Studies on leptospirosis : clinical aspects and pathophysiology - Chapter 12: Summary, discussion and directions for future studies

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Studies on leptospirosis : clinical aspects and pathophysiology

Wagenaar, J.F.P.

Publication date

2010

Link to publication

Citation for published version (APA):

Wagenaar, J. F. P. (2010). Studies on leptospirosis : clinical aspects and pathophysiology.

General rights

It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations

If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

6XPPDU\GLVFXVVLRQDQGGLUHFWLRQV

IRUIXWXUHVWXGLHV



&+$37(5

&KDSWHU

6XPPDU\ GLVFXVVLRQDQGGLUHFWLRQVIRUIXWXUHVWXGLHV  /HSWRVSLURVLVLVDQHPHUJLQJLQIHFWLRXVGLVHDVHRIJOREDOLPSRUWDQFH$VHYHUH FDVHPDQLIHVWVLWVHOIDVDIXOPLQDQWVHSVLVZLWKPXOWLRUJDQIDLOXUHDQGKHPRUUKDJLQJ $OWKRXJKFRQVLGHUDEOHSURJUHVVKDVEHHQPDGHLQXQUDYHOLQJWKHPHFKDQLVPVRI VHSVLVDQGUHODWHGFRDJXODWLRQGLVRUGHUVNQRZOHGJHRQWKHSDWKRSK\VLRORJ\RI OHSWRVSLURVLVUHPDLQVH[WUHPHO\OLPLWHG7KLVWKHVLVVWXGLHGWKHUROHRIVHFRQGDU\ KHPRVWDVLV DQG WKH LQQDWH LPPXQH UHVSRQVH LQ SDWLHQWV VXIIHULQJ IURP VHYHUH OHSWRVSLURVLV,QDGGLWLRQZHKDYHDOVRSUHVHQWHGVRPHHSLGHPLRORJLFDOGDWDDQG WKURZVRPHOLJKWRQWKHXVHIXOQHVVRIDUDSLGGLDJQRVWLFDVVD\0RVWGDWDSUHVHQWHG LQWKLVWKHVLVZDVJOHDQHGIURPSDWLHQWVZKRZHUHDGPLWWHGDQGWUHDWHGLQWKH'U .DULDGLKRVSLWDOLQ6HPDUDQJ,QGRQHVLD

3DUW , RI WKH WKHVLV JLYHV WKH UHDGHU PRUH LQVLJKW LQWR FRDJXODWLRQ GLVRUGHUV LQ OHSWRVSLURVLV ,Q&KDSWHU  WKH OLWHUDWXUH ZDV UHYLHZHG LQ WKLV UHJDUG DQG ZH DGGUHVVHGKHPRUUKDJLFV\QGURPHVLQÁDPPDWLRQSULPDU\DQGVHFRQGDU\KHPRVWDVLV DQG WKH ÀEULQRO\WLF SDWKZD\V 7KH HQGRWKHOLDO FHOO UHFHLYHV VSHFLDO DWWHQWLRQ DV SRWHQWLDOWDUJHWFHOO)URPWKHVWXGLHVUHYLHZHGZHFRQFOXGHGWKDWLQOHSWRVSLURVLV WKH EOHHGLQJ WHQGHQF\ PLJKW EH WKH UHVXOW RI DQ LPEDODQFH LQ WKH KHPRVWDWLF HTXLOLEULXP +RZHYHU DW WKDW SRLQW GHWDLOHG VWXGLHV RQ KRZ WKLV LPEDODQFH LV WULJJHUHG DQG ZKDW LQÁDPPDWRU\ DQG FRDJXODWLRQ SURWHLQV DUH LQYROYHG ZHUH ODFNLQJ 0RUH LQVLJKW LQWR WKH SDWKZD\V LQYROYHG LV JLYHQ LQ WKLV WKHVLV 7KH FDVH UHSRUWLQ&KDSWHUGHVFULEHVDW\SLFDOFDVHRIVHYHUHOHSWRVSLURVLVZLWKSXOPRQDU\ KHPRUUKDJHV)URPWKLVSDWLHQWZHUHFRYHUHGVWUDLQ/DQJNDZLWKDWZDVLGHQWLÀHG DVDQHZVXEW\SHRIVHURYDU/DLZKLFKLVWKHFODVVLFDOVHURYDUDVVRFLDWHGZLWKOXQJ EOHHGLQJ/DQJNDZL,Q&KDSWHUZHKDYHSURYLGHGGDWDRQFRDJXODWLRQDFWLYDWLRQ DQG ÀEULQRO\VLV LQ UHODWLRQ WR EOHHGLQJ DQG SRRU RXWFRPH LQ SDWLHQWV ZLWK VHYHUH OHSWRVSLURVLV 3DWLHQWV GHPRQVWUDWHG VWURQJ DFWLYDWLRQ RI WKH FRDJXODWLRQ V\VWHP DV UHÁHFWHG E\ KLJK SODVPD OHYHOV RI WKURPELQDQWLWKURPELQ 7$7 FRPSOH[HV SURWKURPELQIUDJPHQWDQG'GLPHU3URORQJDWLRQRISURWKURPELQWLPH37DQG DFWLYDWHGSDUWLDOWKURPERSODVWLQWLPHLQWKHVHSDWLHQWVSRLQWWRZDUGVFRQVXPSWLRQ FRDJXORSDWK\ +RZHYHU ZH FDQQRW H[FOXGH WKH SRVVLELOLW\ RI LPSDLUHG IXQFWLRQ



