Analysis of molecular biology techniques for the diagnosis of
human papillomavirus infection and cervical cancer prevention
Análise de técnicas de biologia molecular para o diagnóstico de infecções causadas
por papilomavírus humanos e a prevenção do câncer cervical
Fernanda Nahoum Carestiato
1, Kátia Cristina da Silva
1, Daniela Signorelli Balthazar
1,
Licínio Silva
2, Marisa Marinho
3, Ledy Horto dos Santos Oliveira
1and Silvia Maria Baeta Cavalcanti
1ABSTRACT
The o b j e c tive o f the pr e se nt study was to e valuate the use fulne ss o f mo le c ular me tho do lo gie s to ac c e ss human papillo mavir us ge no me in the ge nital tr ac t. Sample s fr o m 1 3 6 wo me n age d 1 7 to 5 2 ye ar s o ld o b taine d fr o m the Dr. Sé r gio Fr anc o Lab o r ato r ie s b e twe e n 2 0 0 0 and 2 0 0 1 , we r e analyze d b y the hyb r id c aptur e assay and amplifie d b y PCR with ge ne r ic pr ime r s MY0 9 /MY1 1 and spe c ific pr ime r s fo r type s 1 6 , 1 8 , 3 1 , 3 3 , 3 5 , 5 8 . Vir al ge no me was de te c te d in 7 1 .3 % o f the sample s b y hyb r id c aptur e and 7 5 % b y amplific atio n. Whe n c yto patho lo gy was use d as a r e fe r e nc e me tho d fo r sc r e e ning le sio ns, hyb r id c aptur e ( p= 0 ) and amplific atio n ( p= 0 .0 0 2 ) pr e se nte d po sitive asso c iatio n. The 3 me tho ds sho we d ab so lute agr e e me nt whe n c yto patho lo gy c o nfir m e d papillo m avir us infe c tio n and high gr ade intr ae pithe lial le sio n. Disagr e e m e nts o c c ur r e d fo r 1 0 c ase s: se ve n inflammato r y c ase s po sitive b y PCR and ne gative fo r hyb r id c aptur e and 3 lo w squamo us intr ae pithe lial le sio ns po sitive fo r hyb r id c aptur e b ut ne gative fo r amplific atio n. In c o nc lusio n, hyb r id c aptur e was sho wn to b e se nsitive and spe c ific e no ugh fo r use in c linic al r o utine s. Mo r e o ve r, the e valuatio n o f vir al lo ad value s o b taine d b y this me tho d we r e sho wn to b e r e late d to the se ve r ity o f the le sio n and me r it fur the r studie s to analyze the po ssib le asso c iatio n with r isk o f pr o gr e ssio n to malignanc y.
Ke y- wo r ds: Human papillo mavir us. Squamo us intr ae pithe lial ne o plasia. Canc e r. Hyb r id c aptur e . Po lyme r ase c hain r e ac tio n.
RESUMO
O o b j e tivo do no sso tr ab alho fo i avaliar o e mpr e go de mé to do s mo le c ular e s par a c o mpr o var a pr e se nç a do s papilo mavír us humano s no tr ato ge nital. Amo str as de 1 3 6 pac ie nte s c o m idade s e ntr e 1 7 e 5 2 ano s, c o le tadas no s Lab o r ató r io s Dr. Sé r gio Fr anc o e ntr e 2 0 0 0 e 2 0 0 1 , fo r am analisadas pe las té c nic as de c aptur a híb r ida e amplific aç ão pe la r e aç ão e m c ade ia da po lime r ase c o m pr ime r s ge né r ic o s MY0 9 /MY1 1 e e spe c ífic o s par a o s tipo s 1 6 , 1 8 , 3 1 , 3 3 , 3 5 , 5 8 . O ge no ma vir al fo i de te c tado e m 7 1 ,3 % de ssas amo str as pe la c aptur a híb r ida e 7 5 % pe la r e aç ão e m c ade ia da po lime r ase . Quando a c ito pato lo gia fo i usada c o mo mé to do de r e fe r ê nc ia par a r astr e ame nto das le sõ e s, a c aptur a ( p= 0 ) e a amplific aç ão ( p= 0 ,0 0 2 ) de mo nstr ar am assoc iaç ão positiva. Os três testes demonstraram c onc ordânc ia absoluta quando a c itopatologia diagnostic ou proc esso c ompatível c o m papilo mavír us o u le são intr ae pite lial de alto gr au. De z c aso s disc o r dante s o c o r r e r am: se te c aso s de c ito lo gia inflamató r io po sitivo s na r e aç ão e m c ade ia da po lime r ase mas ne gativo s na c aptur a híb r ida e 3 c aso s de le são de b aixo gr au po sitivas na c aptur a híb r ida e ne gativas pe la r e aç ão e m c ade ia da po lime r ase . Co nc luímo s que a c aptur a híb r ida apr e se nto u se nsib ilidade e espec ific idade adequadas para uso c línic o. Avaliaç ão da c arga viral obtida por esta metodologia relac ionou-se c om a severidade da le são e me r e c e e studo s adic io nais a fim de de te r minar se u valo r pr o gnó stic o par a o c ânc e r.
