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John V Lee
Water and Environmental Microbiology Reference Unit, Gastrointestinal Emerging & Zoonotic Infections HPA Centre for Infections, 61 C Colindale li d l A Avenue, L London, d NW9 5EQ Email John.V.Lee@hpa.org.uk
The usefulness and interpretation of the result depends on the quality and timeliness of the sample
Advice on sampling
Standing Committee of Analysts 2005 The determination of Legionella bacteria in water and other environmental samples (2005) - Part 1 - Rationale of surveying and sampling Methods for the Examination of Waters and Associated Materials
Available free from the Environment Agency Website http://www.environmentagency.gov.uk/commercial/1075004/399393/4018 49/?version=1&lang=_e
Cost: 18 Order from: Dr. John V Lee, GEZI, GEZI HPA Centre for Infections, , 61 Colindale Avenue, London, NW9 5HT Email John.V.Lee@hpa.org.uk
Sampling
the result of the analysis of a sample must be representative of the situation when the sample was collected
biocides neutralise if possible transit time & storage conditions analyse ASAP but always within 24 hours of collection
Sampler training
basic microbiological sampling - aseptic technique knowledge of the ecology of the organisms and f t factors affecting ff ti it its occurrence knowledge g of risk assessment labelling and record keeping legal requirements continuity of evidence
Sampling Equipment
Keep a set of sampling equipment and materials ready for outbreak investigations
Interpretation of results
Biocide neutralisation
Oxidising biocides (chlorine, chlorine dioxide, bromine, ozone, iodine)
180 200 mg /L sodium thiosulphate pentahydrate should neutralise up to 50 mg/ L of free and combined chlorine
Safety
Take precautions to minimise exposure
Minimise aerosol formation A id exposure t Avoid to aerosols l
Run taps gently Dont operate sprays spra s and showers sho ers Switch off cooling tower circulation etc
Outbreak investigations
Sampler should not be in a highly susceptible group Sampler may (very rarely) need to be trained to wear RPE anticipate through knowledge of towers on register
Sample as near heat source as possible Neutralise biocides where possible Method with minimum theoretical mathematical detection limit of less than or equal to 100 cfu/l Laboratory should be UKAS accredited for the test and participate in a Legionella EQA scheme
Ideally sample from somewhere on the return to cooling tower Neutralise biocides where possible Method with minimum theoretical mathematical detection limit of less than or equal to 100 cfu/l Laboratory should be UKAS accredited for the test and participate in a Legionella g EQA scheme
Number / ml
<10 104 >104 - 105
Interpretation
Under control Retest immediately - if result is similar review control & risk assessment & carry out remedial actions Implement corrective action - Retest immediately, shot h t dose d with ith bi biocide. id R Review i control t l & risk i k assessment & carry out remedial actions identified
>105
Timing
Routine and problem solving
when (and where) biocide concentration is lowest When counts are likely to be highest
Just after the p pumps p are switched on When warmest
Outbreak
Always note when the last and next biocide additions are i relation in l ti t to th the ti time th the sample l i is t taken k
Sample point
Interpretation
Under control Retest immediately - if result is similar review control & risk assessment & carry out remedial actions ti Implement corrective action - Retest immediately, shot h t dose d with ith bi biocide. id R Review i control t l & risk i k assessment & carry out remedial actions identified
>103
Concentration of L. pneumophila by culture (and microscopy) in cooling towers that were still infectious at the time of sampling
O tbreak Outbreak
BBC London 1988 BAe, Bolton 1988 Wi Wisconsin i Retirement hotel, USA Hotel, Sydney Delaware 1994
cf /litre cfu/litre
106 (109) 105 (107) 106 9 x 106 2.8 x 107 3.4 x 106 2 - 9 x106 1 - 2 x 106
Reference
Westminster Action Committee 1988 Lee, unpublished Addi et Addis t al. l 1989 Breiman et al 1990 Bell et al 1996 (2 towers) Brown et al. 1999
Slide 22 S3
Surman-Lee, 15/02/2007
* in HSE Guidance
site for routine monitoring (not after a TMV) site for investigative monitoring
Outbreak investigation
Sample outlets used by case or that case is likely to have been exposed to.
Expansion vessels
Expansion vessels are pressurised and contain a butyl bladder into which the water can expand Should be sited so that they do not get warm ideally on cold supply after non-return valve Should have valve at bottom to enable sampling
Pressurised system with instantaneous heater, hot water storage, storage and expansion vessel vessel.