6800$5<

&KDSWHU



RU V\QWKHVLV RI FORWWLQJ IDFWRUV &RQFXUUHQWO\ DQWLFRDJXODQW SDWKZD\V ZHUH GRZQ UHJXODWHGDVUHÁHFWHGE\GHFUHDVHGOHYHOVRIWKHDQWLFRDJXODQWPDUNHUVSURWHLQ& DQGDQWLWKURPELQ3DWLHQWVDOVRGHPRQVWUDWHGHYLGHQFHRIDFWLYDWLRQDQGLQKLELWLRQ RIÀEULQRO\VLVZKLFKZDVUHÁHFWHGE\HOHYDWHGSODVPLQDQWLSODVPLQ3$3FRPSOH[HV DQGSODVPLQRJHQDFWLYDWRULQKLELWRUSODVPDOHYHOV7KHVHGLVWLQFWLRQVZHUHPRUH SURQRXQFHGLQSDWLHQWVZLWKDIDWDORXWFRPH$VXEDQDO\VLVEHWZHHQSDWLHQWVZLWKDQG ZLWKRXWVHYHUHEOHHGLQJUHYHDOHGDPRUHSURQRXQFHGLPEDODQFHRIWKHFRDJXODWLRQ V\VWHPLQSDWLHQWVZLWKVHYHUHEOHHGLQJ7KLVZDVUHÁHFWHGE\DVLJQLÀFDQWDVVRFLDWLRQ ZLWK37DQGWKH7$73$3UDWLRDQLQGLFDWRURIWKHEDODQFHEHWZHHQFRDJXODWLRQDQG ÀEULQRO\VLV$OVR WKURPERF\WRSHQLD ZDV DVVRFLDWHG ZLWK EOHHGLQJ7KH DEVHQFH RI PDUNHGGLVRUGHUVLQSDWLHQWVZLWKPLOGEOHHGLQJFRPSDUHGWRWKRVHZLWKRXWDQ\VLJQV RIKHPRUUKDJLQJSRLQWVWRWKHLQYROYHPHQWRIRWKHUSDWKRSK\VLRORJLFDOPHFKDQLVPV 2YHUWGLVVHPLQDWHGLQWUDYDVFXODUFRDJXODWLRQZDVDSSDUHQWLQRXWRISDWLHQWV EXWZDVDVVRFLDWHGZLWKQHLWKHUEOHHGLQJQRUSRRURXWFRPH,Q&KDSWHUWKHUROH RIHQGRWKHOLDOFHOOG\VIXQFWLRQLQUHODWLRQWREOHHGLQJLVFKDUDFWHUL]HGE\VWXG\LQJ WKHG\QDPLFVRIWKHHQGRWKHOLDOFHOOVSHFLÀFPDUNHUVVROXEOH(VHOHFWLQV(VHOHFWLQ DQGWKHYRQ:LOOHEUDQGIDFWRUY:)6ROXEOH(VHOHFWLQDQGY:)OHYHOVZHUHVWURQJO\ HOHYDWHGRQDGPLVVLRQDQGGHFUHDVHGGXULQJIROORZXSEXWWKH\GLGQRWUHYHUWWR QRUPYDOXHV,QDVXEJURXSDQDO\VLVEHWZHHQSDWLHQWVZLWKDQGZLWKRXWEOHHGLQJ SODVPDOHYHOVZHUHQRWVLJQLÀFDQWO\GLIIHUHQW1RVWDWLVWLFDOGLIIHUHQFHZDVREVHUYHG LQHLWKHUV(VHOHFWLQRUY:)SODVPDFRQFHQWUDWLRQVEHWZHHQSDWLHQWVWKDWVXUYLYHG DQGWKRVHWKDWGLGQ·W:HFRQFOXGHGWKDWPDUNHUVRIHQGRWKHOLDOFHOODFWLYDWLRQDUH VWURQJO\HOHYDWHGLQSDWLHQWVVXIIHULQJIURPVHYHUHOHSWRVSLURVLVUHJDUGOHVVRIEOHHGLQJ PDQLIHVWDWLRQVRURXWFRPH7KHÀQDOFKDSWHURIWKLVSDUWRIWKHWKHVLV&KDSWHU GHÀQHVWKHDFWLYDWLRQRIWKHFRQWDFWV\VWHPLQSDWLHQWVZLWKVHYHUHOHSWRVSLURVLV7KH DFWLYDWLRQRIWKHFRQWDFWV\VWHPZDVGHWHUPLQHGE\PHDVXULQJIDFWRU;,);,IDFWRU ;,,);,,DQGDFWLYDWHGSURWHLQLQKLELWRU,1+FRPSOH[HV);,D&,+);,,D&,+ NDOOLNUHLQ&,1+DQG);,D$7,1+LQSDWLHQWVZLWKVHYHUHOHSWRVSLURVLV,QDGGLWLRQ PDUNHUVRIFRDJXODWLRQDFWLYDWLRQ7$7SURWKURPELQIUDJPHQWDQGÀEULQRO\VLV 3$3ZHUHPHDVXUHG2YHUDOOWKHPHGLDQSURWKURPELQWLPH37ZDVKLJKQRUPDO ZKHUHDVWKHDFWLYDWHGSDUWLDOWKURPERSODVWLQWLPH$377ZDVPDUNHGO\SURORQJHG 7ZHQW\VL[SDWLHQWVKDGDSURORQJHG$377DQGDQRUPDO373DWLHQWVKDGVLJQLÀFDQWO\

