Lecture3 Drug Resistance - Evolutionary...

Theevolu)onofdrugresistance

Drugresistancedescribestheabilityofaparasiteorpathogentoovercomeoura9emptsto

controlthem.

Drugresistancehasevolvedtobecomeaworldwidehealththreat.Itistheresultof

evolu)onbynaturalselec)on.

1Lecture“Evolu)onofhost–parasiteinterac)ons”,DieterEbert

Evolu)onofdrugresistance1.  Formsofresistance(describingthephenomenon)

–  Resistancetodrugs–  Resistancetovaccines(e.g.Marek’sdisease)–  Resistancetodiagnos)ctests

2.  Gene)cmechanismsleadingtodrugresistance–  Pointmuta)ons–  Copynumbervaria)on–  Gene)crecombina)on–  Horizontalgenetransfer

3.  Doesevolu)onofresistancealwaysoccur?4.  Howdoparasitesevolveresistance

–  Ageneralmodel

5.  Resistanceacquisi)on:Withinandbetweenhostprocessofdrugresistance6.  Canwestoptheevolu)onofdrugresistance?(“evolu)onproofing”)

–  Resistancevariesinspaceand)me–  Uselessdrugs;cyclingandmixing–  Reducepopula)onsize(hithardandstrictadherence!)–  Combina)ontherapy–  Specificsugges)ons

7.  Costsofdrugresistance8.  Summingitup

2

What’stheproblem?

Whatisthemechanism?

Onemechanismormany?

Doweunderstandtheprocess?

Whatshouldwestopd

oing?

1.Formsofresistance:Resistancetodrugs

Infec)onscausedbyEnterococcusincludeurinarytractinfec)ons,bacteremia,bacterialendocardi)s,diver)culi)s,andmeningi)s.Sensi)vestrainsofthesebacteriacanbetreatedwithvancomycin.

Year

%vancomycinresistant

EnterococcusisolatesinUShospitals

1.Formsofresistance:Resistancetodrugs

Klebsiellapneumoniaecarbapenemase(KPC)-producingKlebsiellapneumoniaeisapathogencausinganepidemicofcarbapenem-resistantEnterobacteriaceae(CRE)inhealthcaresegngsinmanycountries.StatesoftheUSwithKPC-producingCarbapenem-resistantEnterobacteriaceae(CRE)reportedtotheCentersforDiseaseControlandPreven)on(CDC)asofFebruary2015demonstratetherapidspreadofthesebugs.

InJan.2001onlyNorthCarolinareportedCRE!

1.Formsofresistance:ResistancetodrugsManyparasites/pathogensevolveresistancetodrugs

Malaria•chloroquineresistancewasreportedfrom81of92countries

Tuberculosis(TB)•0-17%ofisolatesshowprimarymul)-drugresistance

Gonorrhoea•5-98%penicillinresistanceinNeisseriagonorrhoeae

Pneumoniaandbacterialmeningi9s•0-70%penicillinresistanceinStreptococcuspneumoniae

Diarrhoea:shigellosis•10-90%ampicillinresistance,5-95%cotrimoxazoleresistance

Hospitalinfec9ons•0-70%resistanceofStaphylococcusaureustoallpenicillinsandcephalosporins

Drugresistanceevolu)oncostsabout50Billion$ayearinUSAalone.Wearepugnglessthan1%ofthemoneythatweputintodrugdiscoveryintoresearchonhowtomakethesedrugsmoresustainableinthefaceofevolu)on.

1.Formsofresistance:Resistancetovaccine

6

Step-wiseevolu)onofvirulenceofMarek’sdiseasevirus(MDV).Rela)onshipbetweenthevirulenceincreaseandtheintroduc)onofdifferentvaccinesisshown.HVT,herpesvirusofturkeys;bivalent,HVTandserotype2(SB-1)vaccines;Rispens,CVI988strain.

year

Con)nuumofMDVvirulence.ComparisonofthevirulenceranksofMDVisolates,groupedintovMDV,vvMDVorvv+MDVpathotypes.

