Sampling for Legionella

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

Milton Keynes October 2009

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

BS 7592 Sampling of water and related materials for Legionella bacteria

Under revision will be a Code of practice to be published later this year

BS 7592:2008 Sampling p g for Legionella Bacteria in Water Systemsy Code of Practice

with its own Webpage on the BSi Website
www.bsigroup.com/bs7592.

HPA DVD Water testing for L i Legionella ll explained l i d

Four short films
1. 2. 3 3. 4. General background Basic sampling techniques S Sampling li i in th the fi field ld Laboratory techniques

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

the sample should represent the greatest risk likely

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

Steps in the examination of water for Legionella species

Sampling Processing
Concentration Detection Identification Typing yp g

Interpretation of results

The purpose of sampling

To determine if the risk is controlled
Sample should be representative
- Of whole system y - Of situation at that time Neutralise biocides at time of sampling (sufficient thiosulphate to neutralise up to 50 mg/l chlorine or more )

Sample if possible under worst case conditions

Minimum biocide concentration The water likely y to be aerosolised The points most likely to be colonised The points most likely to have infected the patient

Protect sample from changes during transport

- Protect from light and heat

Analyse as soon as possible after collection and certainly within 24 hours

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

Silver and copper ?

Possibly EDTA at 10mg / L NOT sodium thioglycollate

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

Routine Legionella testing - cooling towers

Test at least quarterly and more often:
during commissioning; to establish a new treatment plan; p ; if towers cannot be cleaned twice a year; if risk assessment indicates it is necessary (not in ACOP)

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

Sampling cooling towers for Legionella

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

COOLING WATER : Heterotrophic Colony C Count t (A (Aerobic bi C Colony l C Count) t)

Synonyms ACC, TVC, Colony Count,

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

Tested weekly; count incubated at 30oC for at least 48 hours.

Sampling cooling systems - where

Routine samples p based on risk assessment
Warmest point Possibly the pond (furthest away from fresh water inlet ball valve if possible) Any y others suggested gg by y the risk assessment

Investigative samples as for routine and possibly

S Supply l water t Make up water Biofilm (slime) from pack, and other components Foam

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

Sampling cooling systems

Compressor

Sample point

Dip samples and swabs

avoid cross contamination
wear disposable plastic/latex gloves use sterile wrapped bottles or disinfect outside of bottle b f before sampling li change gloves between sample site

In exceptional circumstances RPE during outbreak investigations may be needed

Legionella count in cooling water

Number / ml
102 >102 - 103

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

Tested at least quarterly

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

Sample selection survey of water systems

The choice of sampling point requires detailed knowledge of the lay-out of the water system to be examined, and a thorough understanding of the ecology of the organism prior to taking any sample, Establish the nature of the system and all equipment that utilises water or generates aerosols. In outbreak I b k investigations, i i i there h may be b no information i f i available on the lay-out of the system or of previous risk assessments assessmentstherefore need to do one to support the outbreak investigation and the health and safety interests of sampling staff ! patient/s has/have been Establish which outlets the p exposed to.
S3

Slide 22 S3
Surman-Lee, 15/02/2007

Legionella sampling hot / cold water systems

In hot water systems treated with biocides where temperatures are reduced from the recommended monthly and review after 1 year when frequency may be reduced if confident the efficacy of biocide regime is established In systems where the control levels of the treatment regime (e.g. temperature, biocide levels) are not consistently achieved frequent (weekly) samples .. until the system is brought back under control Outbreaks Hospital wards with susceptible patients

Sampling cold water systems

Cold water storage tank* O tl t - furthest Outlets f th t from f tank* t k* High g risk s a areas eas in hospitals* osp ta s Warmest points WC cisterns

* in HSE Guidance

Hot water samples: 1

C l ifi drain Calorifier d i Calorifier outlet or nearest tap to it Calorifier return or nearest tap to it Furthest from calorifier Coolest Outlets of particular concern - wards with high risk patients S Sample l each h ring i

site for routine monitoring (not after a TMV) site for investigative monitoring

Routine sampling hot water: 2

Routine monitoring
Do not normally sample through thermostatic mixer valve (TMV) controlled taps or showers samples should be representative of the circulating system TMV controlled t ll d outlets tl t may need d sampling li subject bj t to risk assessment

Outbreak investigation
Sample outlets used by case or that case is likely to have been exposed to.

Water Systems in outbreak investigations

Plant / equipment & components associated with system eg pipework,pumps, tanks,valves, showers,chillers, heat exchangers etc Deadlegs, D dl test t t loops, l injection i j ti moulding ldi machines Humidifiers, spa baths, pools, indoor fountains etc Air scrubbers, effluent disposal plants, i i ti systems, irrigation t etc. t

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)

Legionella count in hot / cold water (L8)

No. / litre 102 >102 - 103 Interpretation Under control If only 1 or 2* samples positive, resample then if result is similar review control & risk assessment & carry out remedial actions If majority j it positive iti - consider id disinfection, di i f ti immediately review control & risk assessment & perform remedial actions Retest immediately. Review control & risk assessment & carry out remedial actions identified, possibly including disinfection

>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%

Legionella in hot/cold water

In outbreaks associated with hot / cold water systems the majority of samples are usually positive Results should be interpreted p on the basis of the proportion of samples positive. Suggest that if greater than 30% are positive then disinfection is indicated. Individual high (>1000 cfu/litre) should be investigated further but whole system disinfection may not be warranted subject to risk assessment. Samples p from showers and down stream of TMVs will often be positive there is no control that works consistently at these points.