6XPPDU\

GLVFXVVLRQDQGGLUHFWLRQVIRUIXWXUHVWXGLHV



ORZHU OHYHOV RI );,, DQG );, RQ DGPLVVLRQ FRPSDUHG WR WKH FRQWUROV );,, DQG );, OHYHOVZHUHLQYHUVHO\FRUUHODWHGZLWK$377SURORQJDWLRQ1RHYLGHQFHIRUFRQWDFW DFWLYDWLRQZDVREVHUYHGVLQFHWKHPHGLDQ);,,D&,+);,D&,1+DQG);,D$7,1+ FRPSOH[HVZHUHQRWVWDWLVWLFDOO\GLIIHUHQWIURPWKHFRQWUROVDQGNDOOLNUHLQ&,1+ FRPSOH[HV ZHUH XQGHWHFWDEOH 7KHUH ZDV QR DVVRFLDWLRQ EHWZHHQ HLWKHU VHYHUH EOHHGLQJ RU PRUWDOLW\ DQG FRQWDFW DFWLYDWLRQ PDUNHUV 0DUNHG DFWLYDWLRQ RI WKH 7)DQGÀEULQRO\WLFSDWKZD\ZDVGHPRQVWUDWHGHVSHFLDOO\LQVXEMHFWVZLWKVHYHUH EOHHGLQJVDQGLQWKRVHZKRZHQWRQWRGLH,QFRQFOXVLRQSDWLHQWVVXIIHULQJIURP VHYHUHOHSWRVSLURVLV\LHOGHGUHGXFHGOHYHOVRI);,,DQG);,WKDWH[SODLQHGLVRODWHG $377 SURORQJDWLRQ EXW QRW EOHHGLQJ RU SRRU RXWFRPH$OWKRXJK WKH ORZ OHYHOV RI );,,DQG);,PD\LQGLFDWHFRQVXPSWLRQRIWKHVHFRDJXODWLRQIDFWRUVQRHYLGHQFHRI FRQWDFWV\VWHPDFWLYDWLRQZDVREVHUYHG

7KHVHFRQGSDUW3DUW,,RIWKLVWKHVLVIRFXVHVRQLQÁDPPDWLRQ,Q&KDSWHUWKH ORQJSHQWUD[LQ37;LVH[SORUHGLQSDWLHQWVZLWKOHSWRVSLURVLV3HQWUD[LQVDUHDVXSHU IDPLO\ RI SURWHLQV FKDUDFWHUL]HG E\ D PXOWLPHULF XVXDOO\ SHQWDPHULF VWUXFWXUH 7KHDLPRIWKHVWXG\ZDVWRHYDOXDWHWKHXVHIXOQHVVRI37;LQPRQLWRULQJSDWLHQWV VXIIHULQJ IURP VHYHUH OHSWRVSLURVLV 7KH UHVXOWV ZHUH FRPSDUHG ZLWK WKH ZLGHO\ XVHGVKRUWSHQWUD[LQ&UHDFWLYHSURWHLQ&53DQGWKHSURLQÁ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DPROHFXOHLQYROYHG LQWKHUHJXODWLRQRIWKHLQQDWHLPPXQHUHVSRQVH7ROOOLNHUHFHSWRUV7/5VSOD\DQ LPSRUWDQWUROHLQWKHLQLWLDWLRQRIDQLQQDWHLPPXQHUHVSRQVH7/5VDUHLQKLELWHG E\ QHJDWLYH UHJXODWRUV LQFOXGLQJ WKH PHPEUDQHERXQG 67 P67 UHFHSWRU V67 FDQ LQKLELW VLJQDOLQJ WKURXJK P67 7KH DLP RI WKLV VWXG\ ZDV WR GHWHUPLQH WKH GHJUHHRIV67DQGSURLQÁDPPDWRU\F\WRNLQHUHOHDVHLQSDWLHQWVDGPLWWHGZLWK

