R ev ist a d a S o c ie d ad e B r as ile ir a d e M e d ic in a T r o p ic al 2 9 ( 3 ) :2 5 9 - 2 6 9 , m aio - ju n , 1 9 9 6 .
SPf66 V A CCINE TRIA L IN BRA ZIL: CO N CEPTUA L
FRA M EW O RK STUD Y D ESIGN A ND
A N A LYTICA L A PPRO A CH
M. Urdaneta, A. Prata, C.J. Strachiner, C.E. Tosta, P. Tauil and M. Boulos
T his p a p e r d e s c r ib e s t he st u dy p o p u lat io n a n d t h e st u dy de s ig n o f t he p h a s e III f i e l d t r ial o f t h e S P f6 6 v ac c in e in B r az il. A ssess m en t o f v alidit y a n d p r e c is io n p r in c ip le s n e c e s s ar y f o r t h e ap p r o p r iat e e v alu at io n o f t h e p r o t e c t iv e e ffe c t o f t he v ac c in e a r e disc u sse d, as w e ll a s t h e resu lt s o f t h e p r e lim in ar y an aly s e s o f t h e g a t h e r e d d at a. T he a n aly t ic al a p p r o a c h f o r t he e s t im at io n o f t he p r o t e c t iv e e ffe c t o f t h e v ac c in e is p r e s e n t e d .
T his p a p e r p r o v id e s t he c o n c e p t u al fr a m e w o r k f o r fu t u r e p u b lic at io n s . K e y - w o r ds : M alar ia v ac c in e . S P f6 6 v ac c in e t rial. V ac c in e e ffic ac y .
The m ain o b jectiv e o f a v accine p hase III trial is to estim ate the d irect p ro tectiv e effect o f the v accine in the ind iv id ual (v accine efficacy ) o r, in o th e r w o rd s, th e alte ratio n o f th e v ac c in ate d in d iv id u al’ s su sc e p tib ility to infectio n4 22 2<s. In o rd er to o b tain a v alid and p recise estim atio n o f the efficacy o f a v accine th at allo w s fo r a b io lo g ic a l m ean in g fu l interp retatio n, sev eral co nd itio ns m ust b e satisfied in a field trial.
W i t h t h e p u r p o s e o f p r o v i d i n g th e co ncep tu al fram ew o rk o f the p hase III trial o f SPf66 v accine ag ainst the asexu al blo o d -stag es o f P l a s m o d i u m f a l c i p a r u m in Braz il, w e w o rk e d b a s e d u p o n the s e q u e n c e o f p atho g enic p ro cesses that w o uld lead to the end p o int(s) o f interest the v accine is sup p o se to p rev en t o r m o d ify as illu strated b y Struchiner et al.21. Such fram ew o rk divid es the p ro cess in three seq u ences, each o ne w ith im p o rtant issues o f v alid ity and p recisio n
Esco la N acio n al d e Saúde Púb lica, Fun d ação O sw ald o C ruz.
M ed icina T rop ical, Facu ld ad e d e M ed icina d o Triângulo
M ineiro. Lab o rató rio d e M alária, N ú cleo d e M ed icina
T ropical e N u trição e D ep artam en to d e Saúde C oletiva,
U n iv ersid ad e d e Brasília. D ep artam en to d e D o en ças
In f eccio sas e Parasitárias, Facu ld ad e d e M ed icina,
U niversid ad e d e São Paulo.
S ou rces o f su p p o rt: D r. M .E. Patarro y o , w h o p ro vid ed th e
S P Í 6 6 v accin e and p laceb o . N ational H ealth Foundation, M inistry o f H ealth, Brazil (g ran t:0 0 8 / 9 1 ) and Brazilian
Research C ouncil (C N Pq). Pan-A m erican H ealth O rganization
( g ra n ts :0 0 9 / 9 1 ,1 6 7 / 9 1 and H D P/H D R /R G /BR A / 119).
A d d re s s to : Prof. A luizio Prata. M ed icina Tropical/FM TM . C aixa Po s tal:118, 3 8 0 0 1 - 9 7 0 U b erab a, M inas G erais, M G,
Brasil. Fax:5 5 - 3 4 - 3 1 2 7 7 2 2 Ram al: 12 7 9 .
R eceb id o p ara p u b licação e m 2 2 / 1 2 / 9 5 .
p rincip les: 1) selectio n o f a su scep tib le study p o p ulatio n, d eterm inatio n o f an ad eq u ate sam p le size, d efinitio n and m easu rem ent o f relev ant facto rs (co v ariates), and the rand o m v accine allo catio n; 2) d eterm inatio n o f the am o unt o f exp o su re to infectio n the study p o p u latio n is su b jec te d to ; and 3) the d efinitio n o f a resp o nse m o d el.
S t u dy a r e a a n d p o p u l a t i o n . The field trial w as carried o ut in the rural settlem ents o f the m unicip ality o f Co sta M arques, Ro nd o nia in the A m azo n b asin o f Brazil. Th e reg io n is characteriz ed b y a ho t hum id , eq u ato rial clim ate, w ith seaso nal d istributio n o f rains (d ry seaso n fro m M ay to Sep tem b er and a rainy seaso n fro m O cto b er to A p ril). Th e v eg etatio n is c o m p o sed o f d ense tro p ical fo rest9.
U rd anet a M , P rat a A , S t ru c h in e r Cf, To sta CE, T aull P, B o ulo s M . S P / 6 6 v a c c in e t rial in B ra z il: co n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a na ly t ica l a p p ro a c h. R ev ista d a So cie d a d e B ra s ileira d e M e d ic in a T ro p ical
2 9 : 2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
river, the ap p ro xim ate p o p ulatio n siz e w as 2,625, and alo ng the ro ad , the p o p ulatio n w as, 5,975 fo r a to tal o f 23,045 inhabitants (FNS: u np ublished d ata).
