Sampling for Legionella

John V Lee

Water and Environmental Microbiology Reference Unit,Gastrointestinal Emerging & Zoonotic InfectionsHPA Centre for Infections,61 C li d l A L d NW9 5EQ61 Colindale Avenue, London, NW9 5EQ

Email John.V.Lee@hpa.org.uk

Milton Keynes October 2009

The usefulness andThe usefulness and interpretation of the result depends on the quality and timeliness of the sampletimeliness of the sample

Advice on samplingAdvice on samplingStanding Committee of Analysts 2005 The determination of Legionella bacteria indetermination of Legionella bacteria in water and other environmental samples (2005) - Part 1 - Rationale of surveying and sampling Methods for theand sampling Methods for the Examination of Waters and Associated MaterialsAvailable free from the Environment Agency Website

http://www.environment-agency.gov.uk/commercial/1075004/399393/401849/?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 gfor Legionella Bacteria in Water Systems- Code of yPractice

with its own Webpage on the BSi WebsiteBSi Website

www.bsigroup.com/bs7592.

HPA DVD – Water testing for L i ll l i dLegionella explained

Four short filmsFour short films1. General background2. Basic sampling techniques3 S li i th fi ld3. Sampling in the field4. Laboratory techniques

Cost: £18 Order from:Dr. John V Lee,GEZIGEZI,HPA Centre for Infections,61 Colindale Avenue, ,London, NW9 5HTEmail John.V.Lee@hpa.org.uk

SamplingSampling

the result of the analysis of a sample must bethe result of the analysis of a sample must be representative of the situation when the sample was collectedwas collectedbiocides – neutralise if possible

transit time & storage conditions

analyse ASAP but always within 24 hours of collectionanalyse ASAP but always within 24 hours of collection

the sample should represent the greatest risk likelylikely

Sampler trainingSampler training

basic microbiological sampling - aseptic techniquetechnique

knowledge of the ecology of the organisms and f t ff ti itfactors affecting its occurrence

knowledge of risk assessmentg

labelling and record keeping

legal requirements – continuity of evidence

Sampling EquipmentSampling Equipment

Keep a set of sampling equipment and materials ready for outbreak investigationsmaterials ready for outbreak investigations

Steps in the examination of water for Legionella species

Sampling

ProcessingConcentrationConcentration

Detection

Identification

Typingyp g

Interpretation of results

The purpose of samplingThe purpose of sampling

To determine if the risk is controlledTo determine if the risk is controlled• Sample should be representative

- Of whole systemy- 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 to be aerosolisedy- The points most likely to be colonised- The points most likely to have infected the patient

• Protect sample from changes during transportProtect sample from changes during transport- Protect from light and heat

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

Biocide neutralisationBiocide neutralisation

Oxidising biocides (chlorine, chlorine dioxide, bromine, ozone, iodine)bromine, ozone, iodine)180 – 200 mg /L sodium thiosulphate pentahydrate should neutralise up to 50 mg/ L of free and combined chlorineneutralise up to 50 mg/ L of free and combined chlorine

Silver and copper ?Possibly EDTA at 10mg / L

NOT sodium thioglycollateNOT sodium thioglycollate

SafetySafety

Take precautions to minimise exposureTake precautions to minimise exposureMinimise aerosol formation

A id t lAvoid exposure to aerosols• Run taps gently

Don’t operate spra s and sho ers• Don’t operate sprays and showers• Switch off cooling tower circulation etc

Outbreak investigationsOutbreak investigationsSampler 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 towersTest at least quarterly and more often: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 possibleNeutralise biocides where possible

Method with minimum theoretical mathematical detection limit of less than or equal to 100 cfu/ldetection limit of less than or equal to 100 cfu/l

Laboratory should be UKAS accredited for the test and participate in a Legionella EQA schemeparticipate in a Legionella EQA scheme