Nair2005.TheVeterinaryJournal

1.Formsofresistance:resistancetodiagnos)ctestNewvariantofChlamydiatrachoma9s

Chlamydiatrachoma9sisanobligateintracellularpathogenandcancausesevereinfec)onsinhumans(urethri)s,proc))s(rectaldiseaseandbleeding),trachoma,infer)lity).Itismostlysexuallytransmi9ed.AnewvariantofC.trachoma9s(nvCT)wasdiscoveredinSwedenin2006thathada377-bpdele9oninaplasmid.Thedeletedareaincludedthetargetsequenceusedbygene)ctestsforC.trachoma9smanufacturedbytwocommercialcompanies,Abbo9m2000(Abbo9)andAmplicor/TaqMan48(Roche).Thisdele)onresultedinseveralthousandfalse-nega)veresults,andthepropor)onofnvCTrecordedwasbetween20%and64%inswedishcoun)esusingtestsystemsthatwereunabletodetectnvCTin2006/2007.Adifferentgene)ctestusedinSweden,wastheProbeTecsystemfromBectonDickinson(BD).ThissystemhasalwaysbeenabletodetectnvCT,becauseanothertargetregiononthecryp)cplasmidisused.Thepropor)onofnvCTincoun)esusingBDwasbetween7%and19%duringthesame)meperiod.Themuta)onwhichhadleadtothe377bpdele)onwasadap)veforthebacterium,asitescapeddetec)onandthereforetreatment.Inswedishcoun)eswherediagnos)ctestsfailedtodetectnvCT,prevalenceincreaseddras)cally.

Klintetal2010.ClinicalMicrobiol&Infec9on

1.Formsofresistance:resistancetodiagnos)ctestNewvariantofChlamydiatrachoma9s

%nvCT

year2007 2008 2009

Counties using always

BD test.

Counties using only since end 2007 BD tests.

Aserthenon-func)onaltestswerereplacedinlate2007bynewtests(BectonDickinson=BD)theincidenceofnvCTdeclined.

2.Gene)cmechanismsleadingtodrugresistancePointmuta)ons

9

Muta)onsleadingtoresistancetodrugsusedagainstPlasmodium.

Modifiedfrom:h9p://www.tulane.edu/~wiser/protozoology/notes/drugs.html

2.Gene)cmechanismsleadingtodrugresistancePointmuta)ons

10

Typicalmolecularmechanismsofresistance:

•  Altera)on,circumven)onoftargetprotein

•  Overexpressionoftargetprotein•  Degrada)onofdrug•  Effluxpumps

•  Changingpermeability

Evolu)onarytheorycanpredictthatevol.ofdrugresistancewilloccur,butcannotpredictthenatureoftheresistancemechanism.

2.Gene)cmechanismsleadingtodrugresistanceCopynumbervaria)on

Nairetal.2008.PLoSGene)cs

Laos:Fansidarhardlyusedun)l2006

Thailand:Fansidarused

intensivelysince1970

IncreasedcopynumberofthegchgeneincreasesresistanceagainstFansidar.

2.Gene)cmechanismsleadingtodrugresistanceGene)crecombina)on

12

Gene)crecombina)onallowsthenewcombina)onofmuta)onsfromdifferentindividuals.Itisdetectablebytheabsenceofcongruenceingenetrees.

Manyvirusesundergofrequentrecombina)on(osencalledre-assortment)e.g.HIV,InfluenzaA

Highrecombina)onrates:noclonalstructuredetectable

e.g.Neisseriagonorrhoeae,Helicobacterpylori

Intermediaterecombina)onrates:clonalstructuredetectablee.g.Neisseriameningi9dis,Staphylococcusaureus,Streptococcuspyogenes,Strep.pneumoniae

No/Verylowratesofrecombina)on:clonalstructurelonglas)ng

e.g.Salmonellaenterica

Feiletal.2001.PNAS

2.Gene)cmechanismsleadingtodrugresistanceHorizontalgenetransfer

13

Resistancetovancomycinwasfirstreportedin1988forEnterococcusfaecium.ResistanceismediatedbyTn1546amobilegene)celements.Highlevelvancomycinresistanceappeared10yearslaterinStaphylococcusaureus.Gene)canalysessuggeststhetransferofvancomycinresistancetoamethicillin-resistantS.aureusoccurredinvivobytransferofTn1546fromEnterococcus.