The nu m b er o f cases rep o rted b y the FNS, sho w ed a d ram atic and p ro g ressiv e increase d uring the 80’s, ap p arently related to the m ig rato ry influx. In 1983, 3,125 cases w ere rep o rted in co ntrast to 16,029 cases in 1987. The d ram atic increase in the num ber o f cases and the p ro g ressiv e o ccu p atio n o f the fo rest, stim ulated the establishm ent o f sp ecializ ed centers alo ng the ro ad w here free d iagno sis and treatm ent is o ffered to the p o p ulatio n. H ealth ag ents w ere trained to refer to the m ain health c enter in the v illag e all p o ssib le sev ere cases. Biannual sp raying o f all ho u ses w ith i n s e c ti c i d e s o f r e s id u a l a c tio n an d th e treatm ent o f p resum p tiv e cases d etected by activ e search in the field w ere m aintained . A red u ctio n in the num ber o f cases w as then o bserv ed , p ro bab ly d ue to the streng thening o f the activ e and p assiv e c ase d etec tio n p ro ced ures, as this co ntro l m easure has an im p act o n transm issio n (7,532 cases w ere rep o rted in 1990). A ltho u g h m alaria is co nsid ered the m ain health p ro blem in the m unicip ality, m o rtality is lo w p ro bab ly d ue to the easy ac c ess b y the p o p u latio n to d iagno sis and sp ecific treatm ent24.
M alaria cases hav e a seaso nal d istributio n, b eing hig her during the b eg inning and the end o f the rainy seaso n. During the early co lo niz atio n stag e and b efo re the streng hthening o f co ntro l m easures, the p rev alence o f m alaria v aried b etw een 8 to 26% , acco rd ing to the area. In the co m m unities alo ng the ro ad the p rev alence rang ed b etw een 14 to 26%, and in the v illage b etw een 8 to 9% , b eing the ratio o f P. f a l c i p a r u m and P . v i v a x infectio ns 2:1. Sp leen
rates in child ren 2 to 10 y ears o ld rang ed fro m 17% to 67% (d ep end ing o n the y ear seaso n), b o th in resid ents o f the urban and rural area, a m eso end em ic transm issio n lev el15. Cases w ere o b serv ed in all ag e gro up s and p arasitem ia w as usually acco m p anied by the classical sy m p to m s b ein g su b clinical infectio ns, an excep tio nal find ing 17. Th e p rev alence in infants w as o f 9% and the intensity o f clinical m anifestatio ns had n o ap p arent relatio nship w ith ag e. Malaria infectio ns during p reg nancy d id n o t relate w ith p arity . In ad d itio n, c o m p lic atio n s asso c iate d w ith p arasite m ia during g estatio n w ere rare, as w ell as co ng enital
m alaria, p ro b ab ly d ue to the freq u ent therap eutic use o f antim alarials. Ig G antibo d ies against asexu al b lo o d fo rm s o f P . f a l c i p a r u m w ere d etected in 42% o f the p arturients at v ery lo w titers and sho w ed a rap id d ecay 23. In the last p ast y ears, P. v i v a xinfectio ns p red o m inate and a red uctio n o n p rev alence w as o bserv ed , achiev ing 10% in a p relim inary surv ey fo r the v accine trial d o ne in resid ents alo ng the ro ad in 1990.
T h e FN S th e r a p e u ti c a l s c h e m e s f o r treatm ent o f m alaria cau sed by P . f a l c i p a r u m hav e chang ed freq uently during the p ast few y ears, d ue to the o ccu rrence o f multid rug resistance, as sug g ested b y i n v iv o stud ies16. C hlo ro q u ine is u sed ex c lu siv ely fo r the tr e a tm e n t o f P . v i v a x in f e c ti o n s , s in c e sensitiv ity o f P. f a l c i p a r u m to it w as 18.2% . The sensitivity to sulfad o xine-p y rim etham ine w as 53-4% , to q uinine o f 81.8% and to q uinine plus tetracy cline o f 93% . The p resent treatm ent schem e is b ased o n the co m b in atio n o f quinine plus tetracy cline in p atients o ld er than sev en y ears o f ag e and no n-p reg nant w o m en. During p reg nancy , the first line treatm ent is quinine in a 10 d ays sched u le. M eflo q uine is av ailable and p rescrib ed in case o f resistance to the co nv entio nal schem e.
V rd a ne t a M , P ra t a A , S t ru c h i n e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c in e t rial in B ra z il: c o n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a na ly t ica l a p p ro a ch. R ev ista d a S o c ie d a d e B ras ile ira d e M e d ic in a T ro p ica l
2 9 :2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
salivary glands (TA Klein: personal communication, 1989).
The p o p u latio n o f interest fo r the field trial w as that settled alo ng the ro ad , as it is co m p o sed m ainly o f m igrants. A fter arriving to the area, the m ain activ ities o f the im m igrants w ere the build ing o f tem p o rary shelters, clearing the fo rest and , gro w ing subsistence cro p s. They are v ery unstable, since their p erm anence d ep end s m ainly o n eco no m ic su ccess and health co nd itio ns. O nce settled, the m ain o ccu p atio ns are agriculture and lum bering 15.
Th e clinical, sero lo g ical and ento m o lo g ical data, as w ell as the p o p ulatio n d ynam ics, characteriz e Co sta M arques as an “agricultural fro ntier”25 o f m ed ium end em icity , w ith p erio d s o f hig her and lo w er transm issio n, o ccu p ied recently b y no n-im m une ind ivid uals.
S P / 6 6 v a c c i n e . The sy nthetic v accine SPf66 co nsisted o f a seq u en c e o f three fragm ents d erived fro m m ero z o ite p ro teins linked b y a s e q u e n c e o f th e te tr a p e p tid e o f th e circum sp o ro z o ite p ro tein o f P . f a l c i p a r u m . O ne m illiliter o f the p ro d uct co ntained 4m g o f the sy nthetic p ep tid e so lubiliz ed in saline so lutio n ab so rb ed o nto 2m g o f alum inum hy d ro xid e1. It w as p ro d uced in the Instituto de Inm uno lo gia, Universidad N acio nal de Co lo m bia by the research g ro up o f Dr. M.E. Patarro yo . The v accine, as w ell as the p laceb o (tetanus to xo id fo r 1st and alum inum hy d ro xid e fo r 2nc* and 3rc* d o ses), w ere b o ttled in clear glass recip ients co ntaining 10 d o ses eac h and co d ed w ith the letters S and L in C o lo m bia. The v accinatio n sched u le w as d efined as the su bcu taneo us ap p licatio n o f 0.5m l o n days 0, 30 and 18018.