Sampling cooling towers for Legionella

Ideally sample from somewhere on Ideally sample from somewhere on the return to cooling towerthe 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 EQA schemeg

COOLING WATER : Heterotrophic Colony C t (A bi C l C t)Count (Aerobic Colony Count)Synonyms – ACC, TVC, Colony Count,

Number / ml Interpretation<104 Under control10 Under control

>104 - 105 Retest immediately - if result is similar review control & risk assessment & carry out remedialcontrol & risk assessment & carry out remedial actions

>105 Implement corrective action - Retest immediately, h t d ith bi id R i t l & i kshot dose with biocide. Review control & risk

assessment & carry out remedial actions identified

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

Sampling cooling systems - whereSampling cooling systems where

Routine samples based on risk assessmentpWarmest point

Possibly the pond (furthest away from fresh water inlet ball valve ifPossibly the pond (furthest away from fresh water inlet ball valve if possible)

Any others suggested by the risk assessmenty gg y

Investigative samples as for routine and possiblyS l tSupply water

Make up water

Biofilm (slime) from pack, and other components

Foam

TimingTiming

Routine and problem solvingRoutine and problem solvingwhen (and where) biocide concentration is lowest

When counts are likely to be highest• Just after the pumps are switched onp p• When warmest

OutbreakOutbreak

Always note when the last and next biocide additions are i l ti t th ti th l i t kin relation to the time the sample is taken

Sampling cooling systemsSampling cooling systems

Compressor

Sample point

Dip samples and swabsDip samples and swabs

avoid cross contaminationwear disposable plastic/latex gloveswear disposable plastic/latex gloves

use sterile wrapped bottles or disinfect outside of bottle b f libefore sampling

change gloves between sample site

In exceptional circumstances RPE during outbreak investigations may be neededoutbreak investigations may be needed

Legionella count in cooling water

Number / ml InterpretationNumber / ml Interpretation

102 Under control10 Under control

>102 - 103 Retest immediately - if result is similar reviewcontrol & risk assessment & carry out remedial

tiactions

>103 Implement corrective action - Retest immediately, h t d ith bi id R i t l & i kshot dose with biocide. Review control & risk

assessment & carry out remedial actions identified

Tested at least quarterly

Concentration of L. pneumophila by culture (and microscopy) in cooling towers that were(and microscopy) in cooling towers that were still infectious at the time of sampling

O tbreak cf /litre ReferenceOutbreak cfu/litre ReferenceBBC London 1988 106 (109) Westminster Action Committee

1988BAe, Bolton 1988 105 (107) Lee, unpublished

Wi i 106 Addi t l 1989Wisconsin 106 Addis et al. 1989

Retirement hotel, 9 x 106 Breiman et al 1990USA

Hotel, Sydney 2.8 x 107 Bell et al 1996 (2 towers)

3.4 x 106

Delaware 1994 2 - 9 x106

1 2 x 106

Brown et al. 1999

1 - 2 x 106

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. I b k i i i h b i f iIn outbreak investigations, there may be no information available on the “lay-out” of the system or of previous risk assessments-risk assessmentstherefore need to do one to support the outbreak investigation and the health and safety interests ofinvestigation and the health and safety interests of sampling staff !Establish which outlets the patient/s has/have been pexposed to. S3

Slide 22

S3 Surman-Lee, 15/02/2007

Legionella sampling – hot / coldLegionella sampling hot / cold water systemsIn hot water systems treated with biocides where temperatures are reduced from the recommended –monthly and review after 1 year when frequency may bemonthly and review after 1 year when frequency may be reduced if confident the efficacy of biocide regime is establishedIn 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 controlOutbreaksHospital wards with susceptible patients

