Background: Tuberculosis is an ancient disease, which still remains one of the major ills faced by mankind in the 21st century.
In recent decades, new technologies employing the knowledge gained from molecular biology studies have allowed for more accurate detection of tuberculosis and increased investigation of the etiology and epidemiology of the disease.
Aim: Evaluating the degree of similarity among strains of Mycobacterium tuberculosis provided by the Phthisiology Sector of Centro de Saúde Navegantes (Navegantes Health Clinic) in Porto Alegre, RS, Brazil.
Method: A retrospective study was performed involving RFLP typing of 55 sputum samples from outpatients examined at the Centro de Saúde Navegantes. The results of the genotyping were correlated to the conventional epidemiology d a t a .
Results: A single pattern was seen in 39 (70.9%) of the isolates, whereas 16 isolates (29.1%) showed clustering patterns and were grouped into 8 clusters of 2 patients each. An epidemiological link was found for 6 (37.5%) of the 16 patients in the clusters.
Conclusion: The appropriate combination of conventional epidemiology and genotyping of M. tuberculosis contributes to a better understanding of the dynamics of tuberculosis transmission even when such a study is performed in a single, isolated health clinic.
Key words: Mycobacterium tuberculosis. Transmission. Polymorphism, Restriction Fragment Length. Epidemiology.
*St u d y ca rried o u t a t t h e Cen t ro d e Desen vo lvim en t o Cien t ífico e Tecn o ló g ico (Cen t er fo r Scien t ific a n d Tech n o lo g ica l Develo p m en t ) o f t h e Fu n d a çã o Est a d u a l d e Pro d u çã o e Pesq u isa em Sa ú d e (St a t e Fo u n d a t io n fo r Hea lt h Resea rch ), Po rt o Aleg re, RS, Bra zil
Co rre sp o n d e n ce t o : Mich e le Bo rg e s. Av. Ip ira n g a , 5 4 0 0 , Ba irro J a rd im Bo t â n ico CEP: 9 0 610 - 0 0 0 . Po rt o Ale g re , RS, Bra sil. Em a il: m ich e b o rg e s@ ya h o o .co m .b r
Fin a n cia l su p p o rt p ro vid ed b y t h e Co n selh o Na cio n a l d e Desen vo lvim en t o Cien t ífico e Tecn o ló g ico (CNPq , Na t io n a l Co u n cil fo r Scien t ific a n d Tech n o lo g ica l Develo p m en t ) a n d t h e Red e Bra sileira d e Pesq u isa em TB (Red e- TB, Bra zilia n Tu b ercu lo sis Resea rch Net wo rk)/ Gra n t n o . 6 2 .0 0 5 5 / 01 - 4 - P ACDT- Mile n io .
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Abbreviations used in this paper:
CSN– Ce n t ro d e Sa ú d e Na ve g a n t e s (Na ve g a n t e s He a lt h Clin ic )
EMB– Et h a m b u t o l ETH– Et h io n a m id e
HIV– Hu m a n im m u n o d eficien cy viru s INH– Iso n ia zid
IS– In sert io n seq u en ce
Mt b – Myco b a ct eriu m t u b ercu lo sis PZA– Pyra zin a m id e
RMP– Rifa m p icin
RFLP– Rest rict io n Fra g m en t Len g t h Po lym o rp h ism TB– Tu b ercu lo sis
INTRODUCTION
Tu b e rcu lo sis (TB) h a s n o fla g , u n ifo rm o r h o m elan d . It h as b een a lo n g - t im e co m p an io n o f m a n , p e rh a p s e ve n sin ce we b e ca m e b ip e d s. Rep o rt s h ave sh o wn evid en ce o f TB in p reh ist o ric hu man bon es(1). Recen t molecu lar st u dies show t hat
t h e d is e a s e - c a u s in g a g e n t , Myc o b a c t e r iu m
t u b ercu lo sis (Mt b ), h a s resid ed in t h e h u m a n
organ ism for approximat ely 10,000 years, sin ce t he t im e at wh ich h u m an s first b eg an t o d o m est icat e an d h erd an im als(2). Mo re t h an a h u n d red years
aft er Ro b ert Ko ch id en t ified t h e d isease- cau sin g ag en t (in 1 8 8 2 ), TB st ill h as o n e o f t h e h ig h est rat es o f m o rb id it y an d m o rt alit y am o n g all su ch ch ro n ic in fect io n s.
