Cad . Saúd e Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998
A case-cont rol study of human papillomaviru s
and cervical squamous intraepit helial
lesions (SIL) in Harris County, Texas:
d i ff e rences among racial/ et hnic gro u p s
Um e stud o d e c aso -c o ntro le d e infe c ç ão p o r
p a p i l l o m a v i rus humano e c ânc e r d e c o lo ute rino
e m Harrys Co unty, Te x a s
1 De p a rt m en t of Gy n e c o l o g i c On c o l o gy Un i versi ty of Tex a s M . D. An d erson Ca n c e r Ce n t e r, Ho u s t o n , Tex a s 7 7 0 3 0 - 4 0 9 5 ,U S A . 2 Nation al Cen ter for In f ectiou s Di s e a s e s , Cen t ers f or Disease C o n t rol a n d Pre ve n t i o n . Ma il Stop G-18, 1600 Cli fton R d . , At l a n t a ,G A ,3 0 3 3 3 ,U S A . 3 App lied Re s e a rch Bra n c h , Di vision of Can cer Con tro l an d Popu la tion Sci en ce, N a ti on a l Ca n cer In s t i t u t e . 6130 Exe c u t i ve Bl vd . ,M S C 7 3 4 4 , Be t h e s d a , Ma ry l a n d 2 0 8 9 2 - 7 3 4 4 ,U S A .
Gu illerm o To rt o l e ro - Lu n a 1
M ich ele Follen Mit ch ell 1
Da vid C. Sw an 2
Ru th An n Tu ck er 2 Lou ise W i d e roff 3
Josep h P. Icen ogle 2
Abst ract W e con d u cted a case-con t rol stu d y of t h e a ssocia tion bet w een SIL a n d H PV a m on g w h ites (W ) , African Am erican s (AA), an d Hisp an ics (H) in Ha rris Cou n ty, Tex a s . Ca ses w ere iden -t ified a -t M .D. An d erson Can cer Cen -t er Col p osco p y Clin ic. C o n -t rols w ere id en -t ified a m on g w om en obta in in g rou tin e Pap screen in g at tw o Harris Cou n ty Hea lth De p a rtm en t Clin ics. H P V w as d etected by a PCR- based flu orescen t a ssay. Dich otom ou s an d p olytom ou s logistic re g re s s i o n m od el s w ere u sed t o estim a te ad ju st ed od d rat ios (AOR) an d 95% con fid en ce in terva ls (CI) for SIL am o n g ra cia l/et h n ic grou p s an d grad e of d isea se. Pre v alen ce of HPV in fection w a s 64% in l ow gra d e SIL (LSIL), 84% in h igh gra de SIL (HSIL), an d 19% in con tro l s . Risk of SIL w as high er in H t h a n in W an d AA, AOR 29 .5 (12.4 -70.5), 15.3 (6.0- 33 .8), a n d 5.8 (2.6- 12 .6), re s p e c t i ve l y. Si m i l a rl y, racial/eth n ic d ifferen ces w ere observed for bot h LSIL (AOR = 16.6, 7 . 7 , a n d 4.3, re s p e c-t i vely) a n d H SIL (AOR = 78.6, 3 4 . 6 , an d 14.2, re s p e c c-t i ve l y ) . Fin d in gs su p p orc-t c-the associac-tion be-tw een SIL an d HPV an d d ifferen ces in th e stren gt h of th e association w ith LSILs an d HSILs. Da t a a lso su ggest a h igh er risk for H an d a low er risk for AA.
Key words Pa p i l l o m a v i r u s ; Ce rvix Ne o p l a s m s ; Eth n ic Gro u p s ; Ca s e - C o n t rol St u d i e s
Re sumo Foi realiz ad o u m est u d o ca so con trole p a ra an alisar a associação en t re lesões in t ra -ep it el iais escam osas d o colo u terin o (SIL) e H PV en t re m u lh eres b ra n c a s , n e g ras e la tin as em Harris Cou n ty, Tex a s . Os casos fora m id en tifica d os n a M . D. An d erson Can cer Cen ter Colp oscop y C l i n i c , e os con troles foram ob tid os rea liz an d o- se ex a m e d e Pa p a n icola u em d u a s clín ica s d o De p a rta m en to de Saú d e. O HPV foi d etectad o p or m eio d e en sa io d e PCR (p rim er M Y09/M Y11).
Fo ram con stru íd os m odelos d e re g ressão logística p ara estim ar as o d d s ra t i o sa ju stad as (AO R ) , e
seu s in tervalos d e con fia n ça d e 95% (IC) d e SIL en tre os gru p os étn icos e grau s d a doen ça. A p re-valên cia d e HPV n as SIL de b aixo grau (LSIL) foi de 64%; n as d e alto grau (HSIL), 8 4 % ; e 19% n os c o n t ro l e s . O risco d e SIL foi m aior em m u lh eres latin as q u e em bran cas e n egra s , sen d o observ a-d a s , re s p e c t i va m e n t e , as segu in tes AO R : 29,5 (12,4-70,5); 15,3 (6,0-33,8); e 5,8 (2,6-12,6). De for-m a sifor-m ilar, f o ra for-m ob serva d as d iferen ças p ara afor-m bos LSIL (AO R , re s p e c t i va for-m e n t e , d e 16,6; 7,7 e 4,3) e HSIL (AOR d e 78,6; 34,6 e 142). Os resu ltad os ap óiam a existên cia de a ssociação en tre SIL e H P V, d i f e ren ças n a força de associa çã o com SILs e H SILs, e su gerem risco m ais eleva d o para m u -l h e res -lat in as e m en or pa ra m u -lh eres n egra s .
T O RTO LERO - LUNA, G. e t al. 1 5 0
Cad . Saúde Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998
I n t ro d u c t i o n
Du rin g t h e la st d eca d e, s eve r al ep id e m io lo gi-c al an d exp erim en ta l stu d ies h a ve d o gi-cu m en ted a str on g a sso cia tio n b etween cervic a l n eo -p lasia an d h u m a n -p a -p illom aviru s (HPV) in fec-tio n (Mu ñ oz, 1997). Th e a ss o cia fec-tio n h a s b een o b s e r ved fo r b o th in va s i ve a n d p re - i n va s i ve c e r vica l n eo p las ia s, a n d th e resu lts h a ve b een co n sisten t wo rldwid e (Bo sch et a l., 1993; 1995; Sh iffm an et al., 1993; Becker et a l., 1994; Liaw et al., 1995; Olsen et al., 1995). Alth o u gh in ciden ce a n d m o r ta lity fro m cer vical ca n cer in th e U.S. h a ve d eclin ed d u rin g th e la st 50 ye a r s, ra c i a l / eth n ic differen ces rem a in . Wo m en from u n d erre p erres en ted ra c ia l/ eth n ic gro u p s an d low i n -com e wom en con tinu e to carry a h igh er bu rd e n f rom th is n eop la sm . In th e U.S., in ciden ce ra t e s are h igh er am on g Vietn am ese, Hisp an ic, African A m e rican , Na t i ve Ala skan , an d Ko rea n wo m en ( Becker et a l., 1992; Miller et a l, 1996). Si m i l a r disp arities in in ciden ce an d m ortality rates from c e rvica l can cer a re re p o rted in Te x a s. In c i d e n c e rates in Texa s are h igh er in Hi s p a n i c s, in term e-d iate in Africa n Am erican , a n e-d lower in wh ites. Desp ite th is, few stu dies h a ve exa m in ed th e ro le of ra cia l/ eth n ic d ifferen ces in risk fa cto rs a ss o ciated with cervica l n eo p las ia . It is n ecess a r y to d et er m in e if d iffe ren c eecess in th e p re va -le n ce o f th es e fa ct o rs a n d th eir a ss o cia tio n with th e cer vical n eo p la sia m igh t h elp exp la in t h e ra cia l/ e th n ic d is p a rities in in c id en c e a n d m o r tality ra tes (Na p o l e s - Sp rin ger et a l., 1996). Little is kn own ab o u t d ifferen ces in th e p re va -le n ce of b io lo gic al fa c to rs s u ch a s H PV in fe c-t io n a n d o f H PV c-typ es a m on g ra c i a l / e c-t h n i c g rou p s an d th eir associatio n with cervical n eo-p la sia . Becker et a l. (1994) re eo-p o r ted differe n c e s b e t we e n H is p a n ic a n d n o n -Hisp a n ic w h ite wom en in New Mexico in th e risk o f HSIL associa ted with HPV in fectio n . A stro n ger a ss o associa -t io n b e-t ween H PV a n d h igh -gra d e d ys p la s ia , p a r tic u lar ly a m o n g H PV 16 a n d 18 p o s it ive wo m en , wa s o b se r ved fo r Hi s p a n i c s. In ad d ition , Bo sch et a l. (1993; 1995) h a ve sh own geo -g ra p h ic d ifferen ces in th e p re va le n ce o f H PV in fectio n an d HPV typ es.