&KDSWHU



VHYHUH OHSWRVSLURVLV $OO SDWLHQWV KDG HOHYDWHG V67 ,/ ,/ DQG ,/ OHYHOV RQ DGPLVVLRQ 6ROXEOH 67 OHYHOV ZHUH IRXQG WR EH FRUUHODWHG ZLWK DGPLVVLRQ ,/  ,/ DQG ,/ FRQFHQWUDWLRQV 6ROXEOH 67 OHYHOV ZHUH VLJQLÀFDQWO\ DVVRFLDWHG ZLWK VHYHUH EOHHGLQJ 7KLV DVVRFLDWLRQ ZDV XQLTXH VLQFH QRQH RI WKH F\WRNLQHV VKRZHG WKLV FRUUHODWLRQ )XUWKHUPRUH V67 ZDV DVVRFLDWHG ZLWK SRRU RXWFRPH :KHQ HLWKHU ZKROH EORRG RU LVRODWHG SHULSKHUDO EORRG PRQRQXFOHDU FHOOV 3%0&V ZHUH VWLPXODWHG ZLWK /HSWRVSLUD LQ YLWUR QR V67 SURGXFWLRQ FRXOG EH GHWHFWHG *LYHQWKHVWURQJDVVRFLDWLRQZLWKEOHHGLQJLQFRPELQDWLRQZLWKWKHUHVXOWVRIWKH

LQ YLWUR H[SHULPHQWV ZH FRQFOXGHG WKDW V67 FRXOG EH DQ LQGLFDWLYH PDUNHU IRU

WLVVXHGDPDJHLQSDWLHQWVVXIIHULQJIURPVHYHUHOHSWRVSLURVLV&KDSWHUGHVFULEHV DVHWRILQYLWURH[SHULPHQWVIRFXVLQJRQWKHLQQDWHLPPXQHUHVSRQVHWRGLIIHUHQW

/HSWRVSLUD VHURYDUV WZR VDSURSK\WLF WZR SDWKRJHQLF UHIHUHQFH DQG WZR KRVW

DGDSWHGYLUXOHQWVWUDLQV:HXQGHUWRRNNLOOLQJDQGVWLPXODWLRQH[SHULPHQWVXVLQJD KXPDQPRQRF\WLFFHOOOLQH7+3KXPDQ3%0&DQGZKROHEORRG6DSURSK\WLFDQG SDWKRJHQLFUHIHUHQFHVWUDLQVEXWQRWYLUXOHQWKRVWDGDSWHG/HSWRVSLUDZHUHNLOOHG E\ZKROHEORRGDQGVHUXP/LYH/HSWRVSLUDLQGXFHGDYLJRURXVVHURYDUGHSHQGHQW F\WRNLQHUHVSRQVHZLWKDGLIIHUHQWSDWWHUQFRPSDUHGWRWKHUHVSRQVHREWDLQHGZLWK KHDWNLOOHG EDFWHULD7KH VWURQJHVW F\WRNLQH UHVSRQVH WR /HSWRVSLUD ZDV REVHUYHG LQ ZKROH EORRG IROORZHG E\ 3%0&V DQG 7+3 FHOOV ,QKLELWLRQ H[SHULPHQWV ZLWK DQWL7/5DQGDQWL7/5DQWLERGLHVLQZKROHEORRGVKRZHGWKHLQYROYHPHQWRIERWK 7/5DQG7/57KHVHÀQGLQJVDUHXQLTXHVLQFHSUHYLRXVZRUNVKRZHGWKDW7+3  UHFRJQL]HG KHDWNLOOHG /HSWRVSLUD DQG OHSWRVSLUDO /36 H[FOXVLYHO\ WKURXJK 7/5 LQVWHDGRI7/5,QDGGLWLRQRXUGDWDUHYHDOHGWKDW7/5VLJQDOLQJZDVDGGLWLYHLQ WKHFDVHRIWKHSDWKRJHQLFUHIHUHQFHVWUDLQDQGV\QHUJLVWLFLQWKHFDVHRIWKHKRVW DGDSWHGYLUXOHQWVWUDLQ

3DUW,,,RIWKLVWKHVLVJLYHVVRPHLQVLJKWLQWRGLDJQRVWLFDQGHSLGHPLRORJLFDODVSHFWV ,Q&KDSWHUWKHVHURSUHYDOHQFHRIDQWL/HSWRVSLUDDQWLERGLHVDQGWKHXWLOLW\RI WZR UDSLG WHVWV IRU WKH VHURGLDJQRVLV RI WKH GLVHDVH ZHUH VWXGLHG LQ %LQK 7KXDQ DQDUHDLQVRXWKHUQ9LHWQDP,QDQLQLWLDOVXUYH\EORRGVDPSOHVIURPSDWLHQWV ZLWKDFXWHXQGLIIHUHQWLDWHGIHYHUDQGKHDOWK\VXEMHFWVZHUHH[DPLQHGIRUDQWL