The p o ssib le d irect effects o f SPf66 m alaria v ac c in e are th e m o d ific atio n o f c lin ic al m anifestatio ns, includ ing d uratio n, since its m ain m echanism o f actio n w o uld b e the ind u c tio n o f the p ro d u ctio n o f sp ec ific antibo d ies ag ainst antig ens o f the asexual fo rm s o f P . f a l c i p a r u m , cap ab le o f lim iting their m ultip licatio n19. The v accine m echanism o f actio n w o u ld m im ic the acq uisitio n o f partial im m unity o bserv ed naturally in highly end em ic areas.
Incid ence o f clinical cases co uld b e red uced b y ind irect m eans. The p ro tectiv e ag ent co uld
in te r f e r e w ith th e h e p a tic s c h iz o g o n y , p rev enting the d ev elo p m ent o f p arasitem ia sim ulating the effect o f a v accine that p revents infectio n. If the p ro tectiv e effect o f the v accine is m anifested b y the p resence o f subclinical p arasitem ias, a red u ctio n in the rep o rted num ber o f clinical c ases w ill b e no ticed and/ or, if the d uratio n o f p arasitem ia is d im inished , there co u ld b e an effect o n the p ro d u ctio n o f the sexu al fo rm s altering transm issio n7 20 26.
S t u d y d e s i g n . The o b jectiv e o f this trial w as to ev aluate the efficacy , im m uno g enicity and safety o f the SPf66 v accine in no n-im m une resid ents o f a Braz ilian end em ic reg io n. The m ain sp ecific o b jectiv es w ere: first, to estim ate the o v erall p ro tectiv e d irect effect o f the v accine fo r all ep iso d es, as w ell as crud e and stratum sp ecific estim ates fo r the first and seco nd ep iso d es sep arately fo r P . f a l c i p a r u m and P . v i v a x infectio ns. A futher o b jectiv e w as to d etect p o ssible d ifferences in asexu al b lo o d - stag e p arasite d ensities. Seco nd , to assess the im m uno g enicity o f the v accine and its relatio n w ith m alaria infectio ns, as w ell as its p o ssib le relatio n w ith p ro tectio n, and third, to d escribe and d eterm ine the freq u ency o f im m ed iate o r d elay ed sid e-effec ts asso c iated w ith the ap p licatio n o f SPf66 v accine.
The stud y co nsisted o f a rand o m iz ed , d o uble-blind ed , p laceb o -co ntro lled , efficacy trial. A ll v o lunteers, m ale o r fem ale, ag ing 7 to 60, resid ent in the rural settlem ents alo ng km 52 to 112 o f the ro ad BR 429 w ere rand o m ly assig ned to a num ber b etw een o ne to 800. Ind iv id u als su ffe rin g o f ac u te o r se v e re d iseases, w ith histo ry o f allerg ies and p reg nant w o m en w ere exclu d ed . A ll p articip ants w ith an o d d nu m b er receiv ed the p rep aratio n lab eled L and tho se w ith an ev en nu m b er the p rep aratio n lab eled S, ship p ed “read y fo r u se”, by the C o lo m bian labo rato ry .
U rdanet a M , P ra t a A , S t ru c h i n e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c i n e t rial in B ra z il: co n ce p t u a l f ra m e w o rk , s t ud y d e s ig n a n d a na ly t ica l a p p ro a c h. R ev ista d a So cie d a d e B ra s ileira d e M e d ic in a T ro p ica l
2 9 : 2 5 9 - 2 6 9 , m a i- jun , 1 9 9 6 .
ad d ing a d esig n fac to r o f 2, a to tal o f 358 su b jects in eac h treatm ent g ro up seem ed ap p ro p riate. W e inclu d ed 400 ind iv id uals in each gro up .
Study p articip ants w ere reg istered w ith an indiv id ual id entificatio n num ber. The fo llo w ing info rm atio n w as reco rd ed fo r each o ne: nam e, sex, ag e, site o f resid ence, birth p lace, p lace o f last resid ency , d ate o f arrival to the study area, p rev io us histo ry o f m alaria ep iso d es (num ber, usual sy m p to m s and co m p licatio ns, d ate o f last infectio n), and clinical histo ry o f allergies o r o ther d iseases.
V accine and p laceb o p rep aratio ns w ere ap p lied in three d o ses: the l s^ o n d ay 0, the 2nd o n d ay 30 and the 3rd o n d ay 180. The reco m m end ed d o se o f 0.5m l w as ap p lied su b cu taneo u sly in the d elto id regio n.. To facilitate the d etectio n o f lo cal reactio ns, the first and third d o ses w ere ap p lied o n the rig ht arm and the seco nd , o n the left.
T ria l e n d - p o i n t s : d e t e rm i n a t i o n a n d m e a s u re m e n t : Co nsid ering the o b jectiv es o f the trial, sev eral end -p o ints w ere d eterm ined and m easured : 1) incid ence o f m alaria cases b y sp ecies, 2) d ensity o f asexu al b lo o d -stag e p arasitem ias, 3) d eterm inatio n o f sp ecific Ig G antibo d ies ag ainst blo o d -stag e antig ens and v ac c in e p e p tid e s an d , 4) f re q u e n c y and d escrip tio n o f sid e effects.