TheplasmidpLW1043withtheinser)onsiteandgene9celementofTn1546(innercircleinlightgreen).

9genesareencodedontheTn1546transposon.Thevangenesprovidehighlevelvancomycinresistance:ORF1andORF2aretransposaseandresolvaseenzymes,respec)vely,requiredformobiliza)onofthetransposon.

3.Doesevolu)onofresistancealwaysoccur?

14FromJ.Antonovicsetal.(unpublishedmanuscript).

Plasmodiumfalciparumdevelopedresistancetochloroquineinthe1950s.P.vivaxdidnotevolveresistancetochloroquineun)l40yearslaterinspiteofextensiveuse.

Penicillinhasbeenanan)bio)cagainstTreponemapallidum,thebacteriumcausingsyphilis,forover60years,butthereisnorecordthatthebacteriumhasevolvedpenicillinresistance.Ithashoweverevolvedresistancetoothermorerecentlyintroducedan)bio)cs.

WhileTreponemalacksgenesfortheclassicalβ-lactamasesthatgivepenicillinresistanceinotherbacteria,itdoeshaveaprotein,Tp47,thatcleavespenicillin,butitisineffec)vebecausetheproductsofthiscleavageinhibitfurtherenzymeac)vity(Chaetal.2004).WhyTreponemahasnotacquiredmuta)onstoavoidsuchend-productinhibi)onremainsamystery,evenmoresoasthedomainthatisinhibitedisnottheac)vecleavagesite.

Smallpoxwasaninfec)ousdiseaseuniquetohumans,causedbyVariolamajorandVariolaminor.Inthe1970sitwaseradicatedwiththehelpofaworldwidevaccina)oncampaign.

Thereseemtobelimitstoevolu)on!!!

3.Doesevolu)onofresistancealwaysoccur?

Smallpoxisoneoftwoinfec)ousdiseasestohavebeeneradicated,theotherbeingrinderpest,whichwasdeclarederadicatedin2011.Neithervirusevolvedtoovercomethevaccine.

4.Howdoparasitesevolveresistance?

Emergence First appearance of resistant genotype in focal population

Establishment Rise of resistant genotype beyond danger of immediate extinction

Increase Increase of resistant genotype to high levels in population

Equilibrium (extinction) Extinction of sensitive wild-type strain

Equilibrium (coexistence) Long-term coexistence of resistant and wild-type strains

Stochastic process︎Depends on mutation (and immigration) rate and population size. Because of rareness it is sensitive to chance extinction. ︎

Stochastic process︎Depends on selection coefficient, and effective population size. The smaller the selective advantage and the smaller the population size the more likely chance extinction may result. ︎

Deterministic process︎Depends predominantly on selection coefficient. Once the frequency is out of the “danger zone” (i.e. the genotype is frequent enough) it will spread due to selection. ︎ Deterministic process︎

Depends on various factors, such as costs of resistance, alternative resistance mechanisms, migration, etc. ︎

Deterministic process︎Depends on selection coefficient, and effective population size. Faster with stronger selection and smaller pop. size. ︎

ModifiedaserzurWieschetal.2011.LancetInfect.Dis.

Strong immigration may bypass the Emergence and establishment phase︎



Stochastic process︎Depends on mutation (and immigration) rate and population size. Because of rareness it is sensitive to chance extinction. ︎

Resistantgenotypesarearesultof1.   Muta9on(mostcommon,e.g.M.tuberculosis),2.   recombina9on(insomeviruses,e.g.Influenza)or3.   horizontalgenetransfer(e.g.S.aureus)

Emergenceofresistancedependsontherateswithwhichresistantgenotypesareproducedandonthepopula)onsizeofthepathogen.