Sampling cold water systemsSampling cold water systems

Cold water storage tank*

O tl t f th t f t k*Outlets - furthest from tank*

High risk areas in hospitals*g s a eas osp ta s

Warmest points

WC cisterns

* in HSE Guidance

Hot water samples: 1

C l ifi d iCalorifier drainCalorifier outlet or nearest tap to itnearest tap to itCalorifier return or nearest tap to itnearest tap to itFurthest from calorifierCoolestOutlets of particularOutlets of particular concern - wards with high risk patientsS l h i

site for routine monitoring (not after

Sample each ring a TMV)

site for investigative monitoring

Routine sampling hot water: 2Routine sampling hot water: 2

Routine monitoringRoutine monitoringDo not normally sample through thermostatic mixer valve (TMV) controlled taps or showers samplesvalve (TMV) controlled taps or showers – samples should be representative of the circulating system

TMV t ll d tl t d li bj tTMV controlled outlets may need sampling subject to risk assessment

Outbreak investigationSample outlets used by case or that case is likely toSample outlets used by case or that case is likely to have been exposed to.

Water Systems in outbreakWater Systems in outbreak investigations

Plant / equipment & components associated with system eg pipework,pumps, tanks,valves, with system eg pipework,pumps, tanks,valves, showers,chillers, heat exchangers etc

D dl t t l i j ti ldiDeadlegs, test loops, injection moulding machines

Humidifiers, spa baths, pools, indoor fountains etc

Air scrubbers, effluent disposal plants, i i ti t tirrigation systems, etc.

Expansion vesselsExpansion vessels

Expansion vessels are pressurisedExpansion 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 and expansion vesselhot water storage, and expansion vessel.

For plate heat exchanger p gheated systems with large hot water demands a hot water storage vessel (buffer)water storage vessel (buffer) may still be required – these can be a focus for legionella growth just like storage calorifiers and should be treated accordinglytreated accordingly

Expansion vessel

Buffer / hot water storage vessel

Plate heat exchangerPlate heat exchanger

Drain valve (use for sampling)

Legionella count in hot / cold water (L8)

No. / litre Interpretation

102 Under control102 Under control

>102 - 103 If only 1 or 2* samples positive, resamplethen if result is similar review control & riskassessment & carry out remedial actions

If j it iti id di i f tiIf majority positive - consider disinfection,immediately review control & riskassessment & perform remedial actions

>103 Retest immediately. Review control & riskassessment & carry out remedial actionsidentified, possibly including disinfectionidentified, possibly including disinfection

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 mixerwater 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 waterLegionella in hot/cold water

In outbreaks associated with hot / cold water systems the majority of samples are usually positive

Results should be interpreted on the basis of the pproportion 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 from showers and down stream of TMVs will poften be positive – there is no control that works consistently at these points.

Diagram of aDiagram of a typical spa pool indicating gpotential sampling points

Potential sample point

Spa poolsSpa pools

As a minimum always sample the pool and sample the pool and the balance tank (where fitted)( e e tted)

When definitely i i i t d lincriminated also consider biofilm

l f ithisamples from within pipes including air lilines

Legionella counts in spa poolsLegionella counts in spa pools

No. / litre InterpretationNo. / litre Interpretation

102 Under control

>102 - <103 Disinfect, drain, clean. Review control & risk assessment & carry out remedial actions identified. R fill d t t t d d 2 4 k l tRefill and retest next day and 2-4 weeks later

I di t l i f CCDC di i f t d i>103 Immediate closure, inform CCDC, disinfect, drain,clean. Review control & risk assessment & carry outremedial actions identified. Refill and retest. Keepclosed until negative results and satisfied riskclosed until negative results and satisfied riskassessment is satisfactory (training, maintenance,record keeping etc)

Sampling private dwellingsSampling private dwellings

Sampling of households for Legionella speciesPrepared by Dr John V Lee & Dr Susanne B Prepared by Dr John V Lee & Dr Susanne B Surman

htt // h k/i f ti /t i /l ihttp://www.hpa.org.uk/infections/topics_az/legionella/sampling.htm