We con d u cted a m u lti-eth n ic, clin ic based , c a s e-c o n t ro l stu dy to assess th e associatio n be-t ween sq u a m o u s in be-tra ep ibe-th elia l lesio n s (SILs ) a n d H PV D NA sta tu s a n d o th er r is k fac to rs a m on g wom en fro m th ree racial/ ethn ic gro u p s ( w h i t e, African Am erica n , a n d Hisp a n ic) re s i d-in g d-in Ha r r is Co u n t y, Te x a s. In th is re p o rt , we foc u s o n th e ro le o f H PV st a t u s am o n g wh ite, A f ri c a n - A m e rica n , a n d Hisp an ic wo m en in th e risk o f SIL (low - g ra de SIL [LSIL] a n d h igh -gra d e SIL [HSIL]).
M aterial and methods
Study population
Th is stu dy wa s co n du cted in Ha r ris Co u n t y, TX b e t ween Sep tem ber 1991 an d Augu st 1994. El igib le ca s es we re id en tified a m o n g wo m en re f e r re d t o th e Un i ver s ity o f Texa s M. D. An d er -so n Ca n c er Ce n ter (U TM DACC) Co l p o s c o py Clin ic fo r fu rt h e r eva lu a tio n o f a n a b n orm a l Pap sm ea r. Th is clin ic p rovides co lp oscop y services for m in o rity a n d eco n om ically disa dva n ta ged wo m e n wit h ab n o rm a l Pa p sm e a rs re -f e r red -fr o m H ealth De p a rtm en t clin ics in Ha r-ris Co u n t y. On ly wo m en with a co n firm ed h is-to lo gical d ia gn o sis o f squ am o u s in tra e p i t h e l i a l lesio n (SIL) we re in clu d ed a s ca ses. Co n t ro l s we re selec ted a m o n g wo m en a tten d in g fa m ily p la n n in g an d s creen in g services a t two Ha r ri s Co u n t y Hea lth D e p a rt m e n t (H CH D ) clin ics. Th e s elec tion o f th e t wo H CH D c lin ics fo r re -c ru itm en t o f -co n tro l su bje-cts was b ased on th e l a rge a n d m u lti-e th n ic p o p u la t io n to wh ich th ey p r ovid e m ed ica l ca re. Wo m en eligib le fo r th e co n trol gro u p we re in clu ded in th e stu dy if th e c yto lo gic a l s m e ar a t th e t im e o f re c ru i t -m en t wa s re p o r ted a s n o r-m a l. Bo th ca ses a n d c o n t ro ls we re n o n p regn a n t wh it e, Afri c a n -A m e rica n , o r His p a n ic wo m en a ge d 18 ye a r s an d o lder, a n d residen ts o f Ha r ris Co u n ty a t the tim e o f th e s tu d y. Oth er e ligib ilit y c ri t e ria in clu ded n o p revio u s h isto ry o f cervica l n eo p la -sia a n d / o r t rea tm en t fo r c er vic a l n e o p la s ia , c a n c e r, o r h ystere c t o m y. In ad dition , wo m en in th e co n tro l grou p h ad a n ega tive h istor y o f a b -n o rm a l Pap sm ear o r cervica l bio p sy.
Data collection
e-Cad. Saúd e Púb lic a, Rio d e Jane iro , 14(Sup. 3):149-159, 1998
rio d ); sm o kin g h isto r y (s m o kin g st atu s, d u ra -tio n , a n d in ten sity o f sm o kin g h ab its); co n trac e p t i ve h isto r y (h ist o r y o r trac u rre n t u s e o f traco n -t ra c e p -t i ve m e -th o d s , -t yp e o f co n -tra c e p -t i ve m eth o d u sed , an d du ra tio n o f u se); Pa p sm ea r f re q u e n c y; an d m edica l h istory (sexu ally tra n sm itted d isea ses a n d ism sm u n o su p p re s s i ve d is o rd ers). Th e seco n d p a rt of th e in te rview co n -sist ed o f a co m p u ter-a s -sist ed fo o d fre q u e n c y q u e s t i o n n a i re u sin g th e “Health Habits an d Hi s t o ry Qu e s t i o n n a i re : Diet Hi s t o ry an d Ot h e r
Risk Fa c t o r s” (Blo ck, 1989). Wom en we re asked
th eir a ve ra ge freq u en cy o f co n su m p tio n o f 98 foo d item s du r in g th e yea r p rio r to en ro l l m e n t . Ca s es h a d a co m p lete p h ys ic a l a n d p e lvic exa m in a tion , rep ea t Pap sm ea r, co lp o s co p ic exam in ation , co lp oscop ically d irected b io p sies o f a b n o r m a l a re a s, c o lle ct io n o f t wo ce rv i c a l sam p les for H PV testin g by Vi ra Pa p / Vi ra Ty p e™ (stan dard care at th e UTMDCC) an d p olym erase ch a in rea ction (PCR). Th e m ed ical sta ff o f th e colp oscop y clin ic (a gyn ecologic on cologist, two g y n e c o l o g i s t s, two fa m ily p h ysic ia n s, a n d two c o l p o s c o p y t ra in ed n u rs e p ra ctit io n e rs) c o n -du cted exa m in a tio n s a n d sp ecim en collectio n . Wom en in th e c on t ro l gr ou p a lso h a d a c o m -p lete -p h ys ic a l a n d -p elvic exam in a tion , Pa -p s m e a r, a n d co llec tio n o f two c ervic a l sa m p les fo r H PV tes tin g b y Vi ra Pa p / Vi ra Ty p e™ a n d PCR. Exa m s a n d sp ec im e n co llec tio n we re p e rf o rm ed by two n u r se p rac tition ers tra i n e d a t th e UTMDACC. Sa m p les fo r Vi ra Pa p / Vi-ra Ty p e™ t estin g we re c o llec ted wit h a co tto n swa p a n d p re s e r ved in th e m e d ia t ra n s p o r t p rovided b y th e m an u fa c tu re. Th e sa m p les fo r PCR an alysis were collected with cervical b ru s h-e s. Ch-e rvical bru sh h-es wh-e rh-e storh-ed in vials an d rh-e- re-f ri g e ra ted im m e d ia tely in d r y ice a n d la t er a t -7 0oC u n til H PV testin g by PCR was co n du cted. In a d d it io n , a 20c c b lo o d s a m p le w as o b -ta in e d fr o m a ll s tu d y p a r ticip a n t s fo r se ru m d e t e r m in a tio n s o f retin o l an d β- c a ro ten e a n d o t h e r s ero lo gic s tu d ie s. Blo o d s a m p les we re collected in EDTA va c u ta in er tu b es, p ro t e c t e d f ro m exp osu re to ligh t with alu m in u m fo il, a n d s t o re d im m ed ia te ly in d r y ice. With in 4 h o u r s a fte r ve n i p u n c t u re, th e s am p les we re cen -t rifu ged a n d seru m wa s s-tored in 1m l a liqu o -ts at -70oC u n til a n a lysis.
Cytological and histological diagnosis
Cyto lo gic al a n d h isto lo gic al sp ecim en s for a ll c a ses we r e in te rp re ted a t th e U TMD ACC D e-p a rtm en t o f Pa t h o l o g y. Two in dee-p en d en t re a d-ers a t th e UTMDACC re v i e wed ea ch Pap sm ear an d b io p sy. A co m m ittee o f sta ff m em b ers in -clu d in g th e D i recto r of th e Co l p o s c o p y Clin ic
(MFM) re v i e wed discrep an t cases m o n th ly a n d re a ch ed a fin a l d ia gn o s is. Cytologic al sp ec i-m en s fo r th e c o n t ro l gro u p fo llo wed t h e n o r-m a l p r o ce ss a t th e Ha r r is Co u n ty He a lt h Dep a r tm en t. All sDep ecim en s we re rea d a n d in ter -p ret ed a t a cen t ra l i ze d la b o ra t o r y a t th e Sa n An to n io Ch est Hosp ita l, in Sa n An to n io, Te x a s. A c o p y o f th e c yto lo gy re p o rt wa s o b t a in ed f rom th e H CHD clin ics for a ll p a tien ts wh o h a d co m p leted a n in ter view in o rd er to d eterm i n e fin a l eligibility.
O ve rall, 1,776 wom en we re screen ed for eli-gib ility (1,264 a t UTMDACC a n d 512 a t H CH D c lin ic s). Six h u n d re d a n d twe n t y-six p a t ien t s (36%) we re n o n -eligib le b eca u se o f re s i d e n c y o u t o f Ha r r is Co u n t y. Ad d i t i o n a l l y, 329 we re n on -eligible b eca u se of: p rior h istory an d t re a t-m en ts o f cervica l a b n o rt-m a lity (n = 105); p re g-n ag-n cy (g-n = 104); a ge less th ag-n 18 yea rs (g-n = 60); Hy s t e rec tom y (n = 13), an d o th e r rea s o n s (n = 40). At UTMDACC 659 wo m e n wh o m et e ligi-b ility a t t h e t im e o f th e fir st visit a cce p ted t o p a rt i c i p a t e. Sixt y fo u r p ercen t (399 wo m e n ) m eet th e eligibility cri t e ria o f h istolo gica l dia g-n o sis of SIL. After exclu dig-n g 45 add itiog-n al ca ses fo r la ck o f H PV d a ta an d 29 for in co m plete da -ta , th e ca se gro u p in clu d ed 325 wo m en . At th e H CHD clin ics, 328 wo m e n we re eligible an d ac-cep ted to p a rt i c i p a t e. Twe n t y-six wom en we re later exclu ded beca u se th ey did n o t m eet cyto-lo gica l cri t e ria (n egative cyto cyto-logy) for in clu sion a n d 32 we re exc lu d ed b ec a u s e o f th e la c k o f H PV d a ta . T h e re f o re, 270 c o n tr o ls we re a va i l-ab le for th e stu dy.