Pa la vr a s- cha ve s: Papilo mavír us humano s. Ne o plasia intr ae pite lial e sc amo sa. Cânc e r. Captur a do híb r ido . Re aç ão e m c ade ia da po lime r ase .
1 . Lab o r ató r io de Diagnó stic o Vir o ló gic o do Depar tamento de Mic r o b io lo gia e Par asito lo gia do Instituto B io médic o da Univer sidade Feder al Fluminense, Niter ó i, RJ. 2 . Departamento de Estatístic a da Fac uldade de Matemátic a da Universidade Federal Fluminense, Niterói, RJ. 3 . Setor de B iologia Molec ular do Laboratório Sérgio Franc o, Rio de Jane ir o , RJ.
Tr ab alho r e alizado no Lab o r ató r io de Diagnó stic o Vir o ló gic o , Instituto B io mé dic o da Unive r sidade Fe de r al Flumine nse e financ iado pe lo CNPq .
Addr e ss to : Dr a. Silvia M. B . Cavalc anti. Lab . Vir o lo gia/De pto Mic r o b io lo gia e Par asito lo gia/IB /UFF. Rua Er nani Me lo 1 0 1 , Ce ntr o , 2 4 2 1 0 - 0 3 0 Nite r ó i, RJ . Te l: 5 5 2 1 2 6 2 9 - 2 4 3 1 ; Fax: 5 5 2 1 2 6 2 9 - 2 4 1 3
Human papillomavirus ( HPV) infec tion is the major c ause
of most c ervic al c anc er and c ervic al intraepithelial neoplasia
( CIN) worldwide1 7. Nearly 5 0 0 ,0 0 0 c ases of c ervic al c anc er arise
eac h year and 2 0 0 ,0 0 0 women eventually die of the disease. In
B r azil, ute r ine c anc e r is the thir d c ause o f de ath due to
neoplasia1 4. Consequently, there is strong motivation to evaluate
the use of HPV testing in cervical cancer screening, which requires
further improvements in the standardization of testing methods.
HPV detec tion has generally been c onduc ted by hybridization
and po lyme r ase c hain r e ac tio n ( PCR) me tho ds. Ho we ve r, neither researc h assays nor c ommerc ial kits ( dot blot or in situ hybridization) have been shown to be adequate for c linic al use.
An assay for routine c linic al use requires reliable and ac c urate
detec tion of the broad range of pathogenic HPV types infec ting
the genital trac t9.
Diagnosis of c ervic al disease, indic ating the presenc e of
ab no r m al c e r vic al e pithe lial c e lls, is usually o b taine d b y
mic rosc opic examination of Papanic olaou stained smears. This
has been the method of c hoic e sinc e the 1 9 5 0 ’s, proving valuable for mass screening and enabling detection of lesions early enough
to be treated effec tively. The Pap smear, however, has limited
sensitivity in detec ting c anc er prec ursors, giving a false-negative
rate ranging from 2 0 to 3 0 %8. Henc e, c omplementary methods
that may enable the improvement of c ervic al disease diagnosis
have been studied for the past two dec ades. Rec ently developed,
the sec ond generation of the hybrid c apture assay ( HCA II) for
HPV DNA detec tion from Digene Diagnostic s ( Silver Spring, Md) , is a nonradioac tive, relatively rapid, liquid hybridization assay
designed to detec t eighteen HPV types divided into high-risk and
low-risk groups. A possible unique advantage c ompared with
other available HPV test kits, the hybrid c apture test is also designed to provide quantitative estimates of viral load, whic h
may c orrelate with the grade and natural history of c ervic al
pathology6 2 2.