6XPPDU\ GLVFXVVLRQDQGGLUHFWLRQVIRUIXWXUHVWXGLHV  'UL'RWDQGDODWHUDOÁRZDVVD\/HSWR7HN/DWHUDO)ORZ,QWKH(/,6$VDPSOHV IURPRIWKHKHDOWK\VXEMHFWVDQGRIWKHIHEULOHSDWLHQWVZHUHIRXQGWRKDYH WLWHUVRIDQWL/HSWRVSLUD,J0RIDWOHDVW,QWKHFRQYDOHVFHQWVDPSOHRQO\RQH RIWKHIHEULOHSDWLHQWVVKRZHGWKHPDUNHGLQFUHDVHLQ,J0WLWHUWKDWLVLQGLFDWLYHRI DFXWHOHSWRVSLURVLV$OWKRXJKWKHSRVLWLYHUHVXOWVRIWKHODWHUDOÁRZDVVD\²DSSOLHG WRZKROHEORRGDQGVHUXP²FRUUHVSRQGHGZLWKWKRVHRIWKH(/,6$WKH UHVXOWVRI WKH ODWHUDOÁRZ DVVD\ ZHUH RIWHQ LQGHWHUPLQDWH 7KH FDUGDJJOXWLQDWLRQ WHVW ZDV PRUH VSHFLÀF7KH RYHUDOO DJUHHPHQW EHWZHHQ WKH UHVXOWV RI WKH UDSLG WHVWV DQG WKRVHRIWKH(/,6$ZDVJHQHUDOO\SRRU,WDSSHDUHGWKDWKLJKVHURSUHYDOHQFHVRIDQWL

/HSWRVSLUD ,J0 DQG ORZ LQFLGHQFHV RI DFXWH OHSWRVSLURVLV LQ WKLV SDUW RI 9LHWQDP

OLPLWWKHGLDJQRVWLFYDOXHRIWKHUDSLGWHVWVWKDWZHUHLQYHVWLJDWHG7KHODWHUDOÁRZ DVVD\ZDVQRWVSHFLÀFHQRXJK7KHFDUGDJJOXWLQDWLRQWHVWLVSRVVLEO\EHWWHUEXW EHFDXVHRIWKHORZLQFLGHQFHLWVVHQVLWLYLW\FRXOGQRWEHHYDOXDWHGDGHTXDWHO\,Q &KDSWHUZHVWXGLHGWKHRFFXUUHQFHRIDFXWHULFNHWWVLDOGLVHDVHOHSWRVSLURVLVDQG GXDOLQIHFWLRQVDPRQJSDWLHQWVZLWKDFXWHXQGLIIHUHQWLDWHGIHYHU$8)LQ6HPDUDQJ ,QGRQHVLDZKHUHULVNIDFWRUVIRUERWKGLVHDVHVDUHOLNHO\WREHSUHVHQW&RQVHFXWLYH RXWSDWLHQWV ZHUH UHFUXLWHG DW WZR SULPDU\ KHDOWKFDUH FHQWHUV DQG KRVSLWDOL]HG SDWLHQWV ZHUH UHFUXLWHG DW D JRYHUQPHQWDO UHIHUUDO FHQWUH 'U .DULDGL 8QLYHUVLW\ +RVSLWDO'HSDUWPHQWRI,QWHUQDO0HGLFLQH7KHEORRGRIIHEULOHSDWLHQWV KRVSLWDOL]HG SDWLHQWV DQG  RXWSDWLHQWV ZDV WHVWHG $ FRQYDOHVFHQW VDPSOH ZDV DYDLODEOH LQ   SDWLHQWV 1LQH SDWLHQWV   GLVSOD\HG VLJQV RI DQ DFXWHLQIHFWLRQZLWK5W\SKLPXULQHW\SKXV0XULQHW\SKXVFRXOGEHGLDJQRVHGLQ RIKRVSLWDOL]HGSDWLHQWVIRURXWSDWLHQWVWKLVSHUFHQWDJHZDV $QRWKHU QLQH FDVHV   VKRZHG LQFRQFOXVLYH 5 W\SKL VHURORJ\ (YLGHQFH RI DFXWH LQIHFWLRQ ZLWK 2ULHQWLD WVXWVXJDPXVKL VFUXE W\SKXV LQ PHPEHUV RI WKH VSRWWHGIHYHUJURXSULFNHWWVLDZDVQRWIRXQG/HSWRVSLURVLVZDVGLDJQRVHGLQ SDWLHQWVRIWKHVHZHUHSRVLWLYHRQO\E\3&5(OHYHQOHSWRVSLURVLV SDWLHQWV ZHUH UHFUXLWHG LQKRVSLWDO VR WKDW WKH SHUFHQWDJH RI $8) FDXVHG E\ OHSWRVSLURVLVLQKRVSLWDOL]HGSDWLHQWVZDVDQGLQRXWSDWLHQWV 1RGXDOLQIHFWLRQVZHUHGHWHFWHGKRZHYHUWKUHHOHSWRVSLURVLVSDWLHQWV VKRZHGWLWHUVLQWKH5W\SKLDVVD\,QWKLVVWXG\ZHKDYHUHSRUWHGIRUWKHÀUVWWLPH RQKRZPXULQHW\SKXVDQGOHSWRVSLURVLVDUHLPSRUWDQWFDXVHVRI$8)LQ6HPDUDQJ

