A Look at COVID 19 in Terms of: 9. Using ATP & the Mycometer® for Assessing Cleaning Completeness

Abstract

ATP and Mycometer instrument theory and operational aspects to test surrogates for cleaning efficiency for COVID 19.

Full text

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ALookatCOVID19inTermsof:9.UsingATP&theMycometer®forAssessing
CleaningCompleteness

AndrewA.“Tony”Havics,CIH,PE
pH2,LLC
5250EUSHighway36,Suite830
Avon,IN46123
(317)7218‐7020Office
(317)409‐3238Cell

Introduction
ThisisacontinuationofaseriesofpapersonCOVID19withrespecttohealth&safety.Itfocuseson
twospecifictestingmethodsthatmightbeusedtoevaluatetheefficacyofcleaningprocessesforCOVID
19.Liketheotherpapers(mostinpreparation),thispaperisintendedtoprovidethefactsandresearch
supportwhereavailable,andthendrawfromthataswellastheauthor’sexperiencetoprovide
recommendationsbasedontheweightingoftheevidence.Thedataprovidedhere,andthe
recommendations,cannotbemaintainedinavacuum,thusotherCOVID19topicalpapersareintended
tofollowthisone,buteachwillhopefullybesufficientlyself‐containedtobeusefulandreliable.The
intendedaudienceofthispaperisprofessionals.Thisincludesindustrialhygienists,occupationaland
publichealthprofessionals,healthandsafetypractitioners,andmedicalpersonnel.Itisnotintendedfor
thegeneralpublic,thoughmanyindividualsmaybenefitfromreadingit.
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ATPReactionforTesting
AdenosineTriphosphate(ATP)isanenergycarryingmolecule(seeFigure1)inalllivingcells1andcanas
suchbetakenasindirectmeasureforcelldensity.VirusesdonotstoreATPandthusarenotabletobe
directlyevaluatedbyATPtests(ItisinterestingtonotethattheydohaveproteinmotorsthatactasATP
enzymes(4)).ATPforpurposeshereismeasuredasabioassay,wherealuciferaseenzyme(fromfirefly
Photinuspyralis)catalyzesanoxidationbyO2ofD‐luceferinusingATPasenergysource.Thus,reducing
AdenosineTriphosphatetoAdenosineDiphosphateandreleasingthefreedenergyaslight.

1Therearemanyinterestingdebatesoverwhethervirusesareliving(seeforinstance1 Forterre,P.:Tobeornotto
bealive:Howrecentdiscoverieschallengethetraditionaldefinitionsofvirusesandlife.StudiesinHistoryandPhilosophyof
SciencePartC:StudiesinHistoryandPhilosophyofBiologicalandBiomedicalSciences59:100‐108(2016),2 Brüssow,H.:The
notsouniversaltreeoflifeortheplaceofvirusesinthelivingworld.PhilosophicalTransactionsoftheRoyalSocietyB:
BiologicalSciences364(1527):2263‐2274(2009),3 Villarreal,L.P.:Arevirusesalive?ScientificAmerican291(6):100‐105
(2004).).Formypurposes,theydonotproduceATPandwillbeplacedinthenon‐livingcategoryforconvenience.

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Figure1.MolecularstructureofAdenosineTriphosphate(ATP)(afterCofitzen,2006)
Thelightfromthisreaction(seeFigure2)ismeasuredinaluminometer,typicallyasRelativeLightUnits
(RLU)andconvertedtoATPvaluesbyacalibrationcurvebasedonATPstandardsalt.Anumberof
commercialluminometersandreagent‐kitsexistonthemarked.Thesecanvarybyapplication,butall
includethefollowingsteps:a)additionofanextractionreagenttothesample,b)anextractionperiod,
c)additionoftheluciferase/luciferinreagents,d)anintegrationperiod.Themeasurementcanbe
usuallybeperformedinminutes.
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Figure2.ATPluminecencereaction(afterCofitzen,2006).
TheATPvaluecannotbeconverteddirectlytonumberofbacteriaorfungi,sincetheATPcontentofthe
singlecelldependsuponbacteria/fungitypeanditsgrowthphase.However,withinaspecific
environment,pragmaticcorrelationscanbedrawn.
Mycometer®forBacteria
FortheMycometer®,twomethodsareused,oneforbacteriaandoneforfungi.IntheBactiQuant
TM
for
bacteriamethodabacterialenzymehydrolase(BH)isusedasindirectmeasureofcelldensity.A
substratecontainingthefluorescentcompound4‐MethylUmbelliferone(MU)(SeeFigure3)bindswith
thespecificenzyme,therebyreleasingtheMU‐ion,whichthencanbedetectedinafluorometer.The
resultisprovidedasrelativefluorescenceunits(FE).