Situations justifying samplingSituations justifying sampling private dwellings

HPA (PHLS) Guidelines for Investigating single cases of legionnaires’ disease

Lee J V and Joseph C 2002 Guidelines for Investigating Single Cases of Legionnaires’ Disease. g g g gCommunicable Disease and Public Health 5(2): 157-162. http://www.hpa.org.uk/cdph/issues/CDPHvol5/No2/guideli 1 dflines1.pdfCase not linked to recognised outbreak

Incubation time suggests infection could have occurred in hospital or home

R d f l d d 2 10 d f d fReturned from travel and symptoms started 2 – 10 days after date of return

Samples from domestic propertiesSamples from domestic propertiesHot water system header tankImmediate 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 thetap if possible) = sample representative of water in the system)ShowerCold water samplesBathroom cold tap• , sample, temperature, water supply (pressure)Incoming mains (usually kitchen cold)

Water closet cistern / cisternsWater closet cistern / cisterns

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

Response to legionella samplesResponse 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.

Don’t keep going back and resampling

“Your system is safe because we have not detected legionellae”HPA EQA Scheme for theHPA EQA Scheme for the detection of Legionella in waterStarted by PHLS in 1993

Laboratory performance in 1993Extremely variable both between and

ithi l b t iy

Over 220 participants: Austria, 13; Belgium, 2; Brazil, 1; Cyprus, 3; Czech Republic 2; Denmark 4; Eire 7;

within laboratories

Some inadequate

Standards of reports extremelyCzech Republic, 2; Denmark, 4; Eire, 7; Finland, 1; France, 2; Germany, 5; Hong Kong, 2; Hungary, 2; Israel, 2;Italy 41; Japan 4; Malta 1; Norway 1;

Standards of reports extremely variable

“No Legionella detected” could mean:Italy, 41; Japan, 4; Malta, 1; Norway, 1;

Portugal, 8; Singapore, 5; Slovenia, 4; South Africa, 2; Spain, 17; Sweden, 5; Switzerland, 7; Turkey, 2; U.A.E, 1; U S A 4 E l d 60 N I l d 1

mean:

• Less than 1 cfu / l to• Less than 60,000 / l

fU.S.A, 4; England, 60; N. Ireland, 1; Scotland, 13.

Performance has improved

BUT

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

16

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median 9,300 cfu/litre

Summary

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Median 9,300 (3.95)Acceptible 1,210 – 26,000 (3.1 – 4.4) 98% reported Legionella94% reported L pneumophila

10

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90t 9 594% reported L. pneumophila

91% reported serogroups 2-14

6

8

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num

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cfu/L (log10)cfu/L (log10)

64

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

52566064

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Log cfu/LSummaryMedian 200 (2.3)Acceptable 51 – 600 (1.7 – 2.75) 52% reported Legionella48% reported L. pneumophila SG. 1

Between lab. variation: - reasonsBetween lab. variation: reasons

Sampling errorsp gTime between sampling and processing

Biocide neutralisationBiocide neutralisation

Laboratory FactorsPoor media QC

Volume processed

Concentration factor

Background flora

Colony recognition

Natural variation - statisticsNatural variation statistics

Reducing variationReducing variationGood laboratories will minimise the variation due to

h d diff b h i l ffmethods, differences between technical staff, poor media, etc by having a good quality system, ensuring staff are adequately trained and their competencestaff 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 HPAsuch as those run by the HPAHowever no matter how good the laboratory is there will still be apparent variation between laboratories bystill be apparent variation between laboratories by chance

Variation in the source waterVariation in the source water

Organisms are distributed at least at random in theOrganisms are distributed at least at random in the original environmental source. If they were strictly distributed at random their distribution would followdistributed 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 a number of factors e.g. clumping y g p gand grazinge.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 mixedEven 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

subsampleRange of count expected 19/20 (95%) times if you p ( ) y

analyse another subsample (i.e. 95% CI)

0 0 - 55 0 - 146 1 - 1610 3 - 2220 9 - 3550 32 - 7250 32 72

So how do you know your laboratory is reliable?