HPV testing
T O RTO LERO - LUNA, G . e t al. 1 5 2
Cad . Saúde Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998
In c., Be ve r l y, MA) at 1000 x g for 30 m in . An a dd itio n a l 2 m l o f water wa s a ddddedd to ea ch re t e n -t a -t e, a n d -th e cen -tr ifu ga -tio n wa s rep ea -ted . Re-ten ta tes we re th en co llected an d dilu ted to 200 µ l wit h wa ter. 10 u l o f th is D NA was u s ed in ea c h flu o ro gen ic PCR rea ctio n . A c on ta m in a -t io n c o n -t ro l fo r PCR rea c-t io n s co n s is -tin g o f 1 m l wa ter was in ser ted a fter eve ry ten th p a tien t s am p le a n d s u b jec ted t o th e en tire extra c t i o n a n d DNA d etectio n p ro tocol. Ea ch DNA sp eci-m en wa s tes te d fo r ove r a ll H PV p o sitivit y by PCR u sin g co n sen su s p rim ers fo llowed by elec-t ro p h o r esis in eelec-th id iu m b ro m i d e - c o n elec-t a i n i n g gels (Ma n o s et al., 1989). H PV- p o s i t i ve sa m p les we re t est ed b y th e q u a n t it a t ive flu o re s c e n t p ro b e a s sa y fo r H PV t yp e s 16, 18, 31, a n d 45. Th is p ap er focu ses o n ly o n th e resu lts from th e co n sen su s p r i m e r.
Pro b e s: Th e flu o rige n ic p r o b e a ss a y is b a s ed o n t h e in c rea s e in flu o res cen t s ign a l wh ich occurs wh en p ro bes are degrad ed by th e 5 ’→3’ exo n u c lea s e ac tivit y of Ta q p olym e ra s e ( F ö r s t e r, 1948). After d egra d a tio n , th e re p o rt e r d ye ((FA M ) 6 c a r b ox y f l u o rescein or (HEX)h exa -c h l o ro - f l u o res-cein ), p resent at th e 5’-en d of ea-ch p ro b e ca n d iffu se a wa y fro m a q u en ch er d ye ( ( TA M R A ) 6 - c a r b ox y- t re t ra m e t h y l - s h o d a m i n e ) p re sen t o n o r n ea r th e 3’-en d o f ea ch p ro b e t h e re by in crea sin g th e flu orescen t sign a l fro m the re p o rter dye. Th e p rob e sequ en ces for ea ch o f th e h igh ris k H PVs we r e se lected a n d syn -t h e s i zed a s d es cr ib ed p revio u s ly (Swa n e -t a l., 1 9 9 7 ) .
Prim ers: Th e p rim er seq uen ces we re select-ed u sin g th e Oligo 5.0 p rim er a n a lysis p ro g ra m ( Na tio n a l Bi o s c i e n c e s, In c., Plym o u th , M N). Th e p r im er p a ir s fo r e ac h o f th e H PV t yp e s we re selected ba sed o n h a vin g a Tm o f ap p rox i-m a tely 65o, p redicted la ck o f c ro s s - h y b ri d i z a-tio n to oth er co m m on HPV typ es, n o p re d i c t e d loo p fo rm ation a n d n o p redicted dim er form a-tion with th e o th er p ri m e r.
Co n t ro l tem p la t es: Co n t ro l tem p la t es fo r H PV t yp e s 16, 18, 31, 33, 35, 45, 51, 52 an d 56 we re p re p a red by PCR a m p lifica tio n o f clo n ed D NA u sin g L1, typ e-s p e cific p r im ers (se-q u en ces a va ilab le o n re(se-qu est) an d p u rified u s-in g Ce n t rico n 100 m ic r o c o n c e n t ra t o r s. T h e DNA co n cen tra tio n s we re d eterm in ed by flu o -ro m e t ry (DyNA Qu a n t 200, Ho e f e r- Ph a rm a c i a Bio tec h , Sa n Fra n c i s c o, CA) u sin g 2 µ l o f DN A so lu tio n in 2 m l fre sh ly p re p a red Ho e c h s t 33258 d ye so lu tion (1 µ g/ m l in 10 m M Tr is p H 7.4, 1 m M ED TA, 200 m M Na Cl) (Ph a rm a c i a Bio tech In c., Piscata wa y, NJ). A 100 µ g/ m l DNA s t a n d a rd wa s u sed .
Ass a y c o n tr o ls co n s is tin g o f a d ilu t io n se-ries of the ho m o logou s tem p late (1x105to 3 x 1 01
co p ies ) a n d a set of h et er olo go u s te m p lat es ( 2 x 1 03cop ies o f HPV typ es 16, 18, 31, 33, 35, 45, 51, 52 an d 56 in sep a ra te tu b es) we re in clu d ed in ea ch r u n . Ea ch co n t ro l sa m p le a ls o c o n -ta in ed 50 n g h u m a n p la cen -ta l DNA.
Fl u o rigen ic PCR. Th e 50 µ l PCR m ixtu re s co n ta in ed 10 m M Tr is p H 8.3, 50 m M KCl, 4.5 m M M gCl2, 200 µ M d NTP’s, 0.3 µ M ea c h p ri m e r, 50 n M ea ch flu o rigen ic p ro b e (FA M -HPV p ro b e a n d H EX-glo bin p rob e), 0.025 U/ µ l Ta q Go ld p o lym e ra se (Pe rk i n - Elm e r Co r p . , No r walk, CT) an d 10 µ l tem p late DNA. Fo l l ow-in g Ta q p o lym er a se a ct ivat io n a n d t em p la te d e n a t u ra tio n fo r 12 m in a t 95o, a m p lifica t io n co n d itio n s we re as fo llows: 40 cycles o f 30 se c a t 94o, 10 s ec at 60oa n d 2 m in a t 65o. Fl u o re scen c e m eas u red in a Pe rk i n Elm er LS 50B lu -m inescen ce sp ectro -m e t e r. Data a cq u isition a nd an a lysis we re perf o rm ed u sin g th e Ta q Man Fl u -o rescen c e Da ta Ma n ager (Pe rk i n - Elm er C-o r p . ) a n d Exc el 5.0 (Mi c ro so ft Co r p o ratio n , Re d -m o n d, WA ) .
Co p y Nu m b er Ca lcu la t io n : Th e s p ill-o ve r f l u o rescen ce fro m the FAM (HPV) ch a n n el in to th e H EX (glo b in ) c h a n n el a n d vice ver sa wa s ca lc u la ted fro m two s ets o f co n tr o l sa m p les ; th e fir st was a s et o f th ree id e n tica l s a m p les co n tain in g b oth p rob es b u t on ly H PV tem p late a n d th e sec o n d wa s a s et o f t h r ee c on t ain in g bo th p rob es b u t on ly glo bin tem p la te. H PV an d glo b in sign a ls we re calcu la ted in p a tien t sa m -p le s a fte r s u b tra ctio n o f th e s-p ill-over sign a l. In clu d ed with each set o f p a tien t sa m ples we re th e assa y co n tro ls a n d a d ilution series of n on -H P V- co n ta in in g h u m a n c ellu la r DNA. Plo ts of th e h o m ologo u s tem p late d ilu tio n series flu o rescen ce versu s lo g (tem p la te co p ies) we re lin -ea r over th e ra n ge 50 to 1x109co p ies ; h e n ce f ro m th e p a tien t flu o rescen c e d a ta it wa s p o s -sib le to ca lcu late H PV co p y n u m b er in p atien t s a m p l e s. Ex t ra p o la tio n o f lo g (co p y n u m b er) ve rsu s flu o resce n ce a b o ve 1x109c o p ie s is o f u n k n own a ccu ra c y; h owe ve r, sam p les with flu -o res ce n c e c-o r res p -on d in g t-o c -op y n u m b ers in th is ra n ge can be p resu m ed to h ave at lea st 109 c o p i e s. Med ia n va lu es of H PV c o p y n u m b er in th e p res en t st u d y d o n o t exceed 108a n d th u s, i n a c c u ra cies in H P V c o p y n u m b e r a b ove 109 will n o t a ffe ct co n c lu s io n s b as ed o n m ed ia n c o py n u m ber va l u e s.