Give n th e fa c t th a t dive r s e c lin ic a l la b o r a to r ie s a r e
c ur r e ntly using this me tho d, the pr e se nt inve stigatio n was c o nduc te d to c o mpar e HCA II with PCR, aime d at pr o viding
to o ls fo r the inte r pr e tatio n o f this ne w availab le pr o c e dur e ,
with the o b j e c tive o f c o ntr ib uting to c anc e r pr e ve ntio n.
MATERIAL AND METHODS
Study population and collection of specimen. The study was approved by the Ethic al Committee of the Fluminense Federal University ( 2 1 /2 0 0 0 ) . The po pulatio n inc luded 1 3 6 wo men
attended at the Sérgio Franco Laboratories, Rio de Janeiro, Brazil, from January 2 0 0 0 to Dec ember, 2 0 0 1 . Women were referred to hybrid c apture after c linic al suspic ion of HPV infec tion during a routine exam. The c ervic al smears were c ollec ted using a c ervic al
c yto b r ush and tr anspo r te d in spe c ime n tr anspo r t me dium ( Digene Diag, Md) .
Cytologic test. The Papanic olaou test was developed and sm e ar s we r e c lassifie d as no r m al fo r no r m al e pithe lium ,
inflammatory for minor alterations of c ervic al c ells, ASCUS for
alterations of squamous c ells of undetermined signific anc e, HPV
fo r alter atio ns suggestive o f HPV infec tio n, LSIL fo r lo w-gr ade
s q u a m o u s i n tr a e p i th e l i a l l e s i o n s , HS I L fo r h i gh - gr a de squamo us intr ae pithe lial le sio ns in situ c ar c ino ma and SCC fo r squamo us c e ll c ar c ino ma.
Human papillo mavir us testing by hybr id captur e. The assay kit detec ts low and high-risk HPV genomes c ombined in
two c oc ktails: group A c ontaining the low-risk group detec ts th e typ e s m o r e c o m m o n l y a s s o c i a te d wi th c o n d yl o m a
ac uminatum: HPV types 6 , 1 1 , 4 2 , 4 3 and 4 4 and gro up B
c ontaining the high-risk types 1 6 , 1 8 , 3 1 , 3 3 , 3 5 , 3 9 , 4 5 , 5 1 ,
5 2 , 5 6 , 5 8 , 5 9 and 6 8 . Ac c ording to the kit protoc ol, spec imens we r e tr e ate d with so dium hydr o xide to hydr o lyze the RNA
spec imen and denature the DNA. The liberated single strand
DNA was hybridized in solution with a RNA probe mix c onsisting
o f the high-r isk o r the lo w-r isk HPV type s. Eac h r e ac tio n mixtur e , c o ntaining any numb e r o f RNA-DNA hyb r ids that
fo r m e d, wa s tr a n s fe r r e d to a c a ptur e tub e c o a te d with
antibodies to the hybrids; c onsequently, immobilizing them.
B ound RNA-DNA hybrids were then reac ted with an alkaline phosphatase-c onjugated antibody direc ted against the hybrids.
Nonreac tive material was removed by washing, and a
dioxetane-b ase d c he m ilum ine sc e nt c o m po und, Lum i- Pho s 5 3 0 , was
adde d as a sub str ate fo r alk aline pho sphatase . The light p r o du c e d b y th e e n s u i n g r e a c ti o n wa s m e a s u r e d b y a
Luminometer. Light measurements were expressed as relative
light units ( RLU) . As a negative c o ntr o l, so nic ated he r r ing
spe r m DNA in Dige ne tr anspo r ting me dium ( 1 0 0
µ
g/ml) wasused. Triplic ate spec imens of HPV 1 6 or HPV 1 1 DNA at 1 0 pg/ml
se r ve d as the po sitive c o ntr o ls fo r high-r isk and lo w-r isk
pr o b e s, r e spe c tive ly. All RLU me asur e me nts fo r spe c ime ns were divided by the mean RLU of the three appropriate positive c o ntr o ls ( PCs) to give a r atio o f spe c ime n RLU/PC. A r atio o f
1 .0 o r gr e ate r was r e gar de d as po sitive fo r HPV DNA, and a
r atio o f le ss than 1 .0 was r e gar de d as ne gative . Sinc e the am o unt o f light pr o duc e d b y the hyb r id c aptur e assay is
the o r e tic ally pr o po r tio nal to the amo unt o f tar ge t HPV DNA,
HCA II c an b e analyze d as a quantitative me tho d8.