In o rd er to d eterm ine the incid ence o f infectio n, tw o m etho d s o f ev aluatio n w ere used : p ro g ram m ed p erio d ic visits and p assiv e and/ o r activ e search d o ne b y the FNS health ag e n ts. P e rio d ic al ac tiv e se arc h e s w e re p ro g ram m ed o n ev ery v accinatio n (d ay s 0, 30 and 180), and at reg ular interv als: ev ery tw o m o nths in the first y ear o f fo llo w -up and ev ery three m o nths in the seco nd (Tab le 1). Blo o d sam p les w ere o b tained fo r p arasito lo g ical exam inatio n in the field . The ad m inistratio n o f an e f f e c ti v e s c h i z o n ti c i d a l d ru g to all p articip ants b efo re the b eg inning o f the trial w as no t d o ne. Sho rtly after the ap p licatio n o f either d o se o f the v accine o r p laceb o , all p articip ants w ith asexu al p arasitem ia w ere ad eq u ately treated . Th e m ic ro sc o p ists in charg e o f the d iag no stic and treatm ent statio ns, as w ell as the field ag ents, w ere resp o nsible fo r the no tificatio n o f all cases o f infectio n id entified in the stud y su bjects d uring the w ho le study p erio d b y p assiv e o r activ e search, sp ecially d uring the interv als b etw een the p ro g ram m ed activ e searches. The su b jects’
T able 1 - A ctiv ity char t.
A ctivity Ev alu atio n
D ay v accinatio n sp lee n sero
-p arasito lo g y
hct leu co g ram
0 1st d o se X X X X
30 2nd d o se X X X X
45 X X
90 X X
180 3rd d o se X X X
195 X X X
240 X X
300 X X
360 X X
450 X X
540 1st y ear X X X
630 X X
720 X X X
3rd d o se: A u g u st, 1991. End o f fo llo w -u p : Feb ru ary ,
1993-nam e, register num ber, d ate and p arasito lo g ical result w ere ad eq u ately reco rd ed in sp ecific no teb o o ks. A w ritten labo rato ry rep o rt w as g iv e n to th e p artic ip an t. T re atm e n t w as sup p lied in case o f the p resence o f asexual p arasitem ia H ealth ag ents w ere instructed to p reserv e all thick b lo o d film s fo r further blind re-exam inatio n at the reference labo rato ry .
In c id e n c e o f m alaria in f e c tio n w as d eterm ined b y the c o n firm atio n o f the info rm atio n o btained fro m each stud y su b ject b y o n e o f the fo llo w ing m eans: w ritten p arasito lo g ical rep o rts g iv en to the stud y subject, m alaria infectio n reco rd s at the FNS statio ns, co nfirm atio n o f asexu al p arasitem ia b y the rev isio n o f thick b lo o d film s in the reference labo rato ry and p arasito lo g ical results o b tained in the p ro g ram m ed activ e searches.
M alaria w as d iag no sed by the m icro sco p ic exam inatio n o f thick b lo o d film s stained as reco m m end ed by the Pan A m erican H ealth O rg anizatio n14. A ll sam p les w ere exam ined b y the FNS m icro sco p ist in the field . N egativ e results w ere d eterm ined after the o bserv atio n o f 200 m icro sco p ic field s. A ll sam p les (p o sitiv e and neg ativ e) w ere p reserv ed and sent to the Malaria Labo rato ry o f the N úcleo d e M ed icina Tro p ical e N utrição o f the Univ ersity o f Brasilia, w here a b lind re-exam inatio n w as carried o ut fo r reliability o f results. A sexual b lo o d -stag e d ensities w ere calcu lated b y co u nting asexu al fo rm s p er 500 w hite b lo o d cells and exp ressed as num ber o f p arasites p er m m 3. Ev aluatio n o f sp leen size w as p erfo rm ed in the p ro ne p o sitio n o n ev ery p ro gram m ed v accinatio n, and o n d ays 540 and 720.
U rd a ne t a M , P rat a A , S t ru c h i n e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c in e trial in B ra z il: co n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a na ly t ica l a p p ro a ch . R ev ista d a So cie d a d e B ra s ileira d e M e d ic in a T ro p ica l
2 9 : 2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
o b tained fro m m em bers o f the co m m unity , ho sp ital reg isters and / o r the FNS reco rd s.
D uring the p ro g ram m ed activ e searches, cap illary b lo o d sam p les w ere co llected fro m the stud y su bjects b y fing er p uncture into three h e p a rin iz e d m ic ro c a p illa ry tu b e s . A fte r centrifug atio n and hem ato crit d eterm inatio n, p lasm a sam p les w ere ad eq u ately p acked , sto red and taken to the Labo rato ry o f Malaria o f the Univ ersity o f Brasilia. Ig G antibo d ies ag ainst asexu al fo rm s o f P. f a l c i p a r u m w ere d e t e r m i n e d u s i n g t h e i n d i r e c t im m u n o flu o rescenc e m etho d , as w ell as an tib o d ies ag ainst the v ac c in e p ep tid es (ELISA ) fo r ev aluatio n o f im m uno genicity.
A surv eillance system fo r d etecting any ad v erse reactio n w as set up d uring ev ery p ro g ram m ed v accinatio n. A ll p articip ants w ere clo sely m o nito red d uring the first 30 m inutes b y the m ed ical inv estig ato r in charg e o f the ap p lic atio n s. Em e rg e n c y e q u ip m e n t and therap eutic p ro ced ures w ere read ily available. Inquiry fo r p o ssible symp to ms and id entificatio n o f lo cal reactio ns w ere p erfo rm ed tw o ho urs after each ap p licatio n, and fo ur w eeks after the 1st d o se and tw o w eeks after the 2nc* and 3 d o ses. Particip ants w ere ad v ised to search fo r m ed ical care in case o f p o ssib le sev ere signs and sy m p to m s seco nd ary to v accinatio n. The freq u en c y and intensity , as w ell as the d esc rip tio n o f any ad v erse effec t, w as sy stem atically reco rd ed .
A s s e s s m e n t o f v a l i d i t y a n d p r e c i s i o n p ri n c i p l e s . A p relim inary step in the analy ses,
is the d escrip tio n and critical ev aluatio n o f the data g athered d uring the study. D uring this p hase, the assessm ent o f valid ity and p recisio n p rincip les is necessary . O nce d eterm ined , it is p o ssib le to d efine relev ant ep id em io lo g ic categ o ries, select p aram eters, d efine statistical m o d els and estab lish the m etho d s m o st ap p ro p riate fo r the estim atio n o f v accine efficacy and/ o r o ther end p o ints o f interest21.