Sample size and statistical analysis
Cad . Saúde Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998
sam p le o f 63 cases an d co n tro ls p er ra c i a l / e t h -n ic gr ou p wo u ld d ete ct a m i-n im u m th re e f o l d risk differen ce between cases a n d co n trols with a t wo -s id ed tes t, a lp h a o f .05 a n d 80% p owe r. Du e t o th e r ac ia l/ eth n ic d is tr ib u t io n o f th e clin ic p o p u la tio n a t th e tim e o f th e stu d y (a p -p roxim ately 60% wh ite, 20% Afri c a n - A m e ri c a n a n d 20% Hisp an ic) a n d stu dy re c ru itm en t p ro -c e d u res (e n ro llm en t p rio r t o fin a l d ia gn o sis) we exp ected to en ro ll a la rger n u m ber o f wh ite wo m en a n d h a d p la n n ed to ra n d o m ly select o n e o f eve ry two o r th ree wh ite ca ses. We co n -du cted a pre l i m i n a r y an a lysis to estim a te cru d e od d s ra tio s (OR) for th e a sso cia tio n b etwe e n SIL co m b in ed an d H PV fo r a ll ra c i a l / e t h n i c g r ou p s com b in ed u sin g t h e to ta l n u m b er o f a vailable wh ite ca ses (n = 147) in th e stu d y s a m -p le a n d a ra n d o m sa m -p le (n = 70) o f th e wh ite cases as or igin ally p la n n ed . Beca use n o im p o r-ta n t d iffe ren c es we re o b s erve d b etwee n b o th risk estim a tes, we p resen t resu lts fo r th e ent i re s a m p l e.
Co m p a r is o n s b etwe en c as es a n d co n tro l s an d a m o n g ra cia l/ eth n ic grou p s we re co n du ct-ed u sin g t h e Pe a r s o n’s X2test fo r ca t ego ri c a l va ria bles (H PV sta tu s, in com e, edu ca tio n , n u m -ber o f sexu al p a rt n e r s, e tc.) a n d ‘t’ test an d on e-way ANOVA fo r co n tin u o u s va r ia b les (a ge a n d ed u catio n ). Odd ra tios a n d 95% con fiden ce in t e rvals (CI) we re u sed to estim a te th e a sso c ia -tio n b etween SIL co m bin ed a n d LSIL a n d H SIL a n d H PV DNA s ta tu s (H PV DN A p o sitive o r n e g a t i ve) a n d o th er kn o wn risk fa ct or s. Tw o typ e s o f lo gist ic r e g ress io n m o d els we re es tim a ted : d ich o to tim o u s lo gistic re g r essio n tim o d -els fo r SILs co m b in ed a n d p o lyto m o u s lo gistic re g ression m o dels fo r grade of lesion (LSIL an d HSIL) (Ho sm er & Lem esh ow, 1989). Th is m odel sim u lta n eo u sly estim ate s th e risk o f LSIL a n d H SIL with re sp ec t t o a co m m o n c o m p a ri s o n g ro u p. Se p a ra te dich o to m o u s a n d p o lyto m ou s m odels we re p er f o rm ed for th e co m b in ed sam -p le an d fo r ea ch racia l/ eth n ic gro u -p.
In a d d itio n t o H PV D NA st at u s a n d a ge, va riables with a p va lu e < 0.05 in th e u n iva ri a t e lo gistic re g ressio n an alysis we re co n sidered fo r in c lu sio n in t h e m u ltiva ria te m o d els. Si m i l a r p ro c e d u re wa s followed fo r th e a n a lysis o f th e co m b in e d s a m p le a n d ea ch ra c i a l / e t h n i c g ro u p sep ara t e l y. Va ria b les in th e fin a l m o d els in c lu d ed : a ge (co n tin u o u s ), H PV DN A sta t u s ( p o s i t i ve, n ega t ive), a ge a t firs t se xu al in t er-co u rs e (<17, 17-19, 20+ [re f e re n ce ca t ego ry ] ) , lifet im e n u m b e r o f s exu a l p a rt n ers (1 [re f e r -en c e ca tegor y], 2-3, 4-5, 6+); sm o kin g h isto r y ( e ve r, n ever), a n d h isto ry o f STDs (ye s, n o ).
Sta tist ica l a n a lyse s we re p erf o r m ed u s in g th e Sta t ist ic a l Pa cka ge fo r th e So cia l Scien ces
(SPSS, 1997) an d STATA (St a t a c o r p., 1997). All p va lu es a re fo r tw o- s id e d t ests, a n d s ta tistic a l sign ifican ce wa s set a t p < 0.05.
R e s u l t s
Fi ve h u n d red n in ety f i ve wo m en we re in c lu d -ed in th is an alysis, 325 ca ses (175 LSIL a n d 150 H SIL) a n d 270 co n tro l s. Am o n g wo m en with a d ia gn o sis o f LSIL, 84 (48%) h a d ko ilo c yto tic a typ ia a n d 91 (52%) h a d c ervica l in tra e p i t h e-lia l n eo p la sia (CIN ) gra d e 1. W h e re a s, a m o n g wo m en with a d ia gn o sis o f H SIL, 70 (47%) h a d CIN 2 a n d 79 (53%) h a d CIN 3. T h i rt y-s ix p er-cen t of a ll stu dy su bjects we re wh ite (147 cases a n d 68 c on t ro ls ), 25% we re Africa n Am er i c a n (75 c a ses a n d 73 co n tro ls ), a n d 39% we re Hi s-p a n ic (103 ca ses a n d 128 con trols). Ni n e t y- n i n e p e rc en t (n = 589) o f wo m en we re ≤45 yea rs o f a ge (m ea n a ge 26.6 year s an d sta n d a rd d evia t io n 6.4). Th e s tu d y s u b jec ts we re o f low s o cioeco n o m ic sta tu s with 86% re p o rtin g a n an -n u a l fa m ily i-n c o m e o f les s th a -n $20,000 a -n d 50% h avin g less th an h igh sch ool edu ca tion .
Co m p a ris o n s o f d em ogr ap h ic ch a ra c t e ri s-tic s b etween ca ses a n d co n tro ls are p re s e n t e d in Table 1. No statistica lly sign ifica n t differe n c e b e t ween SIL cases com bin ed an d co n tro ls we re o b s e rved in m ea n a ge (p = 0.35), edu ca tion lev-el (p = 0.25), a n d in co m e (p = 0.65). Si m i l a r l y, n o sta tistica lly sign ifica n t differen ces in edu ca-tio n an d fam ily in com e we re ob served b etwe e n c o n t rols a nd LSIL an d HSIL. Howe ve r, LSIL ca s-es we re sligh tly yo u n ger th a n c o n tr ols (p = 0 . 0 0 4 ) .
In a d d itio n , ca ses a n d co n tr o ls d iffe red in th e p re valen ce of seve ra l kn own risk facto rs for c e rvica l n eo plasia . Sm o kin g h isto r y wa s h igh er a m o n g c as es (47%) t h a n in co n t ro ls (26%, p < 0.001). Th e ca se-co n tro l differen ce in sm okin g h i s t o r y wa s la rger b e t we en H SIL (57%) th a n b e t ween LSIL (37%) ca ses. Ap p roxim a tely 91% o f ca s es h a d t h eir firs t s exu al in te rc o u rse b e-f o r e a ge 20 co m p a red with 83% o e-f co n t ro ls (p <0.001). Ca ses we re less likely to re p o rt a sin gle lifetim e sexu a l p ar tn er (15%) a n d to re p o rt six o r m o re s exu a l p ar tn er s (35%) th an wo m e n in th e c o n tro l gro u p (36% a n d 16%, re s p e c t i ve l y, p <0.001). Si m i l a r l y, ca se s we re m o re likely t o h a ve a h is to r y o f STDs (33%) th a n c o n tro l s (17%) (p <0.001). No d ifferen ces between ca ses a n d c on tro ls we r e d et ected fo r th e n u m b er o f p regn a n cies a n d ora l co n tra c e p t i ve u se.
T O RTO LERO - LUNA, G. e t al. 1 5 4
C ad. Saúde Púb lica, Rio de Jane iro , 14(Sup. 3):149-159, 1998
to b e wh ite th a n Hisp a n ic o r African Am eri c a n (54%, 47%, a n d 29%, re s p e c t i vely). Wo m e n with HSIL we re m ore likely to b e to less ed u ca ted th a n wo m en with LSIL (< h igh sch o o l ted u -ca tio n 39% a n d 25%, re s p e c t i ve ly), t o h a ve a h i s t o r y of sm o kin g (57% vs. 39%), a n d to h a ve h a d fo u r o r m o r e p r e gn a n c ie s (2 3% vs. 12%,
p = 0.026). Ho we ve r, LSIL a n d H SIL c as es d id n o t d iffer in a ge at first in terc o u r s e, n um b er o f lifetim e sexu al p art n e r s, u se of oral co n tra c e p -t i ve s, a n d h is-tor y of STDs.
Ra cia l/ eth n ic d ifferen ces in th e p re va l e n c e o f se ve ra l r isk fa cto rs we re o b se rved in b o th ca se s a n d c o n tro l s. H is p a n ic ca s es a n d co n -t ro ls we re m o re likely -to b e o ld er, less ed u ca - t-ed , to re p o r t lowe r n u m b er o f lifet im e s exu a l p a rt n e r s, to b e o ld er a t th e tim e o f fir st sexu a l i n t e rc o u r s e, a n d t o h a ve lower ra te s of STDs. Me a n w h i l e, wh ite ca s es a n d c o n t ro ls we re m o re likely to h ave a h isto ry o f sm o kin g, m o re lifetim e sexu al p ar tn ers a n d Africa n Am eri c a n s we re m ore likely to re p o rt an earlier a ge at sex-ua l in tercou rse an d to be n on sm o kers.