Ensure it is UKAS accredited

How informative are it’s reportsHow informative are it s reports

Ask for copies of it’s 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 inRemember 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 importantdistributions that is important.

Typing L. pneumophila

L.pneumoph

Species L. pneumophila

hila

1 2 3 4 5 6 7 8 9 10 11 12 13 1 15 16 Serogroups 1 –16

Monoclonal subtypes3/1 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– sequences of parts of 7 genes

SBT types of England & Wales community acquired clinical and unrelated environmental isolatesclinical and unrelated environmental isolates

Clinical Environmental

Number 167 276

L. p serogroup 1 97.6% 55.8%

Sg 1 mono 3/1 91.6% 8.3%

No. of ST types 42 82yp

ST 47 25.7% 0.4%

ST 37 11.4% 0.7%% %

ST62 9.0% 0

ST1 4 8% 19 6%ST1 4.8% 19.6%

ST79 1.2% 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 - conclusionsSampling conclusions

Samplers need trainingResults need careful interpretation – consult your esu ts eed ca e u te p etat o co su t youenvironmental microbiologistMicrobiological sampling must be done in conjunction g p g jwith other investigationsEssential in outbreaksOf value in monitoring control / preventative measuresUse UKAS accredited labs that participate in the HPAUse UKAS accredited labs that participate in the HPA EQA Legionella scheme or equivalent

Be preparedBe preparedDo 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 tower operatorsg p

Do you have a memorandum of understanding between you, the local Health Protection Unit andbetween you, the local Health Protection Unit and laboratory and the HSE

Do you have staff trained and practiced inDo you have staff trained and practiced in sampling

Be prepared (continued)Be prepared (continued)

Is it possible you will need RPE to sample and if so i h i d RPE d kis there anyone trained to use RPE or do you know where there is?Is your local laboratory accredited for legionellaIs 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 outbreakagreement (SLA) with them (NB in outbreak investigations only HPA laboratories should be used)Do you have contact details for the experts who may be neededDo you have a potential incident room identified and IT backup etc

GuidanceGuidance

Joint HSE & HPA Guidance:

Management of Spa Pools: C t lli th Ri k fControlling the Risk of Infection. London: Health Protection Agency. 2006 ISBN 0 901144 80 00 901144 80 0

110 pages

Can be purchased from theCan 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/europeanguidelines/european guidelines ja_g p _g _j

n05.pdf

WHO bookalso be available on WHO websitealso be available on WHO website

Overflow (deck level) pool with balance tank - suitable for commercial useRequired by UK & mostRequired by UK & most European standards for medium to heavy bathing loads. Overflow (deck-level) spa poolsOverflow (deck-level) spa pools maintain the water level at a constant height with the excess water overflowing into a balance t k th l d htank then replaced when bathers get outWater filtered and continuously ydosed with chlorine Free 3 – 5mg/lCombined: less than 1 mg/lCombined: 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 Diagram of a typical commercial spa pool (from Health Protection Agency /HSE guidelines)(from Health Protection Agency /HSE guidelines)

Why are Spa Pools a bl ?problem?

Elevated temperature 30 - 40C - encourages growthp g gHigh organic load - nutrients washed off bathers by air and water jets .High bather density and water reusedB bbli t l t h d l lBubbling creates aerosol at head levelAir and water circulation systems provide a large surface area for biofilm growth and Pipes often inaccessible and not readily removable for p ycleaning to remove biofilmBiofilm (slime) organisms are more resistant to treatmentAir system often impossible to clean and disinfectB l k i d l l d d f

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 cleaned and often inaccessible for cleaning

Balance tanks inadequately cleaned and Balance tanks inadequately cleaned and q yq yoften inaccessible for cleaningoften inaccessible for cleaning