The first hap p ening that takes p lace in the p ro cess, is the selectio n o f a su scep tible study p o p ulatio n. This is a straig ht fo rw ard co nd itio n fo r the ev aluatio n o f v accine efficacy . The rand o m allo catio n o f the v accine and p lacebo , as w ell as the “d o u ble-b lind ness”, w ere used w ith th e in te n tio n o f h o m o g e n e o u s ly d istributing kno w n and u nkno w n so urces o f h etero g e n eity am o ng “treatm en t” gro up s. Kno w n facto rs (co v ariates), w hich can help d iscrim inate p o ssib le h etero g en e ities o f
exp o sure and/ or suscep tibility to infectio n am o ng the study p o p ulatio n, w ere id entified and quantified d uring the study.
Fo r d eterm ining the su scep tib ility to infectio n o f the study p o p ulatio n, info rm atio n related w ith the num b er o f p rev io us m alaria ep iso d es, sp leen siz e and p resence o f Ig G antibo d ies against the asexu al fo rm s o f P.
f a l c i p a r u m w as o b ta in e d p r io r to th e
b eg inning o f the study. The p resence o f o n e o r all o f these v ariables, ind irectly ind icate that the p articip ant w as exp o sed , w as cap ab le to react inm uno lo g ically and w as su scep tible to infectio n. H o w ev er, their ab sen c e d o es no t n e c e ssarily im p ly re sistan c e to in f e c tio n (ev entho u g h it is p o ssib le), since m o st o f the indiv id uals w ere m ig rants w ith d ifferent tim e since arrival to the end em ic area.
A ltho ugh w e m ay assum e an equal exp o sure to in fec tio n in b o th g ro u p s b e c au se o f rand o m iz atio n, w e can exp ect d ifferences in the am o unt o f exp o sure (hetero g eneity ) w ithin each gro up . Transm issio n is also affected by seaso nality, o ther co nco m itant co ntro l m easures and b y the v accine itself, d ep end ing o n the p ro p o rtio n o f the p o p ulatio n v accinated (herd im m unity). Baseline transm issio n lev els p rio r to the interv entio n and d uring the study, can b e ap p ro x im ate d u sin g in fo rm atio n th at ind irectly d eno tes d ifferences in exp o su re to infectio n. W e co nsid ered ag e, sex, tim e since arriv al to the end em ic area, rep o rted num ber o f p rev io us m alaria ep iso d es, sp leen size, and antibo d ies titers at the b eg inning o f fo llo w -up . A ntibo d y d eterm inatio n and quantificatio n, is a re aso n ab le in d ire c t in d ic ato r n o t o n ly f o r suscep tibility, but also fo r exp o sure to infectio n. D ifferences in exp o su re to infectio n are also related to the num ber o f m alaria ep iso d es, as w ell as ag e and sex. Sp leno m eg aly has b een co nsid ered an ind icato r fo r m alaria end em icity and therefo re an ind icato r fo r exp o su re to infectio n, ho w ev er, its v alue as an ind icato r fo r exp o sure is q u estio nab le in areas w here rapid d iagno sis and sp ecific treatm ent are av ailable to the p o p ulatio n, as p art o f the co ntro l p ro gram s. The p erio d ic m easurem ent o f the co nsid ered facto rs allo w s to track chang es in e x p o s u r e to i n f e c ti o n , as w e ll as in suscep tibility, w hich is altered b y the v accine.
U rd anet a M , P rat a A , S t ru c h i n e r Cf, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c i n e t rial in B ra z il: co n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a n a ly t ica l a p p ro a ch . R ev ista d a So cie d a d e B ra s ileira d e M e d ic in a T ro p ica l
2 9 : 2 5 9 - 2 6 9 , m a i- jun , 1 9 9 6 .
asp ects m ust b e taken into acco u nt: 1) the m o d ificatio n o f the initial co ho rt characteristics u p to th e m o m e n t o f c o m p le tio n o f th e v accinatio n sched u le g iv en the p resence o f tim e-d ep end ent co v ariates, such as, the num ber o f m alaria ep iso d es and hum ural im m une resp o nse, and 2) lo sses to fo llo w -up , w hich w o uld co nstitute an im p o rtant lim itatio n that affects m ainly the p recisio n o f the estim ate o f v ac c in e effic ac y , m aking n ec essary the d eterm inatio n o f its m agnitud e and its causes. Bo th co nd itio ns m ay lead to the lo ss o f the d esired effect o f rand o m iz atio n (co m p arability o f the treatm ent g ro up s) w ith tim e.
Therefo re, the p relim inary analy ses o f data includ es the d escrip tio n o f the the study p o p u l a t i o n w i th r e s p e c t to th e su sc e p tib ility / e x p o su re fac to rs p re v io u sly d iscussed , assesm ent o f rand o m iz atio n and d efinitio n o f lo sses to fo llo w -up .
P re lim in a ry a na ly s e s o f t he d at a . The p resent study started in Janu ary 1991 and fo llo w -up end ed in February 1993- A to tal o f 800 v o lu nteers o f b o th sexes ag ing 7 to 60 years, w ere rand o m ly allo cated to o n e o f the treatm ent g ro up s (400 p articip ants o n each g ro up ). M o st o f them w ere migrants (99-6% ), natural o f no n-end em ic areas o f the co untry and resid ents in sev en d ifferent lo calities o f the rural area o f Co sta M arques.
Ev entho ug h b o th treatm ent gro up s can b e co nsid ered co m p arab le at enro llm ent (Table 2), the data sug g ests that the p o p ulatio n is hetero g eneo u s w ithin each gro up , no t o nly w ith resp ect to ag e and sex, but also related to the p rev io us co ntact w ith m alaria infectio n. A sim p le co rrelatio n analy sis rev ealed that the num b er o f referred p rev io us m alaria ep iso d es as w ell as the antibo d y titers, are p o sitiv ely co rrelated w ith ag e (r = 0.16 and r = 0.19, p <
0.001) and tim e since arrival to the end em ic reg io n (r = 0.37 and r = 0.22, p < 0.001). This suggests d ifferences in exp o su re to infectio n am o ng the p articip ants. W ith resp ect to sex, m ales had in av erag e m o re m alaria ep iso d es than fem ales (4.6 and 3-1, resp ectiv ely ; p = 0.002). H o w ev er, no d ifferences in tim e since arrival and antibo d y titers w ere d etected b etw een sexes. The p rev alence o f m alaria infectio n at the b eg inning o f the trial w as lo w (5.6% ). P. v iv ax (4% ) p red o m inated o v er P. f a lc i p a ru m (2% ) infectio ns. The p ro p o rtio n o f
p articip ants infected in b o th gro up s w ith either
T able 2 - P re- v ac c in at io n c o ho r t charact eristics .