Human papillo mavir us testing by po lymer ase chain r eactio n. The PCR assay was realized from the c linic al samples initially c ollec ted for HCA II. The spec imens were stored at
-2 0 ° C in sodic ac idum until required. First, the spec imens were neutralized using 1 N HCl. DNA extrac tion was performed as follows: 5 0 0 µl of the neutralized spec imen were prec ipitated
on 5 0 µl of the sodium ac etate and 1 ,2 5 0 µl of 1 0 0 % ethanol. Th e s pe c im e n s we r e in c ub a te d a t - 2 0 ° C o ve r n igh t. Th e fo llo wing day, these samples were c entrifuged fo r 3 0 min at 1 4 ,0 0 0 rpm. After c entrifugation, the supernatant was disc arded
a n d th e r e m a in in g p r e c ip ita te d s a m p le wa s wa s h e d in 7 0 % ethanol and c entrifuged for 1 0 min at 1 4 ,0 0 0 rpm. The
prec ipitate was left to dry at room temperature and resuspended
in 5 0 µl of distilled water and stored at -2 0oC.
Polymer ase chain r eaction amplification of gener ic huma n pa pillo ma vir us. Consensus primers MY0 9 /MY1 1 , which amplify 4 5 0 base pair ( bp) DNA sequences within the L1
region of HPV, were used to detect generic HPV DNA. Amplification
2 0 0
µ
M dNTPs, 1 .5 mM MgCl2, 5 0 pmol of eac h primer, 0 .2 5 Uunit of Taq polymerase, and 5
µ
l of sample) with 3 5 c yc les ofamplific ation. Eac h c yc le inc luded a denaturation step at 9 40C
for 1 minute, an annealing step at 5 50C for 2 minutes, and a
c hain elongation step at 7 20C for 2 minutes using a DNA thermal
c yc ler ( Perkin Elmer, CETUS) . The ac tin primers ( 0 .1 pmol eac h) , whic h amplify a 3 6 0 bp region of the human DNA, were
used as an internal c ontrol. PCR produc ts were analyzed on 1 .3 % agarose gel with ethidium bromide staining for visualization of
DNA unde r ultr a vio le t light a nd the ir PM de te r m ine d b y
c omparison with a 1 0 0 bp DNA ladder.
Polymer ase chain r eaction amplificatio n fo r human pa pillo ma vir us using spe cific h uma n pa pillo ma vir us pr imer s. HPV typing was performed by PCR using primers from
the E6 gene DNA sequenc es of HPV 1 6 , 1 8 , 3 1 , 3 3 , 3 5 and 5 81 8.
These primers yielded 2 3 0 , 8 9 , 1 3 4 , 1 1 9 , 9 7 , 1 3 2 , 1 8 6 , and 1 0 0 -bp fragments, respec tively. The PCR inc luded 2 5 c yc les as
desc ribed: 9 4 ° C for 3 0 sec onds, 6 0 ° C for 1 minute, and 7 2 ° C
for 1 minute. The PCR run was c ompleted by extension for 1 0 minutes at 7 2 ° C. Negative controls for background were included.
The primers Ac 1 and Ac 2 , spec ific for the amplific ation of a 3 3 0
bp fragment of the ac tin gene, were used as a c ontrol of DNA extrac tion. Positive c ontrols inc luded SiHa and HeLa c ell c ultures
presenting HPV 1 6 and 1 8 genomes, respec tively.
Sta tistica l a na lysis. The statistic al signific anc e o f the r e sults was analyze d using the SPSS- 8 c o mpute r pr o gr am
( 2 0 0 2 -USA) . Comparisons between PCR and HCA were realized b y the Mc Ne mar te st ( p< 0 . 0 5 ) . Asso c iatio n inde xe s we r e
obtained by the Fisher exac t test. Viral load measurements were
evaluated by the Kruskall-Wallis test ( p< 0 .0 5 ) .
