Download - Legionella Sampling
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 [email protected]
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 [email protected]
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
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
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
nce
med
ian
ne e e le
median 9,300 cfu/litre
Summary
12
14
Ref
eren
ants
med
ian
h pe
rcen
tile
h pe
rcen
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h pe
rcen
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perc
entilSummary
Median 9,300 (3.95)Acceptible 1,210 – 26,000 (3.1 – 4.4) 98% reported Legionella94% reported L pneumophila
10
mer
atio
ns
Parti
cip a5t 10t
90t 9 594% reported L. pneumophila
91% reported serogroups 2-14
6
8
No.
of e
num
2
4
0
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
edia
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24283236
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esu
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12162024N
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048
12
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
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