C haracteristic V accine
n %
Plac eb o
n %
P*
A ge
< 11 57 14 75 19 O.32
11 - 20 131 33 127 32
21 - 50 185 46 168 42
> 5 0 27 7 30 7
Sex
M ale 247 62 245 61 0.88
Fem ale 153 38 155 39
Tim e sin c e arriv al (y ears)
< 1 138 40 162 41 0.95
1 - 2 61 15 62 15
2 - 3 66 16 60 15
> 3 114 29 114 29
Prev io u s m alaria ep iso d es
N o ne 38 10 51 13 0.25
1 - 3 100 25 87 22
sev eral 262 65 262 65
IFA an tib o d ies
p o sitiv e 115 29 115 29 0.88
n eg at iv e 277 71 279 71
Sp leen
p alp ab le 90 23 99 26 0.40
u np alp ab le 297 77 284 74
Prev alenc e o f in fectio n
P. falc ip ar u m 9 2 9 2 O.63
P. v iv ax 16 4 11 3
neg ativ e 367 94 370 95
*p -v alu es f o r y } tests f o r d ifferen c es b e tw e en g ro u p s
sp ecies w as co m p arable. Classic sym p to m s related to m alaria infectio n w ere referred by m o re than 95% o f the p articip ants in b o th gro up s.
Prio r to interv entio n, at least 89% o f the particip ants had b eing exp o sed to infectio n (b y e ith e r s p e c ie s ) and , th e re f o re , w e re su scep tible to it. O nly 29% had d etectable antibo d ies ag ainst the assexual fo rm o f P. fa lc ip a ru m at v ery lo w titers (less than l60).
A fter the c o m p letio n o f the v ac c in atio n sched u le, 93% had at least o ne ep iso d e o f m alaria and 57% w ere sero p o sitiv e.
U rd a ne t a M , P rat a A , S t ru c h i n e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c i n e t rial in B ra z il: co n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a n a ly t ica l a p p ro a ch. R ev ista d a S o c ie d a d e B ra s ileira d e M e d ic in a T ro p ica l
2 9 :2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
Lo sses to fo llo w -u p co u ld b e related w ith the d isease (m alaria ep iso d es b etw een the 1st and 3 d o se), and/ or w ith seco nd ary ad v erse effects o f the v accine/ p lacebo ap p licatio ns during the 1st and 2nd d o ses. To assess these p o ssib le relatio nship s, fo ur v ariables w ere created b ased o n info rm atio n, g athered during th at p e rio d o f tim e: n u m b e r o f tim es th e particip ant had asexual parasitemia ind ep end ent o f the p lasm o d ia sp ecies, num ber o f tim es the ind iv id ual searched fo r d iagno sis, p resence o r no t o f seco nd ary ad v erse reactio ns during the 1 s t a n d / o r 2 n d a p p l i c a t i o n o f th e v accine/ p lacebo , and tim e o f survival until the ap p lic atio n o f th e 3rc^ d o se. C o m p ariso n s w ere m ad e b etw een survivo rs and lo st to fo llo w -u p p articip ants fo r eac h treatm ent g ro up (Tab le 3).
T able 3 - C harac t erist ics o f lost to fo llo w - u p a n d su rv iv ors ivith resp ec t to p o s s ib le re lat e d v ariables.
Lo st Surv iv o rs
C haracteristic v ac c in e p lac e b o P v accine p lac eb o P
n % n % n % n %
Parasitem ia 0.8 0.6
n ev er 54 48 61 53 135 47 145 51
o n c e 33 29 33 29 76 27 75 26
tw ice 18 16 14 12 38 13 37 13
> Tw ice 8 7 7 6 38 13 28 10
D iag n o ses search 0.3 0.9
n ev er 35 31 49 42 85 30 85 30
1 - 2 53 47 41 36 108 38 110 39
3 - 5 16 14 16 14 73 25 69 24
> 5 9 8 9 8 21 7 21 7
A d v erse reactio ns 0.3 <0..001
y es 23 20 17 15 105 37 33 12
no 90 80 98 85 182 63 252 88
Surv iv al tim e (w k s) 0.5 0.8
m ean 12.5 11.5 26.5 26.5
To tal 113 115 287 285 0.9
* p v alu e fo r x 2 test fo r d iffe re n c es b e tw e e n g ro u p s and / o r fo r co m p ariso n o f m ean s (t-stu d en t test).
The p ro p o rtio n o f p articip ants, as w ell as the av erag e tim e o f survival w ere co m p arable b e tw e e n b o th tre atm e n t g ro u p s in the p articip ants lo st to fo llo w -up and survivo rs. No d if f e re n c e s b e tw e e n th e p ro p o rtio n o f v accinated and u nv accinated ind iv id uals w ere d etec ted b e tw een lo st to fo llo w -u p and surv iv o rs, in relatio n to the num ber o f tim es they had d etectab le p arasitem ia o r the num ber o f tim es they searched fo r d iag no sis (relatio n w ith d isease). Ev entho ug h the p ro p o rtio n o f ind iv id uals w ith seco nd ary ad v erse reactio ns
w as significantly g reater in the SPf66 gro up, the p ro p o rtio n o f ind iv id uals lo st to fo llo w -up w ho had seco nd ary reactio ns w as co nsid ered eq u al fo r b o th treatm ent gro up s. Co m p arability betw een lo st and survivo rs w as also m aintained w ith resp ect to all o ther v ariables. Lo sses seem to b e ind ep end ent o f the d isease and o f seco nd ary ad v erse reactio ns.
The m ain causes o f lo sses w ere ab senc e o f the p articip ants and the p resence o f any acute d isease d uring the p ro gram m ed v accinatio ns, and em ig ratio n o f the study area. D eaths w ere no t related to m alaria o r treatm ent (Tab le 4).