Diagram of a typical spa pool indicating g potential sampling points

Potential sample point

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

Legionella counts in spa pools

No. / litre
102 >102 - <103

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

Sampling private dwellings

Sampling of households for Legionella species Prepared by Dr John V Lee & Dr Susanne B Surman http://www.hpa.org.uk/infections/topics_az/legi htt // h k/i f ti /t i /l i onella/sampling.htm

Situations justifying sampling private dwellings

HPA (PHLS) Guidelines for Investigating single cases of legionnaires disease Lee J V and Joseph C 2002 Guidelines for Investigating g g Single g Cases of Legionnaires g Disease. Communicable Disease and Public Health 5(2): 157-162. http://www.hpa.org.uk/cdph/issues/CDPHvol5/No2/guide li lines1.pdf 1 df
Case not linked to recognised outbreak Incubation time suggests infection could have occurred in hospital or home Returned R df from travel l and d symptoms started d 2 10 d days after f d date of f return

Samples from domestic properties

Hot water system header tank Immediate samples from nearest and furthest hot taps Post flush sample from disinfected hot tap (not a mixer tap if possible) = sample representative of water in the system) Shower Cold water samples
Bathroom cold tap
, sample, temperature, water supply (pressure)

Incoming mains (usually kitchen cold) Water closet cistern / cisterns

Ensure samples are representative of whole system e.g. extra bathrooms and outlets used by patient

Response to legionella samples in domestic premises

Think about possible outcomes before undertaking sampling in privately owned single dwellings. Similar to other hot and cold water in principle Advise disinfection, modification of system and its operation as appropriate. Dont keep going back and resampling

Your system is safe because we have not detected legionellae

HPA EQA Scheme for the detection of Legionella in water y PHLS in 1993 Started by Over 220 participants:
Austria, 13; Belgium, 2; Brazil, 1; Cyprus, 3; Czech Republic Republic, 2; Denmark Denmark, 4; Eire Eire, 7; Finland, 1; France, 2; Germany, 5; Hong Kong, 2; Hungary, 2; Israel, 2; Italy 41; Japan, Italy, Japan 4; Malta Malta, 1; Norway Norway, 1; Portugal, 8; Singapore, 5; Slovenia, 4; South Africa, 2; Spain, 17; Sweden, 5; Switzerland, 7; Turkey, 2; U.A.E, 1; USA 4 U.S.A, 4; E England, l d 60 60; N N. I Ireland, l d 1 1; Scotland, 13.

Laboratory performance in 1993

Extremely variable both between and within ithi laboratories l b t i Some inadequate Standards of reports extremely variable No Legionella detected could mean:
Less than 1 cfu / l to Less than 60,000 / l

Performance f has improved BUT

16 14 12

Participa ants median n Referen nce median

Sample G49C L. pneumophila sg1 median 9 9,300 300 cfu/litre

Summary Median 9,300 (3.95) Acceptible 1,210 26,000 (3.1 4.4) 98% reported Legionella 94% reported L. L pneumophila 91% reported serogroups 2-14
5th percentile e 10th percentile e

No. of enum merations

10 8 6 4 2 0

cfu/L (log10)

90th percentile e

95 5th percentil le

Sample G47B L. pneumophila sg 1, median 200 cfu / litre

64 60 56 52 48 44 40 36 32 28 24 20 16 12 8 4 0
90th percentile 10th pe ercentile 5th pe ercentile Participants and r reference me edian 95th percentile

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

Between lab. variation: - reasons

Sampling p g errors
Time between sampling and processing Biocide neutralisation

Laboratory Factors
Poor media QC Volume processed Concentration factor Background flora Colony recognition

Natural variation - statistics

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

Variation in the source water

Organisms are distributed at least at random in the original environmental source. If they were strictly distributed at random their distribution would follow the Poisson distribution but in reality in nature the distribution of organisms is greater than random they are said to be over dispersed. This is caused by y a number of factors e.g. g clumping p g and grazing
e.g. in a study of the incidence of indicator bacteria in natural waters 6 samples were collected 1 meter apart at the surface, 30cm and 100cm. The distribution of results was significantly greater than random. (PHLS
Water Working Group, 1995 Preliminary study of microbiological parameters in eight inland recreational waters. Letters in Appl. Microbiol. 21: 267 - 271. )

Variation within the sample

Even in well mixed samples the organisms are still distributed at least at random

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

So how do you know your laboratory is reliable?

Ensure it is UKAS accredited How informative are its it s reports Ask for copies of its methods Ask what their policy is if the plates are overgrown with other organisms Ask to see its long term performance assessment in the EQA scheme it participates in
Remember the laboratory may occasionally get a sample with a low or high count by chance. It is the overall performance over several distributions that is important important.

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)

Molecular typing (sequence based typing)

sequences of parts of 7 genes

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

http://www.ewgli.org/data/european _g guidelines/european p _g guidelines_j ja n05.pdf

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

Spa pools / hot Tubs

Conventional (rim) types - water level 150mm to 200mm below the top to accommodate bathers. Suitable for domestic use only

Diagram of a typical commercial spa pool

(from Health Protection Agency /HSE guidelines)

Why are Spa Pools a problem? bl ?

Elevated temperature p 30 - 40C - encourages g growth g High organic load - nutrients washed off bathers by air and water jets . High bather density and water reused B bbli creates Bubbling t aerosol l at t head h d level l l Air and water circulation systems provide a large surface area for biofilm growth and Pipes p often inaccessible and not readily y removable for cleaning to remove biofilm Biofilm (slime) organisms are more resistant to treatment Air system often impossible to clean and disinfect Balance B l tanks k i inadequately d l cleaned l d and d often f inaccessible for cleaning A single modern spa pool may contain as much as 75 metres of flexible and fixed pipes with a total surface area of 550m2

Balance tanks inadequately q y cleaned and often inaccessible for cleaning