&KDSWHU  ,QGRQHVLD%RWKGLVHDVHVDUHGLIÀFXOWWRGLDJQRVHRQFOLQLFDOJURXQGVRQO\OHDGLQJWR PLVGLDJQRVLVDQGDEHUUDQWXVHRIDQWLELRWLFVDQGRWKHUSKDUPDFHXWLFDOV7KHUHIRUH UDSLGFKHDSDQGUHOLDEOHWHVWVDUHZDUUDQWHGWRVXSSRUWFOLQLFDOGHFLVLRQPDNLQJ 7DEOH4XHVWLRQVIRUIXWXUHUHVHDUFK 3DUW,&RDJXODWLRQDQGWKHHQGRWKHOLDOFHOO • 'R/HSWRVSLUDKDYHDGLUHFWWR[LFHIIHFWRQSODWHOHWVDQGDUHYLUXOHQW/HSWRVSLUDDEOH WRDOWHUSODWHOHWIXQFWLRQ" • :KLFKPHFKDQLVPEHVLGHVLQÁDPPDWLRQLQGXFHGFRDJXODWLRQDFWLYDWLRQFRQWULEXWHVWR EOHHGLQJ" • $UHFORWWLQJIDFWRUVGHSOHWHGDQGDUHFORWWLQJIDFWRUDQWLERGLHVSUHVHQW" • ,VHQGRWKHOLXPGDPDJHGRUMXVWDFWLYDWHGLQOHSWRVSLURVLV" • &DQ/HSWRVSLUDGLUHFWO\DFWLYDWHRUGDPDJHHQGRWKHOLDOFHOOV" • $UHPDUNHUVIRUHQGRWKHOLDOFHOODFWLYDWLRQGDPDJHDOVRLQFUHDVHGLQPLOGOHSWRVSLURVLV" • :KDWLVWKHUROHRIQHZGUXJVWKDWLQWHUYHQHLQWKHFRDJXODWLRQFDVFDGHLQWKH WUHDWPHQWRISDWLHQWVZLWKVHYHUHOHSWRVSLURVLV" 3DUW,,,QÁDPPDWLRQ • :KDWLVWKHUROHRI7/5QHJDWLYHUHJXODWRU\SDWKZD\VLQOHSWRVSLURVLV" • 7RZKDWH[WHQWLV/36DQLPSRUWDQWIDFWRULQWKHYLUXOHQFHDQGSDWKRJHQLFLW\RI /HSWRVSLUD" • :KLFK7/5OLJDQGVDUHLQYROYHGLQWKHLQQDWHLPPXQHUHVSRQVHWRYLUXOHQW/HSWRVSLUD" • :KDWLVWKHPHFKDQLVPRI7/5HQJDJHPHQWLQYLYR" • $UHWKHUHQHZLPPXQRPRGXODWLQJVWUDWHJLHVWRWUHDWVHYHUHOHSWRVSLURVLV" 3DUW,,,'LDJQRVWLFDQGHSLGHPLRORJLFDODVSHFWV • :KDWLVWKHWUXHEXUGHQRIGLVHDVHIURPOHSWRVSLURVLVZRUOGZLGH" • :KLFKSUHYHQWDWLYHPHDVXUHVDUHPRVWHIIHFWLYHLQUHGXFLQJPRUELGLW\DQGPRUWDOLW\RI OHSWRVSLURVLV" • 7RZKDWH[WHQWZLOOSURPSWGLDJQRVLVHDUO\WULDJHRIVHYHUHFDVHVDQGEHWWHUFOLQLFDO PDQDJHPHQWLPSURYHWKHRXWFRPHRIOHSWRVSLURVLVLQHQGHPLFDUHDV"

6XPPDU\

GLVFXVVLRQDQGGLUHFWLRQVIRUIXWXUHVWXGLHV



7KHGDWDSUHVHQWHGLQWKLVWKHVLVZLOOFRQWULEXWHWRRXUNQRZOHGJHRIOHSWRVSLURVLV EXW FRQVHTXHQWO\ UDLVHV PDQ\ QHZ TXHVWLRQV UHJDUGLQJ LWV SDWKRSK\VLRORJ\ DQG HSLGHPLRORJ\7KHQH[WVHFWLRQGLVFXVVHVWKHPDMRUÀQGLQJVDQGDGGUHVVHVVRPHNH\ TXHVWLRQVWKDWDZDLWIXUWKHULQYHVWLJDWLRQVHHWDEOH