Table 2 shows th e p re valen ce of HPV DNA in ca ses a nd co n tro ls by racial/ eth n ic gro u p. Ove r-all, 73% o f cases an d 19% o f co n trols we re H PV D N A - p o s i t i ve. Pre valen ce wa s h igh er a m o n g HSIL cases (84%) th an am o n g LSIL ca ses (64%). Rac ia l/ eth n ic d iffere n c es in th e p re va len ce o f H PV in fe ct io n we re ob s er ved in th e co n tr o l g r o u p (p = 0.0 17 ) b u t n o t in t h e c a s e c o m
-Tab le 1
Distrib utio n o f d e mo g rap hic c haracte ristic amo ng c e rvic al sq uamo us intrae p ithe lial le sio ns (SIL) c ase s and c o ntro ls
b y g rad e o f le sio n.
Risk fact or S I L
LGSIL H G S I L C o n t ro l s p1 p2
n (% ) n (% ) n (% )
Age (years) 0 . 0 0 4 0 . 2 7 4
Me an (± SD ) 2 5.3 (5.9) 27.7 (8 .1) 26 .9 (5.6)
Race/ Et hnic gro u p < 0.0 01 < 0 . 0 0 1
W h i t e 67 (3 8) 80 (53 ) 6 8 (25)
A f r i c a n - a m e r i c a n 53 (3 0) 22 (15 ) 7 4 (27)
H i s p a n i c s 55 (3 1) 48 (32 ) 128 (47)
E d u c a t i o n 0 . 0 6 0 0 . 8 6 9
> Hig h Sc ho o l 52 (3 1) 36 (25 ) 6 3 (24)
Hig h Scho o l 73 (44) 53 (37) 10 5 (39)
< Hig h Scho o l 42 (2 5) 56 (39 ) 100 (37)
I n c o m e 0 . 9 0 3 0 . 7 3 1
< $ 2 0 , 0 0 0 149 (8 6) 129 (86) 22 8 (85)
$ 2 0 , 0 0 0 24 (14) 21 (14) 41 (15)
M arit al st at us < 0.0 01 0 . 0 1 2
S i n g l e 83 (5 0) 43 (30 ) 8 2 (31)
M a rr i e d 41 (2 5) 64 (44 ) 146 (55)
D i v o rc e d 39 (23) 37 (26) 35 (13)
W i d o w e d 4 (2) 1 (1) 4 (2)
1p value fo r the co mp ariso n b e twe e n LSIL vs. c o ntro l g ro u p . 2p value fo r the co mp ariso n b e twe e n HSIL vs. c o ntro l g ro u p .
Tab le 2
P re vale nc e o f HPV Infe ctio n b y c ase -c o ntro l status, rac ial/ e thnic g ro up
and g rad e o f le sio n.
Race/ Ethnic gro u p S I L
SIL co mb ine d L G S I L H G S I L C o n t ro l s n (% ) n (% ) n (% ) n (% )
W h i t e 1 12 (76 ) 43 (64) 69 (86) 13 (19)
A f r i c a n - a m e r i c a n 51 (68) 34 (64) 17 (77) 22 (3 0)
H i s p a n i c 75 (73) 35 (64 ) 40 (8 3) 17 (13)
A l l 2 38 (73) 112 (64) 126 (8 4) 52 (19)
P value1 N S N S N S 0 . 0 1 7
Cad. Saúd e Púb lic a, Rio d e Jane iro , 14(Sup. 3):149-159, 1998
b in e d gr o u p (p = 0.425). Si m i l a r l y, n o ra c i a l / eth n ic d ifferen ces in H PV p re va len ce we re o b -s e rved a m o n g LSIL ca -se-s (p = 0.99) a n d am o n g H SIL ca ses (p = 0.59). In th e co n tro l gro u p, th e p re va len ce of HPV in fection wa s h igh er am on g A f ri c a n - A m e rica n wo m en (30%), in term e d i a t e am o n g wh ite wo m en (19%), a n d lower a m o n g Hisp an ic wom en (13%).
A s tro n g r isk o f SIL (b o th LSIL a n d H SIL) wa s a s so ciat ed wit h H PV in fectio n ( Ta b le 3). Afte r a d ju stm en t fo r a ge, n u m b e r o f lifetim e sexu al p ar t n e r s, a ge a t first sexu a l in terc o u r s e, a n d h isto r y o f s m okin g a n d STD s th e AOR fo r SIL was 10.8 (95% CI 7.1-16.4). Th e ris k wa s 4 tim es high er fo r HSIL (AOR = 24.8; 95% CI 13.8-44.7) th an fo r LSIL (AOR = 6.6; 95% CI 4.2-10.6).
Hisp a n ics sh owed a fivefo ld stro n ger a sso -ciatio n with HPV in fectio n th an Africa n Am eri-can s an d a twofo ld stro n ger risk tha n whites fo r SIL c o m b in ed , a n d LSIL an d H SIL se p a ra t e l y. Th e AOR for SIL was 29.5 (95% CI 12.4-70.5) fo r Hi s p a n i c s, 5.8 (95% CI 2.6-12.6) fo r Afri c a n A m e ri c a n s, a n d 15.3 (95% CI 6.9- 33.8) fo r w h i t e s. Th e s tren gth o f th e a sso c ia tio n wa s t h ree t o fo u r tim es h igh er fo r H SIL t h a n LSIL. Th e str o n gest a ss o cia tio n wa s o b s erved fo r HSIL a m o n g Hisp an ic wom en (AOR = 78.6; 95% CI 23.8-260.1).
D i s c u s s i o n
Ou r fin din gs su p p ort th e stron g asso ciatio n be-t wee n SIL an d H PV; an a ss o cia be-t io n wibe-t h b o be-th HSIL a n d LSIL b u t fou r fo ld stro n ger for H SILs. Alt h o u gh th is a sso cia t io n wa s o b se rved in a ll racia l/ eth n ic gro u p s, th e da ta su ggest th a t sign ifica sign t d ifferesign ces isign th e stresign gth o f th e a sso -ciatio n am o n g grou p s m a y exist. A stron ger as-so ciatio n wa s o b ser ved a m o n g H isp a n ics fo l-l owed by wh ites an d African Am eri c a n s.
Alth o u gh d irect com p a rison between stu d -ie s is d ifficu lt b e ca u se of m eth o d o lo gic a l d
if-f e re n c e s, th e m agn itu de o if-f th e a sso ciatio n b et ween H PV a n d SIL o b s er ve d in eth is p o p u la -tio n , is co n s isten t with p revio u s stu d ies u sin g PCR tech n iq ues (Mo r rison et al., 1991; Bo sch et a l., 1993, 1995; Sh iffm a n et a l., 1993; Becker et a l., 1994; Ols en et a l., 1995; Lia w et a l., 1995). Mo r r iso n et a l. (1991) d etected a n AOR o f 10.4 fo r th e a ssociatio n between HPV in fectio n an d SIL (am on g in a in n er-city m u lti-eth n ic sam p le in New Yo rk, NY). Si m i l a r l y, in ou r stu dy we o b-s e r ved an AOR of 10.8 (95% CI 7.1-16.4) for SIL c om b in ed . Th ese re su lts a re a ls o co n sist en t with s tu d ie s o f th e a sso c ia tio n b etwe en H PV in fection a n d grad e of lesio n . An in crea sed ri s k fo r bo th low - g rad e a n d h igh -grad e lesion s, bu t s t ro n ger for h igh -gra d e lesio n s, h a ve b e en re-p o rted. Th is stro n ger association with HSIL h a s b een shown to be HPV-typ e sp ecific. (Sh i f f m a n et al., 1993; Brisson et al., 1994; Liaw et al., 1995). Ou r es tim a te s o f th e a s so cia tion b et we e n H PV in fection a n d h igh gra de cervica l in tra e p -ith ela il n eop la sia we re w-ith in ran ge o f re c e n t l y re p o rted stu dies u sin g PCRb a sed a ssa ys (Mu -ñ oz & Bo sch , 1997). AOR r an gin g fr o m 15.5 (95% CI 8.2-29.4) in Co lo m b ia (Bo sc h e t a l., 1993) t o 122.3 (95% CI 38.5- 388.9) in Ta i w a n (Lia w et a l., 1995) h a ve b een re p o rted . In No r-w a y, Ols en et a l. (1995) o b ser ve d a n AOR o f 72.8 (95% 27.6-191.9) a n d in Sp a in , Bosc h et a l (1993) r e p o rt ed a n AOR o f 56.9 (95% CI 24.8-130.6). In th e U.S., Sch iffm a n et al. (1993) in Po rtlan d a n d Bec ker et a l. (1994) in New Me x i-co re p o rted an OR o f 42.0 (95% CI 15.3-124.3) a n d 20.8 (95% CI 10.8-40.2), re s p e c t i ve l y. We o b s e r ved r isk estim a tes fo r HSIL r a n gin g fro m 14.2 (95% CI 3.6- 56.0) a m o n g Afric a n Am e ri-c a n s t o 78.6 (95% CI 23.8-260.1) a m o n g Hi s-p an ics (Ta ble 2).