The in creased in ciden ce of TB over t he last t wo d eca d es h a s b een a t t rib u t ed t o t h e d eclin e o f p u b lic h ealt h syst em s, t o t h e HIV p an d em ic an d t o t he appearan ce of dru g- resist an t st rain s, as well as t o t h e st ro n g co rrelat io n b et ween t h e d isease an d p o vert y(3).
In sp it e o f h a vin g a n a t io n wid e TB co n t ro l program, Brazil report s approximat ely 90,000 n ew ca ses o f t h e d isea se every yea r, a lt h o u g h it is est im at ed t h at t h e act u al n u m b er is 1 2 9 ,0 0 0(4 ). In
t h e st at e o f Rio Gran d e d o Su l, t h e rat e o f TB in cid en ce in t h e yea r 2 0 0 0 wa s 5 5 .7 / 1 0 0 ,0 0 0 in habit an t s, an d t he n u mber of n ew cases report ed w a s 6 , 1 5 2 , 6 5 % o f w h ic h o c c u r r e d in t h e m et ro p o lit an area o f Po rt o Aleg re(5 ).
Gen o t yp in g t ech n iq u es u sed t o d ifferen t iat e among strains of Mtb have improved understanding o f t h e d yn am ics o f TB t ran sm issio n . Th e act ive fo rm o f t h e d isease d evelo p s as co m m o n ly aft er a re c e n t ly a c q u ire d in f e c t io n a s it d o e s a f t e r react ivat ion of an in fect ion acqu ired lon g before(3,6).
In a popu lat ion - based st u dy, it was post u lat ed t hat p at ien t s wit h TB p resen t in g sin g le st rain s sh o u ld b e co n sid ered cases o f TB b y react ivat io n , an d t hose presen t in g isolat es sharin g in dist in gu ishable p at t ern s b e d esig n at ed as b elo n g in g t o clu st ers, rep resen t in g t h e d evelo p m en t o f t h e d isease fro m a recen t ly acq u ired in fect io n(6 ,7 ).
The rest rict ion fragmen t len gt h polymorphism (RFLP) t echn iqu e uses t he 6110 insert ion sequ en ce (IS6110) t o an alyze polymorphism. The RFLP is t he g e n o t y p i n g m e t h o d m o s t w i d e l y u s e d t o differentiate among Mtb strains and has been useful in ca rryin g o u t va rio u s t yp es o f ep id em io lo g ic st u dies(6 ,8 ).
Becau se a bet t er u n derst an din g of t he dyn amics o f TB t ran sm issio n is n ecessary, t h e p resen t st u d y aim ed t o assess t h e d eg ree o f sim ilarit y am o n g st rain s of Mt b provided by t he Pht hisiology Sect or o f t h e Ce n t r o d e Sa ú d e Na ve g a n t e s (CSN, Na ve g a n t e s He a lt h Clin ic ) in P o r t o Ale g r e . Molecu lar biology t echn iqu es were combin ed wit h con ven t ion al epidem iology.
METHODS
Patients and Samples
Th e Mt b iso la t e s u se d in t h e st u d y we re p ro vid ed b y t h e Tu b ercu lo sis Lab o rat o ry o f t h e IPB- LACEN/RS, which receives patient samples from t h e Ph t h isio lo g y Sect o r o f t h e Naveg an t es Healt h Clin ic, an o u t p at ien t clin ic in t h e n o rt h ern p art o f Po rt o Aleg re. We a n a lyzed 5 5 sp u t u m sa m p les o b t a in e d fro m p a t ie n t s e xa m in e d a t t h e CSN b et ween 1 9 9 9 a n d 2 0 0 0 . Ap a rt fro m p rim a ry cu lt u re, t he samples were also t est ed for resist an ce t o an t i- TB d ru g s. Clin ical an d d em o g rap h ic d at a were obt ain ed from pat ien t files, as well as t hrou gh re vie w o f la b o ra t o ry re co rd s a n d d a t a in t h e
Sist em a d e In fo rm ação d e Ag ravo s d e No t ificação
(SINAN, Na t io n a l Ca se Reg ist ry) d a t a b a se. Th e p r e s e n t s t u d y w a s a p p r o ve d b y t h e Et h ic s Reasearch Co m m it t ee o f t h e Fu n d ação Est ad u al d e Pro d u ção e Pesq u isa em Saú d e d o Rio Gran d e do Su l (Rio Gran d e d o Su l St at e Fo u n d at io n fo r Healt h Research).