3DUW,&RDJXODWLRQDQGWKHHQGRWKHOLDOFHOO

7KLV WKHVLV GHPRQVWUDWHV WKDW SDWLHQWV VXIIHULQJ IURP VHYHUH OHSWRVSLURVLV VKRZ FRDJXODWLRQ DFWLYDWLRQ ZLWK FRQFXUUHQW GRZQ UHJXODWLRQ RI DQWLFRDJXODQW V\VWHPV DQG À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,QWKLVUHJDUGVWXG\LQJ SODWHOHWVPLJKWEHDSURPLVLQJNH\WRHOXFLGDWHWKHSDWKRSK\VLRORJ\RIEOHHGLQJLQ VHYHUHOHSWRVSLURVLV/DVWO\ZHVKRXOGH[SORUHQRYHOWKHUDSLHVWKDWLQWHUYHQHLQWKH FRDJXODWLRQFDVFDGHHJDFWLYDWHGSURWHLQ&ZLWKWKHXOWLPDWHJRDORIUHGXFLQJ PRUWDOLW\IURPVHYHUHOHSWRVSLURVLV 3DUW,,,QÁDPPDWLRQ /DWHUHFRJQLWLRQRIVHYHUHOHSWRVSLURVLVFDVHVFRQWULEXWHVWRWKHKLJKPRUWDOLW\UDWH %LRPDUNHUVPD\EHKHOSIXOLQWKHHDUO\GLVFULPLQDWLRQEHWZHHQVHYHUHDQGQRQ VHYHUH FDVHV $GHTXDWH GLVFULPLQDWLRQ EHWZHHQ VHYHUH DQG OHVVVHYHUH FDVHV RI OHSWRVSLURVLVLVYHU\LPSRUWDQWEHFDXVHLWKHOSVWKHFOLQLFLDQLQWKHDOORFDWLRQRIKLJK FDUHIDFLOLWLHVRQO\WRWKRVHSDWLHQWVZLWKDKLJKOLNHOLKRRGRIPRUWDOLW\DQGEOHHGLQJ FRPSOLFDWLRQV7KLVLVSDUWLFXODUO\LPSRUWDQWLQORZUHVRXUFHHQYLURQPHQWV6WXGLHV

&KDSWHU



WKDW LGHQWLI\ VXFK ELRPDUNHUV LQ OHSWRVSLURVLV DUH JUHDWO\ ODFNLQJ ,Q WKLV WKHVLV ZHKDYHLGHQWLÀHGWKHORQJSHQWUD[LQ37;DVDSURPLVLQJFDQGLGDWHPDUNHUDVLW ZDVSURJQRVWLFIRUERWKPRUWDOLW\DQGGLVHDVHVHYHULW\6ROXEOH67ZDVXQLTXHO\ DVVRFLDWHGZLWKERWKEOHHGLQJDQGPRUWDOLW\2XULQYLWURGDWDVKRZHGWKDWEORRG FHOOV ZHUH QRW WKH VRXUFH RI V67 DV ZDV REVHUYHG LQ SUHYLRXV VWXGLHV  :H K\SRWKHVL]HGWKDWV67PLJKWEHDPDUNHUIRUGHHSWLVVXHGDPDJHZKLFKVKRXOGEH HYDOXDWHGLQIXWXUHVWXGLHV

/LWWOHLVNQRZQDERXWWKHLQQDWHLPPXQHUHVSRQVHWR/HSWRVSLUD,QDVHWRIH[SHULPHQWV ZH VKRZHG WKDW WKH F\WRNLQH UHVSRQVH WR /HSWRVSLUD LV VHURYDU GHSHQGDQW :KHQ XVLQJYLDEOHEDFWHULDLQRXUH[SHULPHQWVF\WRNLQHG\QDPLFVFKDQJHGHPSKDVL]LQJ WKHLPSRUWDQFHRIXVLQJIUHVKLVRODWHVLQIXWXUHVWXGLHV:HDUHWKHÀUVWWRUHSRUW WKHLQYROYHPHQWRIERWK7/5DQG7/5HQJDJHPHQWLQDOHSWRVSLURVLVKXPDQZKROH EORRGPRGHO:KHUHDVWKHLQWHUDFWLRQRI7/5DQG7/5LQPHGLDWLQJZKROHEORRG 71)α UHOHDVH DSSHDUV WR EH DGGLWLYH IRU QRQYLUXOHQW SDWKRJHQLF /HSWRVSLUD ZH REVHUYHGWKDWZKROHEORRG71)αUHOHDVHLQGXFHGE\YLUXOHQW/HSWRVSLUDZDVERRVWHG E\ V\QHUJLVWLF DFWLRQV RI 7/5 DQG 7/5 :H K\SRWKHVL]HG WKDW YLDEOH /HSWRVSLUD LQWHUDFWZLWKKRVWFHOOVLQZKROHEORRGLQD7/5DQGGLVUXSWLYHPDQQHUVXFKWKDW HQGRJHQRXV7/5OLJDQGVDUHUHOHDVHGZKLFKHIIHFWLYHO\ERRVWF\WRNLQHSURGXFWLRQ )XWXUH VWXGLHVDUHZDUUDQWHG WRUHYHDOWKHPHFKDQLVPRIDFWLRQRIWKLVREVHUYHG V\QHUJ\DQGWRXQUDYHOWKHLQYROYHPHQWRI7/5GXULQJOHSWRVSLURVLVLQYLYR