Th e racial/ eth n ic differen ces in HPV p re va -len ce in th e co n tro l gr ou p a n d th ere f o re in th e H P V-a ss o c ia t ed r isk fo r SILs in t h e p re s e n t stu d y we re u n exp ec ted . Pa r ticu la rly in t ere s tin g was th e lo w r isk o b ser ved a m o n g Afri c a n
-Tab le 3
Asso c iatio n b e twe e n HPV and SIL b y racial/ e thnic g ro up and g rad e o f le sio n.
Race/ Et hnic gro u p L G S I L H G S I L S I L
C O R1 A O R2( 9 5 % C I ) C O R1 A O R2( 9 5 % C I ) C O R1 A O R2( 9 5 % C I )
A l l 7 . 6 6.6 (4.1-1 0.4) 2 2 . 0 24.8 (13.8- 44.7) 1 1 . 5 10.8 (7.1 -16.4)
W h i t e 7 . 5 7.7 (3.2-1 8.4) 2 6 . 5 34.6 (12.8- 93.8) 1 3 . 5 1 5.3 (6.9 -33.8)
A f r i c a n - a m e r i c a n 4 . 2 4.3 (1.9-9.8) 8 . 0 14.2 (3.6- 56.0) 5 . 8 5.8 (2 .6 -12.6)
H i s p a n i c s 1 1 . 4 16.6 (6.5-42.3) 3 2 . 6 7 8.6 (23.8-2 60.1) 1 7 . 5 29 .5 (12.4-70.5)
1C rude O R; all CO R 95% CIs e xc lud e o ne .
T O RTO LERO - LUNA, G . e t al. 1 5 6
Cad . Saúde Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998
A m e r ica n wo m e n fo r b o th LSILs a n d H SILs. Hisp a nics ha d a fivefold h igh er risk th at A f ri c a n A m e r ic a n s a n d a t wo fo ld h igh er risk th a n w h i t e s. Si m i l a r l y, wh ite wo m en h a d a 2.6 h igh -er risk th an Africa n Am -erican s d id . Ou r risk es-t im a es-t es fo r H SIL am o n g Afric a n Am e ri c a n s c o m p a res to th a t o b served am o n g Co l o m b i a n wom en (Bo sch et a l., 1993), which is am o n g th e l owest risk estim ates re p o rted in recen t stud ies u sin g PCR tech n iq ues fo r HPV testin g (Mu ñ oz , 1997). T h e re is o n ly on e pu blish ed stu dy (Be c k-er et a l., 1993) a n d th e abstra ct p resen ted at th e 1 4t hIn t e r n a tio n a l Pa p i l l o m a v i ru s Co n f e re n c e in Qu eb e c City, Ca n a d a in 1995 (Co ker et a l., 1995) a d d re ss in g ra cia l/ et h n ic d iffere n c es in H P V-a ss o cia t ed risks fo r SILs in t h e U.S. Re-sults fro m th ese stu d ies are co n sisten t with o u r f i n d i n g s. Becker et a l. (1993) o b s erved in Ne w Me x i c o, a two fo ld h igh er r isk a m o n g Hi s p a n i c wo m en (AOR = 25.7; 95% CI 11.0- 60.3) th a n a m o n g n o n -Hisp a n ic wh ites (AOR = 13.3; 95% CI 5.2-34.0) u sin g th e res u lts fro m th e gen eri c PCR pr o b e. Co ker et al. (1995) in So u th Ca ro l i-n a fou i-n d a fo u rfo ld h igh er risk ii-n wh ites (cru d e OR = 21.5) t h a n in Afri c a n - A m e ric an wo m e n ( c ru d e OR = 5.5) th at p ersisted a fter co n tro l l i n g fo r o th er cofacto rs.
De sp it e th e la ck o f in fo r m a tio n a b o u t ra cia l/ eth n ic d ifferen ces in th e H PV- a s s o c i a t e d risk fo r ce rvica l n eo p la sia, th e re is s o m e d a t a th a t s u ggest th at th e p re va len c e o f H PV typ es m a y va r y am o n g d ifferen t p op u la tio n s (Bo s c h et a l., 1995). Th ese differen ces m a y co n tri b u t e, a m o n g ot h e r fa c to rs, to th e geo gra p h ic a n d ra cia l/ eth n ic va riability in cervica l ca n cer in ci-d en ce ra t e s. Bosch et a l. (1995) o bserveci-d a sig-n ifica sig-n t geo grap h ic va riatiosig-n isig-n th e p re va l e sig-n c e o f so m e o f th e les s-co m m o n H PV typ es. H PV-45 wa s m ore p re va len t am o n g cases in We s t e rn A f rica an d HPV-39 a n d HPV-59 we re a lm o st ex-c l u s i vely d eteex-cted a m o n g La tin Am eriex-c an ex-ca s-e s. In su p p ort of th s-e po ts-en tia l ro ls-e o f HPV typ s-e to e xp la in r ac ial/ e th n ic d ifferen ce s in ris k fo r c e r vic a l n eo p la sia, Becke r et al. (1993) fo u n d th e la rgest eth n ic d ifferen ce with p o sitivity to H PV typ es 16/ 18 (by Vi ra Typ e). HPV 16/ 18 p o s-i t s-i ve Hs-isp an s-ic wo m en h a d a 10-fold h s-igh er rs-i s k o f H SIL (AOR 171.0; 95% CI 22.8-1280.5) th a n n o n - Hisp a n ic wh ite wo m en (AOR = 18.0, 95% CI 6.4-50.5).
An o th er p o s sib le exp la n a tio n fo r t h e o b -s e r ved ra cia l/ eth n ic differen ce-s m ay be re l a t e d to va ria bility in the ho st im m u n e fu n ctio n . T h e fa c t th at o n ly a sm all p ro p o r t io n o f wo m en with HPV in fection d evelop ed cer vica l n eo p la-sia an d th e h igh er risk o f cervical n eop la la-sia a n d p ro g res sio n o f d isea se a m o n g im m u n o su p -p res se d wo m e n su gges t a n im -p or t a n t ro le o f
th e im m u n e s yste m in th e p a th o gen es is of H P V-in d u ced d isea se (IARC, 1995). H ost gen et-ic fa c tor s su c h a s h u m a n leu ko cyt e a n t igen s ( H LA) h a ve b e en a lso im p lic ated in th e risk o f c e r vica l n eo p la s ia s. Ap p le e t a l. (1994; 1995) fou n d a sign ifican t asso cia tion between cert a i n H LA cla ss II h a p lo typ es a n d th e risk o f p re i n va -s i ve a n d in va -s i ve cer vic a l le-s io n -s a m on g Hi - s-p a n ic wo m en . Th is asso cia tion wa s ss-p ecifically o b s e rved a m on g HPV16 p ositive wom en . Si m -i l a r l y, a n -in crea sed r-isk fo r cer v-ica l ca n cer h as b een asso cia ted to c erta in H LA cla ss II h a p lo -typ es a m o n g Ca u ca sia n wo m e n (Wa n k et a l., 1993) an d Afri c a n - A m e rica n wo m en (Gre g o i re et al., 1994).
It is a lso p o ssib le th a t ra cia l/ eth n ic d ifferen ces in o th e r ris k fa ct o rs su c h a s so cio eco -n o m ic ch a ra c t e ri s t i c s, sexu a l b eh a vio rs, sm o k-in g h a b it s, re p ro d u c t i ve a n d d ieta r y fa cto r s, h i s t o r y o f STD s, a n d in o t h er c ofa c to r s n o t m e a s u red in th e p resen t stu d y (ch a ra c t e ri s t i c s o f m a le p a rtn ers a n d en d o ge n o u s h o rm o n a l fa cto rs) m igh t exp la in o u r re s u l t s. For exam p le, so cio econ o m ic facto rs m a y o p era te differe n t l y am o n g d ifferen t ra cia l/ eth n ic gro u p s. Level o f ed u ca tion h as b e en fo u n d to b e a n in d e p e n -d en t fac to r fo r c er vica l n eo p la sia . Peters et a l. (1986) fou n d yea rs o f edu ca tio n in versely asso -cia ted with r isk o f ce rvica l ca n cer a m o n g Hi s-p a n ic wo m en in Los An geles Co u n t y. Si m i l a r l y, Be cker et a l. (1993) o b se rved a h igh er r is k o f HSIL a m o n g wom en with lower ed u ca tion al at-ta in m en t. In o u r stu dy sat-ta tis tica lly s ign ifica n t racia l/ eth n ic d ifferen c es in ed u catio n a l leve l s in both ca se s a n d co n tro ls we re ob served; h ow-e vow-e r, wow-e did n o t fin d ow-e du ca tion al low-evow-el to b ow-e a n in d ep e n d e n t r is k fa cto r fo r SIL a m o n g ra c i a l / eth n ic co m b in e d o r a m o n g ea ch gro u p sep a -ra tely (d a ta n o t sh own ).
l-Cad. Saúde Púb lic a, Rio d e Jane iro , 14(Sup. 3):149-159, 1998
ed ge o f fin a l cyto lo gical o r h isto lo gica l d iagn o -sis a n d HPV statu s. In a dd ition , d ata co llectio n i n s t ru m en ts we re t ra n s la t ed to Sp a n is h a n d bilin gu al in terv i e wers we re available in order to i n s u re particip ation a m on g n on -En glish sp ea k-in g Hi s p a n i c s. Th e p re va len c e o f kn o wn ri s k fa cto r s fo r cer vica l n e o p la s ia in b o t h c a s es a n d c o n t ro ls wa s co n sist en t wit h p re v i o u s s t u d i e s. Most factors (su ch a s n u m b er o f sexu al p a rt n e r s, age a t first in terc o u r s e, sm o kin g, an d h i s t o r y o f STDs) we re fo u n d to b e sign ifica n tly asso cia ted with th e risk o f SIL in u n iva r iate lo -gistic m o dels (data n ot sh ow n ) .