Microorganism Culture and Susceptibility Test
Th e Ku d o h & Ku d o h m et h o d wa s u sed t o cu lt ivat e clin ic samples(9 ). Dru g- su scept ibilit y t est s
we re p e rfo rm e d a cco rd in g t o t h e p ro p o rt io n m et h o d . As d escrib ed b y Ca n et t i et a l.(1 0 ), t h e
(RFLP)/ IS6110 Technique
Ge n o m i c DN A w a s e x t r a c t e d u s i n g cet ylt rimet hylammon iu m bromide met hod an d t he IS6110 RFLP wa s ca rried o u t a cco rd in g t o t h e p ro t o co l d evised by Van Em b d en et al.(11 ).
Data Analysis
Ge lCo m p a r so ft wa re , ve rsio n 4 .2 (Ap p lie d Mat h s, Ko rt rijk, Belg u im ) an d visu al an alysis were u sed t o an alyze d ig it ized RFLP im ag es. Ep iIn fo soft ware, version 6.04d (Cen t ers for Disease Con t rol a n d Preven t io n , USA) wa s u sed t o a n a lyze t h e clin ical an d d em o g rap h ic d at a o f p at ien t s.
RESULTS
Am o n g t h e 5 5 Mt b iso lat es an alyzed u sin g t h e RFLP t ech n iq u e, 4 7 d ifferen t b an d p at t ern s were id en t ified . Th irt y- n in e iso lat es (7 0 .9 %) p resen t ed a sin g le p a t t ern , wh erea s 1 6 iso la t es (2 9 .1 % ) presen t ed clu st erin g pat t ern s. Isolat es havin g 6 or less copies of IS6110 represen t ed 5.4% of t he t ot al n u mber of samples an alyzed. The remain in g 94.6% h ad 7 o r m o re co p ies o f t h e in sert io n elem en t . Th e n u m b er o f b an d s ran g ed fro m 2 t o 8 . Th e averag e an d t h e t ren d were 10 co p ies o f IS6110
p er iso lat e.
Iso la t e s p re se n t in g 1 0 0 % sim ila rit y w e re con sidered clu st ers. Ou t of t he 55 isolat es st u died, 1 6 w e r e g r o u p e d in t o 8 d if f e r e n t c lu s t e r s design at ed CL I, CL II, CL III, CL IV, CL V, CL VI, CL VII an d CL VIII. Each clu st er con sist ed of 2 isolat es. Amon g t he st rain pat t erns iden t ified t hrou gh RFLP, t hree pat t ern s belon ged t o t he “A” family fou n d in São Pau lo(8 ) (Figu re 1). Those pat t ern s were visu ally
co m p ared wit h each o t h er an d t h en wit h t h e Mt b referen ce st ra in 1 4 3 2 3 , wh ich wa s u sed a s a n ext ern al m arker in each g el.
Clu st er p at ien t s sh arin g t h e sam e h o u seh o ld , livin g in t h e sam e n eig h b o rh o o d o r b elo n g in g t o t h e sa m e fa m ily we re co n sid e re d t o h a ve a n epidem iological lin k. Su ch an epidem iological lin k was o b served fo r 6 p at ien t s (3 7 .5 %) in 3 o f t h e 8
t ran smission an d t hose resu lt in g from react ivat ion . Bet ween 1999 an d 2000, t he CSN report ed 837 n ew TB cases. In 55 (6.57%) of t hose cases, samples we re o b t a in e d a n d se n t t o t h e Tu b e rcu lo sis Laboratory of the IPB- LACEN/RS. Only samples from p at ien t s n o t resp o n d in g t o Reg im e I were sen t fo r cu lt u re, wh ich exp la in s t h e h ig h p ro p o rt io n o f p revio u sly t reat ed p at ien t s (9 0 .9 %) in t h e st u d y (Tab le 1 ).
Fo rt y iso lat es (7 2 .7 %) h ad d ifferen t resist an ce p ro files an d o n ly 1 5 (2 7 .3 %) were sen sit ive t o all d ru g s u sed in t h e t reat m en t o f TB. Twen t y- t h ree isolat es (41.8%) were resist an t t o on e or more dru g an d 1 7 (3 0 .9 %) were m u lt id ru g - resist an t .