Muta9onrates*

Popula9onsize

HIV 3.5x10-5 107–108inf.cells

Influenza 1x10-5 4x108targetcells

Mycobacteriumtuberculosis 10-6-10-8 108-109

Stapylococcusaureus 10-6-10-10 5x107-4x1010

Plasmodiumfalciparum 10-11-10-20 108-109(1012-1013lethalmalaria)

*Rateofacquiringresistanceperreplica)onModifiedaserzurWieschetal.2011.LancetInfect.Dis.


•  Selec)oncoefficients,s,areosenhigh(>0.1)(s=0.1isafitnessadvantageabout10%).

•  Popula)onsizesareosenhighenoughtoexcludegene)cdris,exceptinmacroparasites.

Establishmentofanewresistantgenotypedependsonitsfitnessadvantage(selec)oncoefficient)andonthepopula)onsizeofthepathogen.


Stochastic process︎Depends on selection coefficient, and effective population size. The smaller the selective advantage and the smaller the population size the more likely chance extinction may result. ︎

Manybeneficialmuta)onsarelostbychance,sincemutantindividualsmaybychancefailtoreproduce.Haldane(1927)calculatedthatabeneficialmutantinalargepopula)on(nostochas)ceffects)hasonaveragechancetoescapebeinglostofabout2s,wheresistheselec)oncoefficient(thisisanapproxima)on).Example:s=0.1,2s=0.2meansthat20%ofmutantmakeit(80%arelost)s=0.2,2s=0.440%ofmutantsmakeit,60%arelost.

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Generations

Alle

le f

requency


Frequencyofthe

favouredmutantgene

s=0.5

s=0.25s=0.1

s=0.05

s=0

Theselec)oncoefficient,s,hastwomeanings:1.  Thestrongertheeffectofamutant(highers)forthedrugexposedcarrier,thequicker

themutantwillspread.2.  Thestrongerselec)on(intensityofselec)on),thequickerthemutantwillspread.A

rarelyuseddrugswillnotselectforrapidspreadofresistancemutants.


Deterministic process︎Depends predominantly on selection coefficient. Once the frequency is out of the “danger zone” (i.e. the genotype is frequent enough) it will spread due to selection. ︎

Strong immigration may bypass the Emergence and establishment phase︎

Oncethefrequencyofamutantishighenoughtoescapestochas)cprocesses,itwillspreadduetoselec)on.



Deterministic process︎Depends on selection coefficient, and effective population size. Faster with stronger selection and smaller pop. size. ︎

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Generations

Alle

le f

requency

Frequencyofthe

favouredmutantgene

s=0.5

s=0.25s=0.1

s=0.05

s=0

Understrongongoingselec)onthemutantwillreplacethewildtype.

Ex)nc)onofdrugsensi)vewildtypeshasneverbeenobserved.Adrugisconsidereduselessmuchbeforethislevelisreached.



Deterministic process︎Depends on various factors, such as costs of resistance, alternative resistance mechanisms, migration, etc. ︎

Resistantanddrugsensi)veformsofpathogensandparasitesarelikelytocoexistforanumberofreasons.

•  Costsofresistance.•  Imperfectusageofdrugs.•  Spa)alvaria)onindrugusageandmigra)on.•  Differentwaystoevolveresistance.







•  muta)onrate•  popula)onsize

•  selec)oncoefficient•  popula)onsize

•  selec)oncoefficient•  intensityofselec)on•  )me•  immigra)on/transmission

5.Resistanceacquisi)onWithinandbetweenhostprocessesofdrugresistance

23

Thefirst)meaparasite/pathogenevolvesresistanceitarisesdenovo.However,lateritmaybeacquiredbytransmissionoftheresistantstraintootherhosts.Thedegreetowhichresistanceevolvesdenovooverandoveragainorisacquiredthroughtransmissionvariesstronglyamongparasites.