RESULTS
The pr e se nt study c o mpar e d the r e sults r e alize d b y PCR
a n d h yb r id c a ptur e HCA I I te s ts fo r dia gn o s in g c e r vic a l infec tio n by HPV in 1 3 6 samples o f c er vic al smear s. The mean
age o f the patie nts was 3 0 .6 r anging fr o m 1 7 to 5 2 ye ar s o ld. The pr evalent inter val fo r HPV infec tio n was fr o m 2 1 -3 0 year s
o ld. Samples were c lassified ac c o rding to the B ethesda system
into inflammato ry ( 4 1 /1 3 6 ) , ASCUS ( 1 1 /1 3 6 ) , HPV ( 2 6 /1 3 6 ) , LSIL ( 5 5 /1 3 6 ) and HSIL ( 3 /1 3 6 ) . A statistic ally signific ant
upwa r d tr e n d in a ge fr o m in fla m m a to r y c a s e s ( m e dium
age = 2 6 .9 ) , ASCUS, HPV and LSIL ( me dium age = 2 8 .3 ) and HSIL ( medium age = 3 9 .5 ) ( p< 0 .0 0 0 1 ) was c learly o bserved.
Tab le 1 sho ws the r e sults o b taine d b y HCA I I : o f the 1 3 6 sam ple s studie d, 9 7 ( 7 1 . 3 % ) we r e infe c te d b y HPV.
High-r isk gr o up B was the mo st pr e vale nt gr o up de te c te d b y
HCA II, ac c o unting fo r 5 1 c ase s alo ne o r mixe d with gr o up A in 3 4 c ases. Henc e, 8 5 ( 6 2 .5 % ) wo men o ut o f the 1 3 6 studied
sho we d high-r isk infe c tio ns.
Tab le 2 pr e se nts the PCR r e sults: o f the 1 3 6 sam ple s studie d, 1 0 2 ( 7 5 % ) wo me n we r e HPV-po sitive : 3 8 ( 3 7 .3 % )
we r e HPV 1 6 ; 2 2 ( 2 1 . 6 % ) HPV 1 8 ; 5 ( 4 . 9 % ) HPV 3 3 ;
5 ( 4 . 9 % ) HPV 3 5 ; a n d 5 ( 4 . 9 % ) HPV 5 8 . Twe n ty s e ve n
( 2 6 .5 % ) c ase s ( HPV X) r e maine d untype d b y PCR.
Table 1 -Detection of low and high-r isk HPV obtained by the hybr id captur e assay accor ding to colpocytology.
Hybrid capture Prevalence Colpocytology Number of Low-risk High-risk Multiple no %
diagnosis patients HPV HPV infection
Inflammatory 41 2 10 5 1 7 /4 1 4 1 .4 ASCUS 11 - 2 - 2 /1 1 1 8 .2 HPV 26 7 9 7 2 3 /2 6 8 8 .5 LSIL 55 3 27 22 5 2 /5 5 9 4 .5 HSIL 3 - 3 - 3 /3 1 0 0 .0 Total 1 3 6 12 51 34 9 7 /1 3 6 7 1 .3 ASCUS: atypical squamous cell of undetermined significance; LSIL: low grade squamous intraepithelial lesions; HSIL: high grade squamous intraepithelial lesions. HPV: human papillomavirus.
Table 2 -Human papillomavir us types detected by the polymer ase chain r eaction accor ding to the colpocytological diagnosis.
Colpocytology Number of HPV types in PCR
diagnosis patients MY 16 18 33 35 58 HPV X Inflammatory 41 24 10 2 2 - - 10 ASCUS 11 3 2 - - - - 1 HPV 26 23 8 6 - 1 - 8 LSIL 55 49 16 13 3 4 5 8 HSIL 3 3 2 1 - - - -Total 1 3 6 1 0 2 38 22 5 5 5 27 No HPV 3 1 was detected. X = untyped. ASCUS: atypical squamous cell of undetermined significance; LSIL: low grade squamous intraepithelial lesions; HSIL: high grade squamous intraepithelial lesions. HPV: human papillomavirus.