Co n cern in g t h e fact o rs t h at m ay b e asso ciat ed w it h t h e d e ve lo p m e n t o f TB, n o sig n if ica n t c o r r e l a t i o n w a s f o u n d b e t w e e n c l i n i c a l / d em o g rap h ic d at a an d clu st er iso lat es (Tab le 1 ). Desp it e wh at h as b een m en t io n ed , d at a o b t ain ed fro m p at ien t s in fect ed wit h resist an t st rain s sh o w t h e sig n ifican ce o f t h ese p at ien t s as lin ks in t h e ch ain o f recen t t ran sm issio n .
DISCUSSION
No t so lo n g ag o , it was virt u ally im p o ssib le t o iden t ify in dividu al Mt b st rain s. Therefore, t rackin g t h e t ran sm issio n o f a g iven st rain in a reg io n o r in st it u t io n was eq u ally d ifficu lt . In recen t years, g e n o t yp in g t e c h n iq u e s h a ve b e e n u s e d t o co m p lem en t t ra d it io n a l m et h o d s o f co llect in g ep id em io lo g ical d at a.
Th ro u g h RFLP an alysis o f Mt b iso lat es, 2 9 .1 % o f p at ien t s evalu at ed in t h e p resen t st u d y were fo u n d t o b e in clu st er, sim ila r t o p ro p o rt io n s report ed in st u dies in volvin g isolat es obt ain ed from p at ien t s in Rio d e J an eiro an d São Pau lo (2 2 % an d 2 3 %, resp ect ively)(1 2 ,8 ). In a st u d y co n d u ct ed
in t he met ropolit an area of Port o Alegre, 34.5% of p at ien t s were fo u n d t o b e in clu st er(1 3 ). Th erefo re,
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Fig ure 1. Den d ro g ra m rep resen t in g t h e va rio u s RFLP p a t t ern s fo u n d in Myco b act eriu m t u b ercu lo sis iso lat es o b t ain ed fro m pat ien t s t reat ed at t he Cen t ro de Saú de Navegan t es. Clust ers were design at ed CL I, CL II, CL III, CL IV, CL V, CL VI, CL VII, an d CL VIII. *Pa t t ern s o f st ra in s b elo g in g t o t h e “A” fa m ily fo u n d in Sã o Pa u lo(8) **M. t u b ercu lo sis referen ce st rain 1 4 3 2 3
**
are very sim ilar t o t h o se o f o t h er st u d ies carried ou t in Brazil (Table 2). As described in variou s ot her st u d ies(1 4 - 1 6 ), p at t ern s wit h an averag e o f 1 0 b an d s
p er iso lat e were o b served .
Even t h o u g h 81 .8 % o f p at ien t s were m ale, t h e rat e o f recen t t ran sm issio n am o n g m ales d id n o t d iffer fro m t h at fo u n d fo r fem ale p at ien t s. Th is in dicat es t hat , alt hou gh male gen der may be a risk f a ct o r f o r t h e d e ve lo p m e n t o f TB, it is n o t n ecessarily co rrelat ed wit h h ig h er rat es o f recen t t ra n sm issio n(6 ,8 ,1 7 ,1 8 ). In a d d it io n , n o sig n ifica n t
race- relat ed differen ce was fou n d in t he percen t age o f clu st er p at ien t s, alt h o u g h t h ere is a g reat er t en den cy amon g black pat ien t s for developin g t he d isease fro m recen t t ran sm issio n . In t h e p resen t st u dy, t he sm all n u m ber of black pat ien t s in volved probably accou n t ed for t he lack of an y sign ifican t differen ce, sin ce blacks represen t on ly 5.2% of t he t ot al Rio Gran de do Su l popu lat ion , whereas whit es rep resen t 8 6 .3 %(1 9 ). It h as b een sh o wn t h at b lacks
are m o re likely t o d evelo p t h e d isease fro m recen t t ran sm issio n(6 ). Alt h o u g h t h is is p ro b ab ly relat ed
t o so cio eco n o m ic st at u s, t h ere h ave b een st u d ies in vest ig at in g t h e p o ssib ilit y t h at g en et ic fact o rs in flu en ce su scept ibilit y(2 0 ).
In t h e p resen t st u d y, 1 4 .3 % o f HIV- p o sit ive p at ien t s d evelo p ed TB fro m recen t t ran sm issio n , which was n ot sign ifican t ly relat ed t he proport ion o f HIV- n eg at ive p at ien t s (3 4 .6 %) wh o d evelo p ed t h e d isease u n d er t h e sam e co n d it io n s . Pro b ab ly d u e t o t h e sm all sam p le size, o u r d at a d iffer fro m wh at h as b een rep o rt ed in t h e lit erat u re, in wh ich HIV- posit ive pat ien t s have been shown t o be more likely t o d evelo p TB – b o t h fro m react ivat io n an d fro m recen t ly acq u ired in fect io n s(3 ).