Resistanceevolvesosendenovo:HIV,M.tuberculosisResistanceisosenacquiredbyhosttohosttransmission:malaria

Forunderstandingtheevolu)onofdrugresistanceandwhatwecandoagainstit,thisdis)nc)oncanbeveryimportant.Acquiredresistancethroughtransmissionoffersaddi)onalop)onstoreducethelikelihoodforspreadingofdrugresistance.E.g.minimizetransmissionfromtreatedpa)ents.

5.Resistanceacquisi)onWithinandbetweenhostprocessesofdrugresistance

24

Mul)pleOriginsandRegionalDispersalofResistantdhpsinPlasmodiumfalciparumMalaria.5independent“de-novo”originsofan)folateresistancemuta)onsinthedihydropteroatesynthase(dhps)genewithuniquegeographicaldistrribu)ons.

Pearceetal.(2009)PLoSMedicine

sulfadoxine-resistance

6.Canwestoptheevolu)onofdrugresistance?

No,butmaybewecanslowitdown!

Thescienceof“evolu9on-proofing”!

Resistancevariesinspaceand9meSpa)alandtemporalvaria)oninlevelsofdrugresistancetes)fiesthatdrugresistanceworksbylocaladapta9on.Bystudyinglocaldifferenceswecanlearnaboutthefactorswhichmightpromoteorslowdowndrugresistanceevolu9on.Understandingthesefactorsisdifficult.Italsoshowsthatsolu)onstotheproblemarenotglobal,butcanbeimplementedlocally.Ontheposi)veside,despiteofglobaltrafficandmigra)on,localsolu)onshavebeenshowntobehighlyeffec)ve.








•  popula)onsize•  selec)oncoefficient


Reducepopula)onsize!

Reduceintensityofselec)on!

Reducetransmission!

Reduceselec)oncoefficient!

6.Canwestoptheevolu)onofdrugresistance?Uselessdrugs

Reduceinten

sityofselec)

on!

Thelessdrugsareused,thelowertheintensityof

selec)on.

PenicillinresistanceofStreptococcuspneumoniae(causespneumoniaand

bacterialmeningi)s)ishighestwereitismostosenused

WHOPolicyPerspec)vesonMedicines,No.010,April2005

Totalan9bio9cuse(DDD/1000popula9on/day)

PenicillinresistantS.pne

umioniae(%

)

6.Canwestoptheevolu)onofdrugresistance?Uselessdrugs

Totalan9bio9cuse(DDD/1000popula9on/day)bycountry

Reduceinten

sityofselec)

on!

Outpa)entan)bio)cusepercountry

6.Canwestoptheevolu)onofdrugresistance?Reducedrugsintheenvironment

Reduceinten

sityofselec)

on!

Examplesoffluoroquinolone*concentra)onsinvariousenvironments

*Fluoroquinolonesarebroad-spectruman)bio)cs(effec)veforbothgram-nega)veandgram-posi)vebacteria)thatplayanimportantroleintreatmentofseriousbacterialinfec)ons,especiallyhospital-acquiredinfec)onsandothersinwhichresistancetoolderan)bacterialclassesissuspected.

6.Canwestoptheevolu)onofdrugresistance?Uselessdrugs:mixingandcyclingofdrugs

Reduceinten

sityofselec)

on!

Whenusingdifferentdrugsagainstthesameparasitetheintensityofselec)onbyeachdrugisreduced.Twomethodsweresuggestedtoincreasetheenvironmentalheterogeneityfortheparasite:Cycling:thescheduledchangesofthepredominantdruginahospital(e.g.weeklyormonthlyaltera)ons)Mixing:assignmentofconsecu)vepa)entstodifferentdrugs.Mixingproducesheterogeneityonafinerscaleandwasthereforesuggestedtobethebe9ermethodforendemicparasites.Forepidemicparasites(e.g.influenza)cyclingmaybebe9er.However,mathema)calmodelsarenotfullyworkedoutandgoodempiricaldataarescarce.












Reducetransmission!


6. Can we stop the evolution of drug resistance? Reducepopula)onsize

Reducepopula

)onsize!

