Team:Baskent Meds/Lpdetect

Standart legionella detection and prevention technics

Protozoans, which are present inside biofilms and residues on warm and moist surfaces, act as a host for Legionella. Especially when hot water systems and aerosolized usage of water are considered, Legionella pneumophila generates serious threat against human health. Therefore, precautionary measures are being taken in order to prevent reproduction and biofilm production. At the new standard health care service regulation, an article about “control of pathogenic biological agents in cooling towers, domestic hot water systems and other water injection systems” is present. Five types of disinfection methods are being used in order to eliminate the risks at pipe systems that include hot water tanks, cooling towers and hot water tubs. Thermal eradication (Superheat-And-Flush) method is executed by heating the system to at least 66.1°C then circulating water in the system. Hyperchlorination method is carried out by applying high concentrations of chlorine to hot water tanks. With ultraviolet light method, legionella bacteriae are eradicated before they enter the water distribution system of building by ultraviolet radiation. In ozonation method, ozone is injected into water system. The most practical way of protection from Legionella bacteriae for domestic hot water systems is copper-silver ionization method. When there is a doubt of contamination or clinical symptoms are seen on individuals who use the building, clinical samples and surfaces should be scanned for Legionella pneumophila.
Diagnostic methods for Legionella species are divided into two categories. These are, methods used for clinical diagnosis of illness and methods used for determining contamination risk at water systems. Especially for identification from clinical isolates, selective medium culture of Legionella pneumophila is required for both diagnosis and advanced subtyping. In standard culture methods, while scanning for Legionella pneumophila, clinical isolates or water samples are inoculated into either charcoal yeast extract (CYE) or buffered charcoal yeast extract (BCYE) agar. Cultures are incubated at 35-37°C for 2 to 3 days. Colonies are distinguished both morphologically and biochemically. In the process of subtyping of clinical isolates, direct immunofluorescence or molecular methods are used. To diagnose the disease, indirect immunofluorescence of serum or urine is also used for certain subtypes. The basic method for scanning of surface or water contamination is culturing of samples in selective medium, but before the culturing step, isolates are filtrated and treated with heat and acid. Then, samples are inoculated into BCYE agar. Serotyping and subtyping of cultures can be done by usage of commercial kits such as direct immunofluorescence and latex agglutination. Alternatively, isolates can be cultured in selective liquid GVPC (glycine – vancomycin - polymyxin - cyclohexamide) medium. DNA is isolated from cultures by using commercial kits, then serotyping and subtyping are done by simultaneous quantitative polymerase chain reaction (PCR) using species specific primer sequences.

Technical problems detected on previous methods

Maintenance of a building’s hot water reservoir, cooling tower and plumbing systems is the basic process which limits Legionella organisms’ to reproduction and spreading, and prevents the illness. Therefore, physical and chemical procedures are being applied. Since thermal eradication method is executed at 66°C, procedure should only be done in very well controlled conditions. Disinfection of domestic hot water systems using chemical methods is neither easy nor safe, because of the risk of consumption of that water by individuals. For example; hyperchlorination method is not safe to apply on drinking water systems and ozonation is a rather expensive method. Considering all these methods, colonization control on potential reproduction surfaces is substantially difficult and costly.
In many countries, environmental Legionella monitorization is advised. Performing standard culture methods and detection of bacteria in samples may last up to 10 days. When detection with simultaneous quantitative polymerase chain reaction is considered, method itself reduces required time. Also, by usage of commercial kits, extraction and DNA isolation steps may be shortened. However, in order this method to give accurate quantitative results at adequate titration, sensitive standardizations must be done for different types of environmental samples. Besides that, for this method to be used, laboratory conditions in which, nucleic acid contamination is minimized to be suitable for routine application of molecular biology techniques, pre and post PCR processes are executed in different physical environments, are required. Because of commercial kits, price of equipment used and qualified personnel, cost per sample is high.

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