Th ro u g h a n a lysis o f t h e clin ica l d a t a , a n ep id em io lo g ical lin k was id en t ified fo r 6 p at ien t s (3 7 .5 %) wh o d evelo p ed t h e d isease fro m recen t t ran smission . The percen t age fou n d in t he presen t s t u d y w a s s im ila r t o t h a t f o u n d in s t u d ie s p erfo rm ed in Ho n g Ko n g (3 0 .2 %) an d in t h e USA (Misso u ri: 4 6 %; Arkan sas: 4 2 %)(2 1 ,2 2 ,2 3 ).
Th e h ig h level o f resist a n ce t o a t lea st o n e d ru g (7 2 .7 % ) o b served in t h e p resen t st u d y is d irect ly rela t ed t o sa m p le select io n . A h ist o ry o f p revio u s t rea t m en t is a p red isp o sin g fa ct o r fo r resist a n t b a cilli select io n(2 4 ), a s evid en ced b y t h e
fa ct t h a t 9 0 .9 % o f t h e p a t ien t s eva lu a t ed in t h e p r e s e n t s t u d y h a d p r e v i o u s l y u n d e r g o n e t reat m en t .
Ra ce
Cau casian 1 3 (3 0 .2 ) 3 0 (6 9 .8 ) 4 3 (7 8 )
Black 2 (3 3 .3 ) 4 (6 6 .7 ) 0.77 6 (11 )
Un kn o wn 1 (1 6 .7 ) 5 (8 3 .3 ) 6 (11 )
Previou s t reat m en t
No 3 (6 0 ) 2 (4 0 ) 0.14 5 (9 .1 )
Yes 1 3 (2 6 ) 3 7 (7 4 ) 5 0 (9 0 .9 )
Resist an ce
No 2 (1 3 .3 ) 1 3 (8 6 .7 ) 0 .1 1 5 (2 7 .3 )
Yes 1 4 (3 5 ) 2 6 (6 5 ) 4 0 (7 2 .7 )
Mu lt id ru g resist an ce
No 11 (2 8 .9 ) 2 7 (71 .1 ) 0 .6 3 8 (6 9 .1 )
Yes 5 (2 9 .4 ) 1 2 (7 0 .6 ) 1 7 (3 0 .9 )
HIV
Neg at ive 9 (3 4 .6 ) 1 7 (6 5 .4 ) 2 6 (4 7 .3 )
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
The correlation between recent transmission and resist an ce was n o t sig n ifican t , alt h o u g h 3 5 % o f pat ien t s presen t in g resist an t st rain s developed TB aft er a recen t t ran smission . Ot her st u dies have also shown that patients with resistant strains contribute sig n ifican t ly t o clu st er fo rm at io n(6 ,8 ,1 5 ,2 5 ,2 9 ), wh ich
co u ld b e relat ed t o t h e d ifficu lt ies en co u n t ered in det ect in g resist an ce an d in t reat in g t hese pat ien t s. In co m p ariso n t o p at ien t s in fect ed wit h sen sit ive st rain s, pat ien t s wit h dru g- resist an t st rain s requ ire t re a t m e n t t h a t is m o re p ro t ra ct e d , t o xic a n d exp en sive, p resen t lo wer cu re rat es, an d t en d t o rem ain in fect ed fo r lo n g er p erio d s(2 6 ).
Even t h o u g h o u r st u d y was lim it ed t o p at ien t s t reat ed at t he CSN, con siderable diversit y was seen am o n g Mt b st rain p at t ern s, an d a h ig h rat e o f ep id em io lo g ic lin ks wa s fo u n d a m o n g p a t ien t s b elo n g in g t o clu st ers. Th is can b e at t rib u t ed t o t h e a p p ro p ria t e co m b in a t io n o f co n ve n t io n a l ep id em io lo g y t ech n iq u es an d g en o t yp in g , wh ich provided an overview of t he t ran smission dyn amics o f t h e d isease at t h is lo cat io n .