For p plate heat exchanger g heated systems with large hot water demands a hot water storage vessel (buffer) may still be required these can be a focus for legionella growth just like storage calorifiers and should be treated accordingly
Expansion vessel Buffer / hot water storage vessel Plate heat exchanger Drain valve (use for sampling)
>103
N.B. Applies to samples of from taps connected directly to the hot / cold water pipes and not to samples collected after a TMV or through a mixer *, L8 says 1 or 2 but suggest <10%
Spa pools
As a minimum always sample the pool and the balance tank (where ( e e fitted) tted) When definitely i incriminated i i t d also l consider biofilm samples l from f within ithi pipes including air lines li
Interpretation
Under control Disinfect, drain, clean. Review control & risk assessment & carry out remedial actions identified. R fill and Refill d retest t t next td day and d2 2-4 4 weeks k l later t Immediate I di t closure, l i f inform CCDC disinfect, CCDC, di i f t drain, d i clean. Review control & risk assessment & carry out remedial actions identified. Refill and retest. Keep closed until negative results and satisfied risk assessment is satisfactory (training, maintenance, record keeping etc)
>103
Ensure samples are representative of whole system e.g. extra bathrooms and outlets used by patient
16 14 12
10 8 6 4 2 0
cfu/L (log10)
90th percentile e
95 5th percentil le
No. of results N
Log cfu/L
Summary Median 200 (2.3) Acceptable 51 600 (1.7 2.75) 52% reported Legionella 48% reported L. pneumophila SG. 1
Laboratory Factors
Poor media QC Volume processed Concentration factor Background flora Colony recognition
Reducing variation
Good laboratories will minimise the variation due to methods, h d differences diff between b technical h i l staff, ff poor media, etc by having a good quality system, ensuring staff are adequately trained and their competence regularly checked, media are prepared correctly and quality controlled, etc. Good laboratories should be UKAS accredited and will participate in an external quality assurance scheme such as those run by the HPA However no matter how good the laboratory is there will still be apparent variation between laboratories by chance
What result will you get if you analyse a second subsample from the same sample bottle?
Count you observed in first subsample p Range of count expected 19/20 ( (95%) ) times if you y analyse another subsample (i.e. 95% CI) 0-5 0 - 14 1 - 16 3 - 22 9 - 35 32 - 72
0 5 6 10 20 50
Typing L. pneumophila
L.pneumoph hila
Species L. pneumophila
10
11
12
13
15
16
Serogroups 1 16
Monoclonal subtypes
3/1
3/1 causes most cases (Helbig et al 2002) a.k.a. Pontiac (Watkins et al 1985 & mAb2+ve (Joly et al 1986)
SBT types of England & Wales community acquired clinical and unrelated environmental isolates
Clinical Number L. p serogroup 1 Sg 1 mono 3/1 yp No. of ST types ST 47 ST 37 ST62 ST1 ST79 167 97.6% 91.6% 42 25.7% % 11.4% 9.0% 4 8% 4.8% 1.2% Environmental 276 55.8% 8.3% 82 0.4% 0.7% % 0 19 6% 19.6% 14.5%
T. G. Harrison et al. 2009 Eur J Clin Microbiol Infect Dis in press, published on web http://www.springerlink.com/content/101941/?Content+Status=Accepted
Sampling - conclusions
Samplers need training Results esu ts need eed ca careful e u interpretation te p etat o co consult su t your you environmental microbiologist Microbiological g sampling p g must be done in conjunction j with other investigations Essential in outbreaks Of value in monitoring control / preventative measures Use UKAS accredited labs that participate in the HPA EQA Legionella scheme or equivalent
Be prepared
Do you know where all the local cooling towers are?
If there is a register is it up-to-date?
Do you have correct up-to-date out of hours contact details for all cooling g tower operators p Do you have a memorandum of understanding between you, the local Health Protection Unit and laboratory and the HSE Do you have staff trained and practiced in sampling
Be prepared (continued)
Is it possible you will need RPE to sample and if so i there is h anyone trained i d to use RPE or do d you know k where there is? Is your local laboratory accredited for legionella analyses in environmental samples if not where is the nearest and is there a service level agreement (SLA) with them (NB in outbreak investigations only HPA laboratories should be used) Do you have contact details for the experts who may be needed Do you have a potential incident room identified and IT backup etc
Guidance
Joint HSE & HPA Guidance: Management of Spa Pools: C t lli the Controlling th Risk Ri k of f Infection. London: Health Protection Agency. 2006 ISBN 0 901144 80 0 110 pages Can be purchased from the HPA www.hpa.org.uk
European Guidelines for Control and Prevention of Travel Associated Legionnaires' Disease
WHO book
also be available on WHO website
Overflow (deck level) pool with balance tank - suitable for commercial use
Required by UK & most European standards for medium to heavy bathing loads. Overflow (deck-level) spa pools maintain the water level at a constant height with the excess water overflowing into a balance t k th tank then replaced l d when h bathers get out y Water filtered and continuously dosed with chlorine
Free 3 5mg/l Combined: less than 1 mg/l
pH
7.0 7.4