Table 3 presents the results obtained by both HCA II and PCR: concordant results were observed for 1 3 2 ( 9 7 .1 %) samples while disagr e e m e nts we r e fo und fo r 1 1 ( 2 . 9 % ) sam ple s: 7 inflammato r y c ase s ne gative fo r HCA b ut po sitive fo r PCR,
1 c ase o f ASCUS po sitive fo r PCR and ne gative fo r HCA II and
3 c ase s o f LSIL po sitive fo r HCA II, b ut ne gative fo r PCR. The two te sts pr e se nte d a go o d asso c iatio n inde x ac c o r ding to
the Fishe r e xac t te st ( p= 0 .0 0 0 2 , Fishe r Te st) and a k appa
c o e ffic ie nt o f k = 0 .7 6 ( 9 5 % c o nfide nc e inte r val) , indic ating
an e xc e lle nt agr e e me nt inde x fo r diagno sing HPV infe c tio n. The prevalenc e o f high and lo w-risk types o f HPV by the hybrid
c a ptur e a s s a y in c o m pa r is o n with th e r e s ults o f PCR is
pr e se nte d in the Tab le 4 . Se nsib ility was highe r fo r HCA II
( 9 9 .2 % ) than PCR ( 9 7 .1 % ) b ut spe c ific ity r ate was lo we r fo r
b o th te c hnique s ( HCA – 8 6 % and PCR – 8 5 .2 % ) .
The mean viral load values measured by the hybrid c apture
assay are shown in Table 4 . An inc rease in the values oc c urred, a c c o r din g to th e s e ve r ity o f th e c yto lo gic te s t fo r b o th
Table 3 - Results and agr eement r ates obtained by PCR and hybr id captur e accor ding to colpocytology.
b e nign ( Gr o up A: inflammato r y = 2 ,1 1 9 to LSIL = 5 8 5 ) and o n c o ge n ic HPV ( Gr o up B : in fla m m a to r y: 1 9 5 . 5 to HSI L:
9 2 3 .1 ) . The Kr usk al-Wallis ( p= 0 .0 5 ) te st sho we d that the r e
was an upwar d tr e nd o f RLU me asur e me nts asso c iate d with
inc reased lesion severity for both the low-risk ( group A) and high-risk ( group B) HPV groups. Viral load values proved to be
statistic ally different between inflammatory and LSIL/HSIL c ases.
DISCUSSION
The initial im pac t afte r the ado ptio n o f Papanic o lao u
sc reening test in Mass Programs for Canc er Prevention was
astonishing, determining a 7 0 % reduc tion in mortality indexes worldwide. Nevertheless, after ten years, indexes stabilized and
even inc reased due to a signific ant group of women failing to
regularly partic ipate in suc h programs. In the last dec ade, HPV
has presented earlier in the female genital tract and the prevalence of infec tion has inc reased, making this virus the agent of the
mo st pr evalent sexually tr ansmitted vir al disease in sever al
c ountries1 0. Moreover, the Papanic olaou test presents intrinsic
rates of false-positives and false-negatives, reinforc ing the need to use c omplementary tests when sc reening for HPV infec tion.
Molec ular biology methodologies, suc h as PCR, have proved their
use fulne ss in de te c ting HPV infe c tio n, thus c o ntr ib uting to
the ear ly diagno sis o f wo men at r isk fo r c er vic al c anc er1 6.
Nevertheless, these tec hniques are expensive, laborious and
frequently reveal c ervic al HPV infec tions that are only transient7.
More rec ently, hybrid c apture assay is c urrently in use for
testing HPV DNA in Brazil and worldwide, however, no studies
have been c onduc ted to test its usefulness in this c ountry. It is not as laborious as PCR, but is still very expensive and its c linic al
relevanc e remains c ontroversial. IARC have proposed that to
reduc e c osts and optimize HCA use in routine diagnosis, the
foc us should be on women over 3 5 years old, usually presenting persistent infec tions or the detec tion of ASCUS in c ytology, due
to their inc reased risk for malignant transformation5.
The present investigation was c onduc ted to c ompare HCA II with PCR, with the aim of providing tools for the interpretation of the usefulness of this newly available proc edure in Brazil. For that purpose, c ervic al samples were studied by both HCA II and PCR assays. The tests were c onc ordant for 1 3 2 ( 9 7 .1 % ) out of 1 3 6 samples. HCA II detec ted HPV infec tion in 7 1 .3 % ( 9 7 /1 3 6 )
of patients, while PCR detec ted HPV DNA in 7 5 % ( 1 0 2 /1 3 6 ) . Disc r epanc ies in the r esults wer e o bser ved: seven c ases o f inflammatory c ytology presented HPV by PCR. Perhaps these
results are due to the greater sensibility of PCR, revealing latent infec tions, but when analyzed using c ytology parameters, they
c ould represent false-positives, sinc e there were no apparent
le s io ns5. Ne ve r the le s s , wo m e n pr e s e nting high- r is k HPV,
espec ially over 3 5 years old, must be followed-up c autiously,
since they may be at a higher risk of developing SILs, as previously
proposed by Koutsky1 3.