ACKNOWLEDGMENTS
We wo u ld like t o t h an k Nu rse Mara Pessin i o f t h e Ph t h isio lo g y Sect o r o f t h e CSN, wh o was kin d
en o u g h t o p ro vid e u s wit h t h e d at a n ecessary fo r t h e co m p let io n o f t h e st u d y. We are also g rat efu l t o t h e Co n selh o Na cio n a l d e Desen vo lvim en t o Cien t ífico e Tecn ológico (CNPq , Nat io n al Co u n cil for Scien t ific an d Techn ological Developm en t ) for p ro vid in g fin an cial su p p o rt .
REFERENCES
1 . Co n d e MB, So u z a GM, Krit ski AL. Tu b e rcu lo se se m m ed o . Ed it o ra At h en eu . 1 ª ed . Sã o Pa u lo : 2 0 0 2 . 2 . Yo u n g DB. Blu e p rin t Fo r Th e Wh it e Pla g u e . Na t u re
1 9 9 8 ; 3 9 3 .
3 . Sep ko wit z KA, Ra ffa lli J , Riley L, Kieh n TE, Arm st ro n g D. Tu b ercu lo sis in t h e AIDS era . Clin Micro b io l 1 9 9 5 ; 8 : 1 8 0 - 9 9 .
4 . Bra sil. Min ist é rio d a Sa ú d e . Ma n u a l t é cn ico p a ra o co n t ro le d a Tu b ercu lo se: Ca d ern o s d e a t en çã o b á sica . Bra sília ; 2 0 0 2 .
5 . SES- RS. Secret a ria d a Sa ú d e d o Est a d o d o Rio Gra n d e d o Su l. Pro g ra m a d e Co n t ro le d a Tu b ercu lo se; 2 0 0 3 . 6 . Sm a ll PM, Ho p ewell PC, Sin g h SP, Pa z A, Pa rso n n et J ,
Ru st o n DC, et a l. Th e ep id em io lo g y o f t u b ercu lo sis in Sa n F r a n c i s c o ; A p o p u l a t i o n - b a s e d s t u d y u s i n g co n ve n t io n a l a n d m o le cu la r m e t h o d s. N En g l J Me d 1 9 9 4 ; 3 3 0 : 1 7 0 3 - 9 .
7 . Ten over FC, Arb eit RD, Go erin g RV. Ho w t o select a n d i n t e r p r e t m o l e c u l a r s t r a i n t y p i n g m e t h o d s f o r ep id em io lo g ica l st u d ies o f b a ct eria l in fect io n s: a review f o r h e a lt h ca re e p id e m io lo g ist s. In f e ct Co n t ro l Ho sp Ep id e m io l 1 9 9 7 ; 1 8 : 4 2 6 - 3 9 .
TABLE 2
Comparison w ith results from other molecular epidemiolog ical studies conducted in Brazil
Co p iesOrig in o f o f Clu st er
iso la t es Lo ca le Perio d n IS6110 p at t ern HIV st at u s Referen ce
Po rt o Aleg re, Ou t p a t ien t 1 9 9 9 - 2 0 0 0 55 2 - 1 8 2 9 .1 % 7 p o sit ive Presen t e Est u do
RS clin ic 2 6 n eg a t ive
2 2 u n kn o wn
RJ Ho sp it a l 1 9 9 3 - 1 9 9 4 19 6 - 1 5 5 3 % 1 5 p o sit ive Iven s- d e- Araú jo et al.(2 7 ) 4 n eg a t ive
RJ Ho sp it a l 1 9 9 0 - 1 9 9 4 32 ——— 2 5 % ——— Lo u ren ço et a l.(2 8 ) Bau ru , SP Ou t p a t ien t 1 9 9 6 - 1 9 9 9 57 3 - 1 7 2 6 .3 % 2 5 p o sit ive Ba p t ist a et a l.(2 9 )
clin ic 1 9 n eg a t ive
1 3 u n kn o wn
Ca m p in a s, SP Ho sp it a l 1 9 9 6 - 1 9 9 9 78 6 - 2 1 2 2 .3 % ——— Ca lu sn i et a l.(1 6 ) RJ Ho sp it a l 1 9 9 3 - 1 9 9 4 1 2 0 3 - 1 8 1 9 % 3 0 p o sit ive Fan d in h o et al. (1 5 )
6 7 n eg a t ive 2 3 u n kn o wn
SP Ou t p a t ien t 1 9 9 5 - 1 9 9 7 2 9 3 2 - 2 0 3 3 % 151 p o sit ive Ferra zo li et a l.(8 )clin ic 1 4 2 n eg a t ive
1 9 6 9 ; 41 : 2 1 - 4 3 .
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