An o th er p oten tial factor affectin g th e in ter-p retation of th e resu lts is th e u n balan ced racial/ eth n ic d is trib u tio n o f ca ses a n d co n tro l s, p a rticu la rly fo r wh ite wo m en ( Tab le 1). As m en -tio n ed in th e m eth o d s sec-tio n , th is im b a la n c e wa s exp e cted given th e ra cia l/ eth n ic d istri b u tio n of th e p a tien t p o p u la tio n a t th e UTM -D ACC co lp osco py clin ic an d th e stu d y re c ru i t-m e n t p ro c e d u re s. We h a d in it ia lly p lan n ed to se lec t a ra n d o m sa m p le o f wh ite c a ses t o o b -ta in a ca se:c o n tro l ra tio o f o n e. In a p re l i m i-n a ry ai-n alysis we estim ated cru de OR fo r th e as-so cia tion b et ween SIL a n d H PV fo r th e to ta l sa m p le (in clu din g a ll wh ite ) a n d for a su bsa m -p le in c lu d in g o n ly a ra n d o m s a m -p le of th e a va ila b le wh ite ca se s. Sim ila r cr u d e risk esti-m ates we re o btain ed with th e total saesti-m p le (OR = 10.0, 95% CI 6.7-15.1) an d th e su bsam p le (OR = 9.9; 95% CI 4.5-21.8). T h e re f o re, th e u n b a l-a n c ed n u m b er o f cl-a s es l-a n d co n tro ls l-a m o n g wh ite wom en d oes n o t seem to ha ve biased th e est im a te o f risk. Fu rt h e rm o re, th e p re va l e n c e o f ris k fac to rs (e d u ca tio n , s m o kin g h is to r y, n u m b e r o f s exu al p a rt n e r s, a ge a t fir st in t er-co u rse a n d h istor y o f STD s) in b o th ca ses a n d c o n t ro ls wa s con sisten t with n a tio n al d ata .
Alth o u gh th e p o ssibility o f in terv i e we r, re-ca ll, a n d in for m a tion b ia s re-can n o t b e elim in a t-ed, a ttem p ts we re m a de to m in im ize th em u s-in g a stru c t u red com p u t er-a ssisted s-in terv i e w, t ra in in g a n d su p er visin g in te rv i e we r s, co n -d u ct in g -d a ta c o llect io n b o th in En glish a n -d Sp a n ish an d p rio r to kn owled ge of th e fin a l di-agn os is a n d H PV st a t u s, a n d b y re m i n d i n g wom en ab ou t t h e co n fid e n tia lity o f th e in fo r-m a tio n a n d th e ir-m p or ta n c e o f th e a ccu r acy o f th eir re s p o n s e s.
Miscla ssificatio n of H PV an d disease sta tu s n eed s to b e a lso c o n sid ered in th e in ter p re t a -tio n . Th e cross-sec-tion al na tu re o f th e HPV testin g m a y n ot a c cu r ate ly re flect th e tr u e p re va -len ce in th e study p op u lation . Desp ite th is lim i-tat io n th e p re valen c e o f H PV o b se rved in th e c u r re n t stu d y, in b o th c as es a n d co n t ro l s, is co n sis ten t wit h p reviou s s tu d ies u sin g PCR
m eth o d s (Mu ñ oz, 1997). Si m i l a r l y, th e ra c i a l / eth n ic d ifferen ces in th e p re va len ce o f HPV in -fec tio n in th e c o n tr ol gro u p is co n sisten t with th e d at a re p o rted by Hild esh eim e t a l. (1993). Th ese a u th o rs a lso fou n d a h igh er p re va l e n c e o f H PV in fe ct io n a m o n g Afric an Am e ri c a n s (43.9%), in ter m e d ia te in wh ite s (32.1%), a n d l ower in Hisp a n ics (25.5%) a m o n g a c lin ic-b a sed p o p u la tio n of wo m en with n or m al cyto-lo gic s m e ar res id in g in th e Was h in gt o n , D C a rea (Hild esh eim et al., 1993).
Th e differen ce in th e a s certa in m en t o f d is-ea se sta tu s in cas es (a t th e De p a r t m en t o f Path o lo gy U TMDACC) an d con tro ls (a t a re f e r-en ce la bo ra t o r y) m a y h a ve in tro du ced m iscla s-sification p articu la rly am on g th e con trol gro u p. Ou r d ec isio n fo r th is d ifferen ce in a sce rt a i n -m en t p ro c e d u res wa s b a se d o n res u lts f r o -m a sm all p ilo t stu dy (n = 100) by on e o f th e au th o rs in th is stu dy (MFM) to com p a re th e agre e m e n t b e t ween th e rea din g a t th e re f e ren ce lab o ra t o-r y a n d th e De p a o-r tm en t o f Pa t h o l o g y. A h igh a g reem en t wa s ob ser ved between th e re a d e r s, ka p p a co efficien t o f 0.85 (p erso n a l c o m m u n ica tion ). Fu rt h e rm o re, m o st wo m en in th e co n -t ro l grou p (98%) h a d a n ega -tive cy-tolo gy a -t -th e tim e o f en try, a ll h ad a n egative h isto ry of cer-vical a bn o rm a l i t i e s, 93% h a ve h ad a Pa p sm ea r with in th e la st ye a r p r io r to en r o llm en t (85% re p o r te d h a vin g an n u a l Pa p sm ea rs ), a n d b a sed on o u r p re l i m i n a r y an a lysis o f H PV typ e 36 of th e 53 HPV p o sitive con trols we re p o sitive fo r n o n h igh -risk H PV t yp e s. Ba sed on t h ese c h a ra c t e ris tics th e likelih o o d o f false n egative resu lts am o n g th e con trols sh o u ld b e low.
T O RTO LERO - LUNA, G . e t a l. 1 5 8
Cad. Saúde Púb lica, Rio d e Jane iro , 14(Sup . 3):149-159, 1998
R e f e re n c e s
APPLE, R. J.; ERLICH , H. A.; KLITZ, W.; MANOS, M . M.; BECKER, T. M. & WHEELER, C. M., 1994. HLA D R- DQ a sso cia tion s wit h ce r vica l ca rc i n o m a s h ow p a pillom avir u s- typ e specificity. Na t u re Ge-n e t i c s, 6:157-162.
APPLE, R. J.; BECKER, T. M.; WH EELER, C. M. & ERLICH , H . A., 1995. Co m p a riso n of h u m a n leu ko -cyt e an tigen DR- DQ d ise ase asso ciat ion s fo un d with cer vica l d ysp lasia an d in va s i ve cervical car-cino m a . Jou rn al of th e Na tion al Can cer In s t i t u t e, 87: 427- 436.
BECKER, T. M.; WHEELER, C. M.; KEY, C. R. & SAMET, J. M., 1992. Ce rvica l ca n cer in c id e n ce an d m o r-ta lit y in New Me x i c o’s Hisp a n ic , Am e rica n In d i-a n s, i-an d n on -Hisp i-an ic wh ites. Western Jou rn al of Me d i c i n e, 156:376-379.
BECKER, T. M.; WH EELER, C. M.; MCGOUGH , S.; PARMENTER, C. A.; JORDAN, S. W.; STIDLEY, C. A.; M CPH ERSON , R. S. & D ORIN, M. H ., 19 94. Sexu a lly tran sm itted d isea ses a n d ot h er risk fa c-t or s fo r cer vical d ysp la sia a m o n g So u c-t h we s c-t e r n Hisp an ic an d n on -Hisp a nic wh ite wom en . J A M A, 2 7 1 : 1 1 8 1 - 1 1 8 8 .
B LOCK, G., 1989. Health Ha bits an d Hi s t o ry Qu e s t i o n -n a i re : Di e t , Hi s t o ry a -n d Oth er Risk Fa c t o r s . Pe r-son al Com puter System Pa c k e t. Washin gton , D. C.: Divisio n o f Can c er Pre ven tion an d Co n t r ol, Na-t io n a l Can ce r In s Na-t i Na-t u Na-t e / NaNa-t io n a l In sNa-t iNa-t u Na-t e s o f He a l t h .