Thelikelihoodofbeneficialmutantsarisingispropor)onaltotheparasitepopula)onsize.Reducingthepopula)onsizefastandtoverylowlevelsslowsdowntheevolu)onofdrugresistance.Thisisinpar)cularimportantincasesofde-novoevolu)onofresistance.Hit-hard:Ahighdrugdosewillreducetheparasitepopula)onfastandtoverydeeplevel.Sub-op)maldrugdosagesreducepopula)onsizenotfastandstrongenoughandmayallowtheoutgrowthofresistantmutants.Non-adherence:Missingadoseofthedrugcanleadtohigherpopula)onsizesandfasterevo.ofresistance,especiallyearlyintreatment.Itisthereforeimportanttofollowtherecommendedtreatmentintervalandtreatmentdura)on.












Reducetransmission!


6. Can we stop the evolution of drug resistance? Combina)ontherapy

Ifresistancetotwo(ormore)drugsisacquiredindependently,combina)ontherapydecreasestheprobabilityofde-novoacquisi)onofresistance,becauseofpathogenwithtwo(ormore)appropriatemuta)onsisunlikelytoexistintheini)alpopula)onandunlikelytoarisespontaneously.Itisimportantthatthedifferentdrugsactatthesame)meandthesameloca)ons.Combina)ontherapyisrecommendasthestandardtreatmentfortuberculosis,HIVandmalaria.

Reduceselec)

oncoefficient!

Combina)ontherapydoesnotaltermuta)onrates,butreducestherela)vebeneficialeffectofmuta)onsthatprovideabenefittoonlyoneofthedrugs.

6.Canwestoptheevolu)onofdrugresistance?Specificsugges)onsforspecificcases

35

2examples:Plasmodium(causingmalaria)isosencontrolledbykillingitsvector.Plasmodiumtakesalong)metocompleteitsvectorstagepartofthelifecycle.Thus,onlyoldmosquitoscantransmitmalaria.Ifwetargetoldmosquitoswithdrugsorinsec)cidesthechancesthatresistanceevolvesisstronglyreduced.Thisisbecausetheforceofselec)onisweakinoldorganisms.Usebiologicalcontroltoreducevectors.Thecontrolagentwillco-evolvewiththevectorandremaineffec)ve.

7. Costs of drug resistance

Ifmutantsconferringdrugresistanceproduceacostfortheparasite(costofresistance),thefrequencyofthesemuta)onsisexpectedtodeclineaserthedrugisnotusedanymore.ThiswasthecaseinMalawiaserchloroquine(CQ)wasreplacedbysulphadoxine/pyrimethamineasfirstlinedrugin1993.

1990 1995 2000

100

75

50

25

0

Frequency of the CQ resistance pfcrt K76T mutation

(Kublinetal.,2003)

(Mitaetal.,2003)

These2studiessuggestthatthepfcrtK76Tmuta)onisabout5%lessfitthanthenormaltypes(assumingfivemalariagenera)onsperyear,andthatnoparasitemigra)onwasoccurring(Has)ngs&Donnelly2005)

Note:Itisunclearifthisisgenerallylikethis.Therearehardlydataoncostsofresistanceinothercases.

Summingup

1.  Evolu)onofdrug/vaccine/diagnos)csresistanceisbasedonnaturalselec)onontheparasite/pathogenpopula)on.Itisanadap)veprocess.

2.  Resistancevariesgeographicallysugges)nglocaladapta)on.3.  Thereissomeevidenceforacostofresistance.4.  Wecannotpreventtheevolu)onofresistance,butwemay

slowitdown.Thecrucialpopula)ongene)cmechanismstobeappliedarereducingpopula)onsize,reducingselec)onintensityandreducingselec)oncoefficients.

5.  Imperfectvaccinesmaycauseevolu)onofhighervirulence.

Evolu)on-proofing

safeslife!

38

Downloadat:h9p://www.who.int/pa)entsafety/implementa)on/amr/publica)on/en/index.html

Publica9ondate:2012Languages:EnglishISBN:9789241503181

ZurWieschetal.2011.LancetInfect.Dis.Vol.11:236-247.

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