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

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DOI: 10.13140/RG.2.2.24131.14884
Cite this publication
Abstract
ATP and Mycometer instrument theory and operational aspects to test surrogates for cleaning efficiency for COVID 19.
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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.
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.
1There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.
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).
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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©2020A.Havics
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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  • Article
    Fungal exposure may induce respiratory symptoms. The causative agents are compounds in the fungal cell wall. Fragments of microbes may be present in air samples but are not measurable using conventional spore counting or by the determination of viable organisms. This study assesses the proportion of fungal cell biomass and endotoxin in different particle size fractions in air samples from homes. Air samples were collected from 15 homes using a cyclone sampler, collecting particles in three aerodynamic size fractions: <1.0, 1.0-1.8, and >1.8 μm. N-Acetylhexosaminidase (NAHA) was determined as a marker of fungal cell biomass. Endotoxin was determined using the Limulus amebocyte lysate method. NAHA and endotoxin in the size range <1.0 μm comprised up to 63% (mean 22.7%) and 96.3% (mean 22.6%) of the total concentrations, respectively. There were significant relationships between the amounts of NAHA and endotoxin in the total amount and in the size fraction >1.8 μm but not in the smaller fractions. The results demonstrate significant amounts of fungal cell biomass and endotoxin in particles <1.0 μm. Homes with reported mold damage had a lower concentration of NAHA in particles <1.0 μm than homes without mold damage. To assess airborne exposure for diagnostic and preventive purposes, measurement techniques that include this fraction should be considered. © 2013 John Wiley & Sons A/S232 April 2013 10.1111/j.1600-0668.2012.00799.x Original Article Original Articles
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