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Figure3.Molecularstructureoffluorescent4‐methylumbelliferoneusedintheBactiQuantTMmethod(afterCorfitzen,2006)
AswiththeATPmethodtheresultcannotbedirectlyconvertedtoabacteriacount,asthecontentof
thespecificenzymedependsonbacteriastrainanditsgrowthstages.Itmustbecorrelatedundermore
specificconditions/applications.
Mycometer®forFungi
TheMycometer®Surfaceformoldmethoduseafluorescentsubstrate,4‐methylumbelliferylN‐acetyl‐b‐
D‐glucosaminide(MU‐NAG)(SeeFigure4)toassesstheenzymaticactivityofN‐acetylhexosaminidase
(NAHA)(SeeFigure5).Thefluorescentcompound(MU)releasecanbedetectedinafluorometer.The
resultisprovidedasrelativefluorescenceunits(FE).
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Figure4.Thefluorescentcompound4‐methylumbelliferylN‐acetyl‐b‐D‐glucosaminide(MU‐NAG)usedinfungaldetection.

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Figure5.N‐acetylhexosaminidase[HexosaminidaseA](NAHA)usedtoassessfungalmass(Source:Wikipedia].
AswiththeATPmethodandtheMycometer®bacteriamethod,thefungalresultcannotbedirectly
convertedtoafungalcount,asthecontentofthespecificenzymedependsonfungaltaxaandits
growthstages.Itmustbecorrelatedundermorespecificconditions/applications.However,significant
correlationsbetweenNAHAandtotalsporecountswerefoundinairsamplesandindustgenerated
frombiomassinabiofuelplant(5,6).Strongcorrelationshavealsobeenfoundbetweenfungalbiomass
(gravimetricweight)andNAHAinfungalspeciesgrownonnutrientagarandbetweenergosteroland
NAHAactivityonmoldcontaminatedgypsumboards(7).AlinearcorrelationbetweenNAHAand
Aspergillusnigerbiomasshasalsobeenreported(8).ThesestudiesdemonstratethatNAHAcanbeused
asamarkerformoldcellbiomassundertheproperconditions.
ATPApplication
ATPtestingbyluminousreactionasahygienictoolhasbeenusedinthefood&beverageindustry(9‐12),
medicalfacilities(13‐17),offices(18),industrialwatertreatmentplants(19,20),manufacturing(21,22)and
schools(23,24).ATPhasbeenusedtoevaluatebacteria(9‐11,13‐17,19‐22,24),mold(7,9,12,21,25),andeven
viruses(18)underasurrogatebasis.Inonestudy(21)itwasfoundtohavealowerdetectionlimitthan
culturingofbacteriaandfungi.
Mycometer®Application
Usingspecificcellularcomponentsandafluorescentresponse,theMycometer®hasbeenusedtoassess
bacteria(19,20,23,26‐30)(viaandfluorescence),andmold(7,26,28,31‐36)(beta‐N‐acetylhexosaminidaseand
fluorescence).SimilartoATPithasbeenusedinfoodindustry(30),commercialofficespace(25,29,31,33),
industrialwatertreatmentplants(19,20,27),animalcarefacilities(32),andhomes(29,31,33‐35).The
manufacturehasrecommendedincreasedsurfaceareaforswabcollectionandusingalongerreaction