It sho uld b e no te d that the pr e vale nc e de sc r ib e d he r e in
is ve r y high, tho ugh the c ur r e nt study was no t de signe d to b e
a pr e vale nc e study o f the ge ne r al po pulatio n. Sample s fr o m wo me n r e fe r r e d fo r fur the r e xams afte r c linic al suspic io n
we r e analyze d. He nc e , high pr e vale nc e was alr e ady e xpe c te d.
Of so me impo r tanc e is the fac t that PCR faile d to de te c t thr e e
sample s c lassifie d as LSIL b y c yto lo gy and po sitive fo r
high-r isk type s o f HPV in HCA II. Muño z1 8 pr e vio usly de sc r ib e d
false ne gative s whe n using MY pr ime r s fo r sc r e e ning HPV
infe c tio n a nd sugge ste d the use o f a n a dditio na l pa ir o f
pr im e r s ( GP5 /6 ) to r e duc e the se e ve nts. We sugge st that sc r e e ning b y MY pr ime r s is still use ful, b ut sample s might
also b e te ste d fo r high-r isk HPV, e spe c ially HPV 1 6 and 1 8 ,
the two mo st o nc o ge nic and pr e vale nt type s de te c te d in Rio
de Jane ir o , B r azil3 1 9. The o the r disc r e panc y was o b se r ve d
fo r an ASCUS sample : HCA II was po sitive , while PCR pr o ve d
ne gative . ASCUS diagno sis is e xc lusio n fo r HPV infe c tio n and
the HPV infe c tio n he r e de sc r ib e d might r e pr e se nt a false
-po sitive b y HCA II. Pr e vio us studie s have alr e ady sugge ste d false po sitive s and the autho r s asso c iate d the c ase s to lo w
vir al lo ads me asur e d b y HCA, sugge sting that the y c o uld b e
so lve d b y c hanging the r e ac tio n c ut o ff fr o m 1 .0 to 3 .0 RLU/
PC1 5 1 6. I n fa c t, th is PCR( - ) ASCUS c a s e wa s po s itive fo r
HCA II, sho wing a vir al lo ad o f 2 .0 9 RLU. Ne ve r the le ss, the
diagno sis o f ASCUS c o ntinue s to pr e se nt pr o b le ms and the
de finitio n o f c r ite r ia is still unde r study, sinc e misdiagno sis
is o fte n s e e n a n d a n o b s e r va tio n o f ASCUS m igh t e ve n
r e pr e se nt HSIL2 1. Mano s e t al1 5 has alr e ady pr o po se d that
ASCUS diagno sis wo uld indic ate te sting fo r HPV DNA, as we ll
as a c o lpo sc o py e xam, in o r de r to disc ar d the po ssib ility o f a
lo st high-gr ade le sio n.
I n this study, sta tistic a l pa r a m e te r s o f se nsib ility a nd
spe c ific ity fo r HCA II and PCR we r e de sc r ib e d, using the c yto patho lo gic al te st as a r e fe r e nc e m e tho d o f sc r e e ning
B r azilian patie nts. Se nsib ility r ate s o f 9 9 . 2 % fo r HCA and
9 7 .1 % fo r PCR we r e o b taine d, while spe c ific ity r ate s we r e a little lo we r fo r b o th te c hniq ue s ( 8 6 % and 8 5 .2 % fo r HCA
a n d P CR , r e s p e c ti ve l y) . Ka p p a va l u e s r e ve a l e d a go o d
asso c iatio n index ( k= 0 .7 6 ) . These results are similar to tho se
de sc r ib e d b y Ille sc as e t al1 2 and sho w that b o th PCR and HCA
II ar e e xc e lle nt te sts fo r sc r e e ning patie nts infe c te d b y HPV
and that might b e at a gr e ate r r isk o f c anc e r.