T able 4 - C au ses o f losses to fo llo w - u p b e fo r e t he c o m p let io n o f t he v ac c in at io n sc hedu le.
C ause V accine Plac eb o To tal
n % n % n %
A b sen ce 59 52.2 58 50.4 117 51.3
D iseases 19 16.8 19 16.5 38 16.7
Em ig ratio n 16 14.1 20 17.4 36 15.8
Preg nancy 8 7.1 10 8.7 18 7.9
W ithd raw al 8 7.1 5 4 3 n 5.7
M o v ed to u rb an area 2 1.8 2 1.7 4 1.7
D eath 1 0.9 1 0.9 2 0.9
To tal 113 100 115 100 228 100
The rand o m iz atio n effect w as m aintained after the co m p letio n o f the v accinatio n sched ule (Table 5), ev entho ug h 111 (19,4% ) o f the 572 p articip ants w ho receiv ed the 3 d o se w ere lo st to fo llo w -up in d ifferent p erio d s o f tim e. The m ain causes o f lo sses w ere the em igratio n o f the stud y area and transfer o f resid ence to th e u r b a n a r e a ( 7 6 . 6 % a n d 1 5 .3 % , resp ectiv ely ). D eaths no t related to m alaria w ere resp o nsible fo r 2.7% and o ther intercurrent d iseases fo r 1.8%. Three p articip ants (2.7% ) w ere exclu d ed fo r using im p reg nated bed nets and 1 (0.9% ) w as v accinated w ith the w ro ng p re p aratio n . Fo u rty six p e rc e n t o f the p articip ants lo st to fo llo w -up w ere v accinated . The gro up s w ere co nsid ered ho m o g eneo us w ith resp ect to the causes o f lo sses (%2; p = 0
.
6).
U rd ane t a M , P rat a A , S t ru c h i n e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c in e t rial in B ra z il: c o n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a na ly t ica l a p p ro a ch . R ev ista d a So cie d a d e B ra s ile ira d e M e d ic in a T ro p ica l
2 9 :2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
T able 5 - C o ho rt a n d lost to fo llo w - u p bas e lin e c harac t erist ic s aft e r t he c o m p let io n o f the v ac c in at io n sc hedu le.
V accine Plac eb o
C haracteristics lo st surv iv o rs lo st surv iv o rs
n % n % p* n % n % P '
A ge
< 11 6 12 45 19 0.52 10 16 53 24 0.52
11 - 20 20 39 73 31 21 35 75 33
21 - 50 20 39 98 42 25 42 76 34
> 5 0 5 10 20 8 4 7 21 9
Sex
M ale 28 55 149 63 0.27 36 60 135 60
Fem ale 23 45 87 37 24 40 90 40
Tim e sin c e arriv al y rs
< 1 24 47 87 37 0.16 30 50 81 36 0.10
1 - 2 10 20 44 19 7 12 49 22
2 - 3 3 6 43 18 7 12 41 18
> 3 14 27 62 26 16 26 54 24
Prev io u s m alaria ep iso d es
n o n e 7 14 17 1 0.18 9 ■ 15 26 12 0.77
1 - 3 15 29 57 24 14 23 53 23
sev eral 29 57 162 69 37 62 146 65
IFA an tib o d ies
p o sitiv e 17 33 72 31 0.69 15 25 66 29 0.51
neg ativ e 34 67 164 69 45 75 159 71
Sp leen
p alp ab le 10 20 6 i 27 0.32 13 22 57 29 0.54
u np alp ab le 40 80 167 73 45 78 159 71
•p - v alu e fo r %2 tests fo r d ifferen c es b e tw e e n p ro p o rtio ns.
ind irectly d iscrim inate b etw een suscep tibility and exp o sure o f the p articip ants to infectio n w ere d eterm ined . They w ill p erm it to create relev ant ep id em io lo g ic categ o ries, necessary in future analy ses, to ad just estim ates o f v accine efficacy o r d escribe effect m o d ifiers.
A na ly t ica l a p p ro a ch f o r es t im at ing v a c cin e e ffica cy . The efficacy o f a v accine has b een e stim ate d fro m 1 - RR, w h e re RR is so m e m easure o f relativ e risk. This co uld b e attack rates, incid ence rates, o r seco nd ary attack rates, am o ng o thers. Such rates sho uld rep resent the instantaneo us p ro bability o f p ro g ress to the end p o int o f interest (infectio n, clinical m alaria o r o thers) o f the v accinated and unv accinated g iv en an eq u al am o u nt o f exp o su re to infectio n21. H o w ev er, ensuring that co m p ariso n p o p u latio ns are su b jec te d to the sam e exp o sure o f infectio n is d ifficult to achiev e in the field . It is usual to enco u nter hetero g eneity o f exp o su re to in fec tio n in the stud y p o p ulatio n, related to facto rs such as ag e, sex, o ccu p atio n and so cio -eco no m ic characteristics, am o ng o thers. Besid es, equal exp o sure to infectio n is no t sufficient to d eterm ine a p recise and m eaning ful estim ate o f the d irect effect, since p aram eter estim ates can vary w ith the am o unt o f exp o sure. Therefo re, the d irect effect m ust b e d efined w ith resp ect to a giv en
am o unt o f exp o su re to infectio n. In field studies o f m o st infectio us d iseases the exp o sure to infectio n canno t b e co ntro lled by the o bserv er. The term field efficacy ap p lies to situatio ns in the field w here the intent is to m easure d irect effects and attem p ts are m ad e to co ntro l fo r the am o unt o f exp o su re. D irect effectiv eness m easures are tho se that includ e so m e interactio n o f ind irect effects w ith the efficacy p aram eter, a situatio n that is co m m o n in field studies o f m o st infectio us d iseases5 6 7 8.