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timealongwithaloweredacceptablelimit(belowdetection)forthosewantingtoapplyittoCOVID
19(37).Thishasreportedresultedina10‐foldincreaseinsensitivity.
ApplicationforCOVID19
Asnotedabove,afewthingsareimportantcaveatsintheapplicationforATPandtheMycometer®to
determininglevelofcleanlinessforaCOVID19cleaningevaluation.ThefirstisthatneitherATPnorthe
Mycometer®directlydetermineviralloadinginanairborneenvironmentoronasurface.Theycan,
eachintheirownway,measureamixtureofbiologicalmaterialthatindicatehumancellularmaterial
(ATPonly),alongwiththatfromavarietyofbacteriaandfungi(24)(bothmethods).Suchmaterial
includesepitheliumfromupperrespiratorymucusmembranes(mouth,throat,nasalpassages)from
salivaandexudatesandassociatedmaterialfromcoughsandsneezesfrompersonswithviralaswellas
bacterialinfections.Becausevirusesareassociatedwithlivingcells(virusesneedthemtoreplicate)ATP
isanoverallgenericmarkerofbiologicalcontamination,anditallowsonetomonitorpotentialviral
contamination(fromviralinfections)indirectly(24).OnestudywithM‐2phage(virus)(18)foundthis
surrogateapproach(ATP)successful.Similarly,theMycometerprovidesawayofmonitoringbiological
load.Ingeneral,TheMycometer®ismorespecificandATPisbroaderinthescopeofbiologicindicators
beingmeasured.Becausebacteriaareorganismsmorelikelytocorrelatewiththepresenceofhuman
shedvirusesthanmoldwouldbe,theuseoftheMycometer®bacteriamethodismoreappropriatethan
thefungalmethodforassessingCOVID19cleaningprocesses.
Cautionarynotesareinorder.Thesemethodsassessbiologicalload,thiscanbebothaliveanddeadfor
ATP;itismostlyaliveforbacteriabytheMycometer®andacombinationofbothaliveanddeadfor
moldbytheMycometer®(37).Usingadisinfectantapplicationmethodthatisintendedtokillthese
organisms“oncontact”doesnotremovethemfromthesurfacenordoesitalterthe
chemical/molecularstructuresufficientlytoeliminatethemortheirconstituentparts.Oneshould
expecttofindresidueoftheseorganismsiftheyarenotphysicallyremovedfromasurface.Ifone
intendstouseATPortheMycometer®forassessmentofcleaning,oneshouldrequirephysicalcleaning.
OneshouldalsobeawarethatmanybiologicalsourceshaveATP,e.g.,wood,plants,insects,starchfrom
gloves,etc.Manysubstanceshavenaturalfluorescence:certainlivingorganisms,minerals,andeven
polymers.Thus,oneshouldrecognizethepotentialforfalsepositivesdependingonthesurface
contaminationakadirtanddebris).
Thesemethodsarebestappliedtoqualitativelyassessperformanceofcleaning,i.e.,don’tusethemto
showthatvirusesarepresent.Norshouldonestatethatapre‐cleaningfindingindicatesaknownrisk.
Thesetestsmay,ifproperlystructured,beabletoassessrelativecleaningeffectiveness(asopposedto
absolute),i.e.,pre:posteffects.Thesemethodsarecorrelatedbyprovidersofthekitsorinstrumentsfor
certaintypesofapplications,certainareasoftesting,andcertaintemperaturesandenvironmental
conditions.Notadheringtothesecorrelatingconditionswilllikelyresultinunusabledata.Onesample
isnotastatistic.Oneneedstocollectasufficientnumberofsamplestoproperlyassesstheexposure
and/orrisk.Theauthorhasfoundthatsomewherebetween5and20samplesfromahomogeneously
contaminatedandcleanedareaoftenprovidessufficientstatisticalpowertomakeinformed
decisions(38).Usersofthesetestsshouldprovideadequatesupportfortheirchoiceofnumberof
samplespriortosampling.
Inconclusion,bothATPandtheMycometer®Bactiquant®testingcanbeusedtoassessCOVID19
cleaningprocesscompletenessinasurrogatefashion,ifappliedproperlyandconsistently.Thesetests

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takelessthananhourtoperforminthefield.Thealternativeistoconductsamplingofsurfaceswith
swabsandhavethemshippedtoalabandanalyzedforparticularRNApatternsbyPCRthatwill
definitivelyidentifySars‐CoV‐22(COVID19)to5viralgenomeunitsperreaction–butthewaitis5‐7
days.

Disclaimers
TheauthorownsaMycometer®andhasuseditforevaluatingmoldandbacteria(includingMRSA),
performedtrialsonfoggingandsurfacecleaning,andhasperformedworkwithATPmeters.Theabove
doesnotconstituteanendorsementforeitherinstrumenttype,butratherapresentationofthedata
availabletotheauthor.Theauthorhasnotreceivedpayment,commission,orpreferentialservicefor
writingthispaper.TheauthordidcontactaMycometer®representativepriortocompletionofthis
papertoverifycertainclaimsmadebyothersabouttheirequipment.Usersshouldeducatethemselves
ontheirinstruments,theassumptionsandlimitationsoftheequipment,theenvironmentforsampling,
andthenmanagetheriskappropriately.
Acknowledgements
ThankstoSteveJahn,ScottArmour,andLisaRogersforquickreviewsofadraftofthispaper.Thanksto
MidwestRemediation,Indianapolis,Indiana,foruseoftheirfacilitiesandperformingmultiplecleaning
techniquesforassessmentbybothMycometer®andATPmeters.ThankstoAaronMankaforhishelp
onATPside‐by‐sidewithMycometerandBrianWilsonforhisworkregardingATPonjointprojects.
Also,DaveMedererforhishelpwithprojectworkusingtheMycometeronMRSA.

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