GU/L for the Legionella pneumophila assay. The efficiency was 91.6% and 96.6% respectively. Both assays were tested on real life water samples from mixed sources (cooling towers, hospital water, schools and private residents). Analysing these randomly collected samples with the qPCR assay targeting Legionella pneumophila and traditional culture good correlation ( N = 43, r=0.77) was found. The assays were also applied in a risk assessment of a newly built residential area with a cluster of Legionnaires´ disease cases. These samples also included sampling before and after interventions such as thermal treatment and hyperchlorination. When all samples from this location were pooled the quantification of Legionella by q-PCR and by culture did not correlate well. However, when the samples were grouped according to their type and how they were collected, such as e.g. ‘circulation water’ and ‘water from first flush from shower hoses’, culture and qPCR showed the same tendencies. Because the ranges of Legionella concentration found by qPCR between and after the thermal treatments overlapped, it was difficult to interpret the specific amount. In samples collected from the first flush from empty apartments, culture and qPCR were inconclusive. The literature studies showed that Legionella is widely dispersed in habitations all over the world, including in Denmark. Different major risk factors were identified: Temperature not sufficient to suppress growth of Legionella. Water tapped from water systems using centralised heating or distant heating was more often colonised than water from systems with instantaneous heaters (no water tank). Most studies showed that copper material suppressed growth of Legionella. Presence of other bacteria and amoebae had a positive effect on growth and survival of Legionella, since Legionella situated inside amoebae were better protected against thermal treatments than free in the water phase. Different treatments can be implemented to overcome Legionella colonisation. Other have shown that more permanent, long-term water treatments, such as copper-silver ionisation, addition of chlorine dioxide or monochloramine to the portable water can be effective against Legionella colonisation, though none of the methods completely eradicated Legionella in all treatet water systems. However, in case of an outbreak/cluster immediate interventions are needed. Two immediate treatments (thermal treatment and hyperchlorination) were investigated in a newly built residential area with a cluster of Legionnaires’ disease cases. The newly built residential area constituted the main area for this PhD study. Raising the temperature in the boilers to 70°C for 24 hours followed by three weeks at 65°C in the boilers, flushing of all taps and shower hoses and hyperchlorination of boilers caused a notable decrease in the number of Legionella. Before the thermal treatment, circulation water contained up to 1.2 * 104 colony-forming units (CFU)/L, but after the treatment, no or very limited Legionella was observed by culture for at least seven months. An important factor when controlling Legionella in a water system is the daily operation of the system. Water should be > 50°C at all taps.
|Place of Publication||Kgs. Lyngby, Denmark|
|Publisher||Technical University of Denmark|
|Number of pages||57|
|Publication status||Published - 2011|
Microbiological risk assessment of urban water. Development of methods for detection and analysis of pathogens with Legionella as model organism
15/04/2008 → 21/09/2011