U rd ane t a M , P ra t a A , S t ru c h i n e r Cf, To sta CE, T a uil P, B o ulo s M . S P / 6 6 v a c c in e t rial in B ra z il: c o n ce p t u a l fra m e w o rk , s t ud y d e s ign a n d a na ly t ica l a p p ro a ch. R ev ista d a So cie d a d e B ras ile ira d e M e d ic in a T ro pica l
2 9 : 2 5 9 - 2 6 9 , m a i- jun, 1 9 9 6 .
effect o f the v accine o r v accine efficacy . Lack o f these info rm atio n lead s to the estim atio n o f co m p o u nd rates that d escribe the transitio n o f the su sc ep tib le stag e in v ac cin ated and u nv accinated to the end p o int o f interest. M e a s u re s o f v a c c i n e e f f i c a c y b a s e d o n co m p o u nd rates tend to und erestim ate the efficacy o f the interv entio n21.
The info rm atio n g athered in this trial is rep o rted as tim e to the ev ent o f interest (m alaria ep iso d es). This w ill allo w us to calcu late incid ence d ensity rates fo r bo th, v accinated and u nv accinated gro up s. Baseline transm issio n, as w ell as su scep tib ility to infectio n o f the study p articip ants, w ill be d eterm ined ind irectly by d efining relev ant ep id em io lo g ic c ateg o ries that d iscrim inate b etw een exp o su re and su scep tib ility to infectio n and hetero g eneities o f b o th in the study p o p ulatio n.
Based o n the clinical characteristics o f m alaria infectio ns in the study p o p ulatio n, d eterm ined in p rev io us stud ies, clinical m alaria can b e easily id entified 17. Therefo re, a case o f m alaria w ill b e d efine as the p resence o f P. fa lc ip a ru m o r P. v iv ax b lo o d- s t a ge parasitemia. A new infectio n cau sed b y either sp ecies w ill b e d e fin e d as a p o sitiv e p arasito lo g ic al exam inatio n in a p articip ant o b serv ed to b e free o f p arasitem ia fo r at least 30 d ays since a p rev io us p o sitiv e slid e. W ith this ap p ro ach w e intend to exclu d e fro m the analy ses p o ssible treatm ent failures, relap ses o r recrud escences. It w ill also p erm it to d efine the tim e at risk fo r eac h p articip ant sinc e b o th sp ec ies o f p lasm o d ia co nsid ered are sensitiv e, up to a certain extent, to the antim alarials used fo r treatm ent fo r either sp ecies.
Estim ates o f the p ro tectiv e effect o f the v accine w ill b e calcu lated fo r the interval b etw een the ap p licatio n o f the 2nc* and 3rc^ d o se and after the co m p letio n o f the v accinatio n sched u le. W e w ill co nsid er an ind uctio n p erio d o f 30 days after the ap p licatio n o f each d o se. W e intend to estim ate the o v erall, as w ell as the crud e and stratum sp ecific estim ates o f the p ro tectiv e effect o f SPf66 fo r the first and seco nd ep iso d es o f P. f a l c ip a ru m and P. v iv ax infectio ns sep arately .
O ne im p o rtant issue w ill b e taken, into ac c o u n t at this p o int. Since no rad ical treatm ent w as ad m inistered to the p articip ants b efo re the ap p licatio n o f the v accine o r p laceb o , w e can exp ect that the im m une
resp o nse to the v accine m ay b e m o d ulated by intercurrent m alaria infectio n. P. f a lc i p a ru m and / o r P. v iv ax infectio ns d etected d uring the v accine ap p licatio ns o r ind uctio n p erio d s, w ill b e co nsid ered as co v ariates in the analy ses.
M easures o f the p ro tectiv e effect o f the v a c c in e in th is trial w ill b e b a se d o n co m p o u nd rates. A ttem p ts w ill b e m ad e in the analy ses to co ntro l fo r hetero g eneities o f exp o sure and suscep tibility to infectio n in the study p o p ulatio n. Therefo re, the estim ated p ro tectiv e effect that w ill b e o b tained w ill b e that o f field d irect effectiv eness. If su bjected to c o rre c tio n u sin g p re v io u s e n to m o lig ic al info rm atio n o f the study area, estim ates w ill b e c lo ser to the id eal c o n c ep t o f b io lo g ical efficacy 21.
RESU M O
O p re s e n t e a rt igo d e s c re v e a p o p u la ç ã o d e es t udo e o d e s e nh o d o e ns a io d e c a m p o d e f a s e III d a
v a c in a s int ét ica S P f6 6 n o B ras il. São a v a liad o s os p rin cí p io s b ás ico s d e v a lid a d e e p re c is ã o , e s s e ncia is p a ra a es t im a çã o a d e q u a d a d a e ficá ci a v a cina i. O s re s ult a do s d a a n á lis e e x p lo ra t ó ria d e d a d o s s ão
d is cut ido s as s im co m o , a a b o rd a g e m a na lít ica p a ra a e s t im a ção d a e ficá ci a v acinai. Es te t ra b a lho f o rn e c e o m a rc o co nc e it ua i p a ra f u t u ra s p u b lica çõ e s .
P alav ras - chav es : V a c in a a nt im a lá rica . E ns a io
d e ca m p o SP f6 6 . E fi c á c ia v acinai.
ETH ICA L CO N SID ERA TIO N S
This trial w as ap p ro v ed b y the ethical co m m ittees fro m the Univ ersity o f Brasilia, Univ ersity o f São Pau lo and Sc h o o l o f M ed icine o f Uberaba. W ritten co nsent w as o b tained o f ev ery p articip ant. Serv ices o f the sam e quality w ere p ro v id ed to accep to rs and no n-accep to rs. Malaria co ntro l m easures w ere kep t u nchang ed d uring the v accine trial.
A C K N O W LED G EM EN TS
U rdanet a M , P rat a A , S t ru c h in e r CJ, To sta CE, T auil P, B o ulo s M . S P / 6 6 v a c c i n e trial in B ra z il: co n ce p t u a l f ra m e w o rk , s t ud y d e s ign a n d a n a ly t ica l a p p ro a ch . R ev ista d a So cie d a d e B ra s ileira d e M e d ic in a T ro p ical
2 9 :2 5 9 - 2 6 9 , m a i- jun , 1 9 9 6 .
and the Braz ilian Research Co uncil (CN Pq) fo r financial sup p o rt.
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