BOSCH , F. X.; MU ÑOZ, N.; DE SANJOSE, S.; NAVA R-RO, C.; MOREO, P.; ASCUNCE, N.; GONZALEZ, L. C.; TAFU R, L.; GILI, M.; LA R R A Ñ AGA, I.; V I LA-DIU , P.; DANIEL, R. W.; ALO N ZO DE RUIZ, P. ; A R I S T I ZABAL, N.; SAN TAMARIA, M.; GU ERR E ERRO, E. & SH AH, K., 1993. Hu m an p ap illo -m a v i r u s a n d ce r vica l in t ra ep it h elial n eo p la sia g ra d e III/ Ca rcin o m a in situ : a case -con trol stud y in Sp ain an d Colo m b ia . Can cer Ep i d e m i o l o gy, Bi o m a rkers an d Pre ve n t i o n, 2:415-422.
BOSCH , F. X.; MANO S, M. M.; MU ÑOZ, N.; SH ER-MAN, M.; JANSEN, A. M.; PE TO, J.; SCHIFFER-MAN, M. H.; MORENO, V. ; KU RMAN, R. & SH AH , K. V, 1995. Pre valen ce of h u m an p ap illom a viru s in cervica l can cer : a wor ld wid e p e rsp ective . In t e rn a -t io n a l Biolo gica l S-t u d y o n Ce r vica l Can c er (IB-S CC). Jou rn al of t h e N a tion a l Ca n cer In s t i t u t e, 8 7 : 7 9 6 - 8 0 2 .
BRISSON, J.; MORIN, C.; FORTIER, M.; ROY, M.; B O U C H A R D, C.; LECLERC, J.; CH RISTEN , A.; G U I M O N T, C.; PE N AU LT, F. & MEISELS, A., 1994. Risk fa cto rs fo r ce r vical in tr ep ith elial n eop la sia: d i f f e re nces b etwee n low-an d h igh- grad e lesio ns.
Am erican Journ al of Ep i d e m i o l o gy, 140:700-710. CO KER, A. L.; KNIGHT, S. M.; JENKINS, G. R. &
PIRISI, L., 1995. Rac ial d iffe re n ces in risk factors for cervical ne oplasia. 14th Intern ation al Pa p i l l o-m av iru s Con fere n c e , Ab s t ra c t s. Qu e b ec: 14th In -t e rna -tion al Pa p i l l o m a v i ru s Co n f e re n c e.
FÖRSTER, V. T., 1948 . Zw i s c h e n m o l e k u l a re e n -e rg i -e w a n d -e r u n g u n d flu o r-e s z-en z. An n als of Ph y s i c s, 2:55-75.
GREGOIRE, L.; LAWRENCE, W. D.; KU KURU GA, D. ; E I S E N B R EY, A. B. & LANCANTER, W. D., 1994. As-sociation b etween HLA-DQB1 alleles an d th e ri s k
of ce r vica l can c er in Afri c a n - A m e r ica n wom en .
In tern ation al Jou rn al of Ca n c e r, 57:504-507. HILDESH EIM, A.; GRAV I TT, P.; SCH IFFMAN, M. K.;
KURMAN, R. J.; BARNES, W.; JONES, S.; TC H A B O, J. G.; BRINTON, L. A.; CO PE LA N D, C.; EPP, J. & M AN OS, M. M ., 1993. De t e rm in a n ts o f gen ita l h u m a n p a p illo m avir u s in fectio n in low - i n c o m e wo m e n in Wash in gt o n , D. C. Sex u a lly Tra n s m i t-ted Di s e a s e s, 20:279-285.
HOSMER, D. W. & LEMESH OW, S., 1989. Applied Lo-gistic Re g re s s i o n. New Yo rk: Jo hn Wiley & So n s. I A RC WORKING GRO U P, 1995. Hu m a n Pa p i l l
o-m a v i r u s e s. IARC Mo n o g ra ph s o n th e Eva l u a t i o n of Ca rcinogen ic Risk to Hu m a n , vol. 64. Lyon : In -t e rn a-tion al Agen cy for Re s e a rch on Ca n c e r. L I AW, K. L.; HSING, A. W.; CHEN, C. J.; SCHIFFMAN,
M . H.; ZHANG, T. Y.; H SIEH , C. Y.; GREER, C. E.; YOU , S. L.; H UANG, T. W.; WU, T. C.; O’LEARY, T. J.; SEIDMAN, J. D.; BLOT, W. J.; MEINERT, C. L. & M AN OS, M. M., 1995. Hu m an p ap illo m a viru s a n d ce r vica l n e op lasia : a ca se- con t ro l stu d y in Taiwan. In tern a tion al Journ al of Ca n c e r, 62:565-5 7 1 .
MANOS, M. M.; TING, Y.; W R I G H T, D. K.; LEWIS, A. J.; B ROKER, T. R. & WO L I N S K Y, S. M., 198 9. Use of p o l y m e rase ch ain rea ction am p lifica tion fo r th e d et e ction o f gen it al h u m an p ap illo m a vir u s e s.
Can cer Ce l l s, 7:209-214.
MILLER, B. A.; KO LONEL, L. N.; BERSTEIN, L.; YOUN G Jr., J. L.; SWANSON, G. M.; KEY, C. R.; L I F F, J. M.; GLOVER, C. S. & ALEXANDER, G. A., 1996. R a c i a l / Eth n ic Pat t ern s of Can cer in th e United States 1988-1992. Bethesd a: Nationa l Ca n -cer In s t i t u t e. NIH Pu b. N 96-4104.
MORRISON, E. A. B.; H O, G. Y. F.; V E R M U N D, S. H .; G O L B E RG, G. L.; KADISH , A. S.; KELLEY, K. F. & BURK, D. B., 1991. Hu m a n p ap illom aviru s in fec-tion an d o th e r r isk fact ors for ce rvical n eop la sia: a case -co n t ro l st ud y. In tern a tion a l Jou rn al of Ca n c e r, 49:6- 13.
MUÑOZ, N., 1997. Hu m a n p a pillom avir u s an d cerv i-cal ca n ce r. In : N ew De velop m en t s in Ce rv i c a l Can cer Screen in g an d Pre ve n t i o n(E. Fran co & J. Mo n s o n e g o, e d s.), p p. 3- 13. Ca m b r id ge : Bl a c k-well Science.
MU ÑOZ, N. & BOSCH , F. X., 1997. Ce r vica l ca n ce r a n d h u m an p ap illom avir us: ep id e m io logica l e vi-d en ce a n vi-d p e rsp e ctive for p re ve n t io n . S a l u d Púb lica de M éxico, 39:274-282.
NAPOLES- SPRINGER, A.; PE R E Z - S TABLE, E. J. & WA S H I N G TON, E., 1996. Risk fa ctor s for inva s i ve c e rvica l can ce r in Latino wom en . Journ al of Me d-ical Sy s t e m s, 20:277-293.
OLSEN, A. O.; GJOEN, K.; SAU ER, T.; ORSTAVIK, I.; NAESS, O.; KIERU L F, K.; SPONLA N D, G. & MAG-NUS, P., 1995. Hu m a n p ap illom avir u s an d cer v i-cal in tra ep ith elia l ne op lasia gra d e II-III: a p op u-la tion -b a se d ca se-c on tr ol st u d y. In t e r n a t i o n a l Jou rna l of Ca n c e r, 61:312-315.
C ad. Saúde Púb lic a, Rio de Jane iro , 14(Sup. 3):149-159, 1998 ROTH MAN, K. J. & GRENLA N D, S., 19 98. Ca s e c o n
-t rol s-tu d ies. In: Modern Ep i d e m i o l o gy(K. J. Ro t h-m a n &ah-mp; S. Gre n la n d , ed s.), p p. 93-114, Ph i l a d e l-p h ia: Lil-pl-p in cott-Ra ve n .
SCHIFFMAN, M. H .; BAUER, H. M.; H OOVER, R. N.; G LASS, A. G., CADELL, D. M.; RUSH, B. B.; SCOTT, D. R.; SH ERM AN, M. E.; KU RMAN, R. J.; WA-CH OLDER, S.; STATON , C. K. & MANOS, M. M., 1993. Ep id em iolo gy e vid e n ce sh owin g th a t h u -m an p ap illo -m aviru s in fection ca uses -m ost ce rv i-cal in trae p ith e lial n e o p lasia . Jou rn a l of t h e Na-tion al Can cer In s t i t u t e, 85:958-964.
SPSS, 1997. Statistical Pack age for th e Social Scien ces for W i n d ow s :Version 8.0.0. Cha p el Hill: SPSS.
S TATACO R P., 1997. Stata Statistical Softw are : Re l e a s e 5 . 0 .Co l l e g e Station : Sta ta Co r p o ra t i o n .
S WAN, D. C.; TU CKER, R. A.; H OLLOWAY, B. P. &. ICENOGLE, J. P, 1997. A se n sit ive, t yp e -sp ecific , f l u o rogen ic p r ob e assa y fo r d e tect io n of h u m an p a p i l l o m a v i ru s DNA. Jou rn al of Clin ical M i c ro b i-o l i-o gy, 35:886-891.