High pr e vale nc e r ate s o f o nc o ge nic HPV infe c tio ns we r e de mo nstr ate d and ac c o unte d fo r 8 5 /9 5 c ase s b y HCA and 7 5 / 9 5 c ase s b y PCR. Twe nty-se ve n HPV X we r e fo und that c o uld r e pr e se nt type s no t te ste d in the PCR use d he r e . HCA sho we d 1 2 lo w-r isk HPV, c o nse que ntly, the se 1 2 lo w-r isk c ase s plus a fur the r 1 5 lo w o r high-r isk HPV type s we r e no t de te r mine d b y PCR. On the o the r hand, it c o uld b e ar gue d that the se ar e high r isk type s due to the high pr e vale nc e o f the se type s b y
HCA. Po lj a k2 0 s ugge s te d th a t th e y c o uld r e pr e s e n t fa ls e
-po sitive r e sults o b taine d b y c r o ss hyb r idizatio n with lo w-r isk Table 4 - Mean vir al load values ( RLU/PC) measur ed by hybr id captur e and its
r elation to the cytological diagnosis and to the HPV gr oup.
HPV Group Cytological diagnosis
o r no nr e pr e se nte d type s in Co c k tail B . The po ssib ility o f
e q u i vo c a l i d e n ti fi c a ti o n o f HP V r e s u l ti n g fr o m c r o s s hyb r idizatio n is c ur r e ntly b e ing r e so lve d b y thir d ge ne r atio n
k it HCA III2 1, sinc e e r r o ne o us diagno sis o f a high-r isk HPV
has r e le vant c o nse que nc e s fo r patie nts, le ading to e r r o r s in
c linic al c o nduc t and c ausing unne c e ssar y pe r so nal str e ss2 2.
In Table 4 , data po ints to a statistic ally signific ant inc r ease in the vir al lo ad me asur e me nts ac c o r ding to the se ve r ity o f
the le sio ns. The use fulne ss o f me asur ing vir al lo ad b y HCA II
has b e e n the o b j e c t o f se ve r al studie s. Clave l e t al2 3 fo und no
signific ant r e latio n b e twe e n me asur e me nts and le sio ns, b ut,
Castle e t al2 1 fo und an str ic t r e latio n b e twe e n high vir al lo ad
a nd SI L le sio ns. Fur the r studie s a r e r e q uir e d to a m plify
c ur r e nt k no wle dge and he lp in e luc idating the me aning o f
HCA vir al lo ad with r e gar d to the dise ase .
In the present work, the age interval of greatest prevalenc e was 2 0 -3 0 , c oinc ident with the mean age of the partic ipants and
with the peak of sexual activity. Most of the infections are transitory
in these periods and, in fac t, prevalenc e dec reased markedly
afte r 3 5 ye ar s o f age . Se ve r al autho r s have sugge ste d that above 3 5 years of age, infec tions might indic ate viral persistenc e,
probably c aused by onc ogenic HPV and thus represent a greater
risk of c anc er for the affec ted women4 1 1.
I n s pite o f b e ing a n e a s ily pr e ve nta b le a nd tr e a ta b le
dis e a s e , c e r vic a l c a n c e r is s till pr e s e n tin g h igh r a te s o f mo r b idity and mo r tality amo ng B r azilian wo me n. Mo le c ular
te c hnique s c apab le o f de te c ting HPV infe c tio n ar e e xpe nsive
and c an no t b e use d in r o utine diagno sis, ho we ve r, the se
wo u l d c o m p l e m e n t c e r vi c a l s c r e e n i n g, wh e n a p p l i e d sub se que nt to the Pap te st, in o r de r to r e duc e the r isk o f
o nc o genesis. In this study, a high prevalenc e rate o f o nc o genic
infe c tio n was fo und, mainly HPV 1 6 . Se ve r ity o f the studie d
lesio ns seemed to be asso c iated with high vir al lo ads o btained b y HCA. B o th te c h n iq ue s s h o we d go o d a gr e e m e n t a n d
r e ve ale d o nc o ge nic infe c tio ns in patie nts o ve r 3 5 ye ar s o f
age, asso c iated with HPV persistenc e in the c ervix. Thus, these
patie nts, as we ll as tho se pr e se nting pr o gr e ssive o r r e c ur r e nt le sio ns, sho uld b e te ste d fo r high r isk HPV infe c tio n, sinc e
the se r e pr e se nt an inc r e ase d r isk fo r o nc o ge ne sis.
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