3DUW,,,'LDJQRVWLFDQGHSLGHPLRORJLFDODVSHFWV

7KH UHDO EXUGHQ RI OHSWRVSLURVLV LV XQNQRZQ ,WV LQFLGHQFH UDWH LV WKRXJKW WR EH JUHDWO\ XQGHUHVWLPDWHG GXH WR LJQRUDQFH PLVGLDJQRVLV DQG ODFN RI DSSURSULDWH GLDJQRVWLF ODERUDWRU\ VHUYLFHV  0LVGLDJQRVLV KDV EHFRPH D FULWLFDO LVVXH LQ UHJLRQV ZKHUH GHQJXH DQG RWKHU LQIHFWLRXV GLVHDVHV ZLWK RYHUODSSLQJ FOLQLFDO SUHVHQWDWLRQ DUH HQGHPLF  ,JQRUDQFH LV ODUJHO\ GXH WR D ODFN RI DGHTXDWH ODERUDWRU\ WHVWLQJ IDFLOLWLHV ZKLFK UHPDLQV WKH PDMRU EDUULHU IRU GLDJQRVLV DQG HSLGHPLRORJLF VXUYHLOODQFH /HSWRVSLURVLV VHURORJ\ LV D ÁDZLQJ PHWKRG IRU SURPSW GLDJQRVLVHVSHFLDOO\EHFDXVHDFRQYDOHVFHQWVDPSOHLVUHTXLUHGDQGWHVWVDUHRIWHQ WRRODERULRXV&RQVHTXHQWO\WKHUHLVDQXUJHQWQHHGIRUUHOLDEOHFKHDSDQGQRQ

6XPPDU\

GLVFXVVLRQDQGGLUHFWLRQVIRUIXWXUHVWXGLHV



ODERULRXVDQWLJHQEDVHGGLDJQRVWLFWHVWV:KHQFRPELQLQJPROHFXODUPHWKRGV3&5 ZLWK VHURORJLFDO WHVWV PRUH PLOG FDVHV ZLOO EH LGHQWLÀHG DV ZDV VKRZQ LQ WKLV WKHVLV,QGHHGHDUO\GLDJQRVLVPD\GHFUHDVHWKHFDVHIDWDOLW\UDWHVLQFHDQWLELRWLF WUHDWPHQW SURYLGHV WKH JUHDWHVW EHQHÀW ZKHQ LW LV LQLWLDWHG HDUO\ LQ WKH FRXUVH RI GLVHDVH  0RUH LQVLJKW LQWR WKH WUXH HSLGHPLRORJ\ RI OHSWRVSLURVLV ZLOO KHOS FRQYLQFHSROLF\PDNHUVWRVXSSRUWDSSURDFKHVIRUIRFXVHGSUHYHQWLRQPHDVXUHVDQG ZLOOUHGXFHWKHDEHUUDQWXVHRIDQWLELRWLFVDQGRWKHUSKDUPDFHXWLFDOVSUHVFULEHGE\ SK\VLFLDQV:KDWUHPDLQVLVWRGHÀQHFOLQLFDOJXLGHOLQHVZKLFKDUHRIWHQDQHFGRWDO UDWKHUWKDQHYLGHQFHEDVHG

&KDSWHU    %KDUWL$51DOO\-(5LFDOGL-10DWWKLDV0$'LD]00/RYHWW0$HWDO/HSWRVSLURVLVD ]RRQRWLFGLVHDVHRIJOREDOLPSRUWDQFH/DQFHW,QIHFW'LV'HFHPEHU   /HYHWW31/HSWRVSLURVLV&OLQ0LFURELRO5HY$SULO   6FKRXWHQ0:LHUVLQJD:-/HYL0YDQGHU3ROO7,QÁDPPDWLRQHQGRWKHOLXPDQG FRDJXODWLRQLQVHSVLV-/HXNRF%LRO1RYHPEHU   &KLHUDNXO:7LHQWDGDNXO36XSXWWDPRQJNRO<:XWKLHNDQXQ93KLPGD./LPSDLERRQ 5 HW DO$FWLYDWLRQ RI WKH FRDJXODWLRQ FDVFDGH LQ SDWLHQWV ZLWK OHSWRVSLURVLV &OLQ ,QIHFW'LV-DQXDU\   YDQ¶W9HHU&YDQGHQ3DQJDDUW36YDQ=RHOHQ0$GH.0%LUMPRKXQ566WURHV(6 HWDO,QGXFWLRQRI,5$.0LVDVVRFLDWHGZLWKOLSRSRO\VDFFKDULGHWROHUDQFHLQDKXPDQ HQGRWR[HPLDPRGHO-,PPXQRO1RYHPEHU   0F%ULGH$-$WKDQD]LR'$5HLV0*.R$,/HSWRVSLURVLV&XUU2SLQ,QIHFW'LV 2FWREHU   .R$,*DOYDR505LEHLUR'RXUDGR&0-RKQVRQ:'-U5LOH\/:8UEDQHSLGHPLF RI VHYHUH OHSWRVSLURVLV LQ %UD]LO 6DOYDGRU /HSWRVSLURVLV 6WXG\ *URXS /DQFHW  6HSWHPEHU

  /D5RFTXH5&%UHLPDQ5)$UL0'0RUH\5(-DQDQ)$+D\HV-0HWDO/HSWRVSLURVLV GXULQJGHQJXHRXWEUHDN%DQJODGHVK(PHUJ,QIHFW'LV0D\