R e v ist a d a S o c ie d a d e B r as ile ir a d e M e d ic in a T r o p ic al 2 8 ( 3 ) : 1 69-177, ju l- set 1995.
A RTIG O S
TH E HEM O RRHA G IC SYN D RO M E O F LEPTO SPIRO SIS: A N EXPERIM EN TA L STUD Y IN G UIN EA PIGS
Jo ão Jo s é Pe re ira d a Silva, B ern ard in o A lves d e Souza N etto, W alter
Iilem b au m , M aria Erb en e A m orim M elo A lvim an d
A lb anita V iana d e O liveira
T he h e m o r r h a g ic s y n d r o m e o f le p t o s p iro s is w as s t u d ie d in g u i n e a p ig s . T he st u d y c o r r e lat e s h e m at o lo g ic al, h is t o p at h o lo g ic al a n d im m u n o h is t o c h e m ic al alt e r at io n s in six t y a n im a ls in o c u la t e d by t h e in t r ap e r it o n e al r o u t e w it h l m l o f t h e c u lt u r e o f v iru len t s t r ain o f Leptospira interrogans s e r o v ar copenhageni. Leptospirae an t ig e n s w e r e d e t e c t e d b y im m u n o p e r o x id as e , c h ie fly in liv er, k id n e y a n d h e a r t m u s c le c ap illar ie s . P o s s ib le p a t h o g e n i c m e c h an is m s r e s p o n s ib le f o r h e m o r r h a g ic s y n d r o m e a r e d is c u s s e d w it h e m p h as is o n t o x ic a n d a n o x i c at t ac k s c au s in g d a m a g e t o e n d o t h e lia, p lat e le t d e p le t io n a n d a l t e r a t io n s t o h e m o s t a s ia r at e s : p r o t h r o m b i n t im e [FT], p a r t i a l t h r o m b o p las t in t im e [ P IT ] a n d fib r i n o g e n c o n c e n t r at io n s . Tide c li n ic al- la b o r a t o iy p i c t u r e is c o m p a t i b l e w it h t h e h i s t o p a t h o lo g ic a l o b s e r v at io n o f d i s s e m i n a t e d
i n t r av as c u lar c o ag u lat io n [D 1C] in m o s t o f t h e g u in e a p ig s f r o m d a y 4 o f in fe c t io n . K e y - w o r d s : L e p t o s p ir o s is . H e m o r r h a g ic s y n d r o m e . P la t e le t d e p le t io n . Im m u n o p e r o x id as e . D is s e m in at e d in t r av as c u lar c o ag u lat io n . D IC .
This serial study aimed to establish a dynamic physio patho lo gical pro file o f the hem o rrhagic p heno m ena that o ccur in lep to spiro sis. The co nstructio n o f such a pro file w as made feasible by the ado ptio n o f an exp erimental mod el that co uld adequately repro duce the morbidity fo und in humans.
Our experimental appro ach w as justified by the present lack o f co nclusive information in the literature, most o f w hich is taken up w ith results o btained from bio psies and necropsies. Su c h fin d in g s d o n o t alw ay s p ro v id e an ad equate v iew o f physio p atho lo gical pheno mena8. Notwithstanding the pioneering exp erimental investigations by A rean2 and A rean & Henry3, and more recently by Higgins & Cousineau16 and Alves et al1, all o f w hich are based o n the o bservatio n o f animals infected w ith a virulent strain o f lepstospira, research o n the hemo rrhagic syndrome has generally b een limited-, in its appro ach; hence our d ecisio n to carry out a study involving a more
co m p lete ev aluatio n o f the o rganic
L ab o rató rio de- Pesq u isa e m A n ato m ia Pato l ó g ica d o H ospital Evandro C hagas d o In stitu to O sw aldo C ruz da Fu n d ação O sw ald o C ruz , Rio d e Jan eiro , RJ, Brasil.
A d d re s s to: 'Prof. Jo ão Jo s é Pereira d a Silva. S erv iço de D o en ças In f eccio sas e Parasitárias d o H osp ital U niv ersitário A n to n io Pe d ro , U n iv ers id ad e Fed eral Flu m in en se. R. M arq uês d e Paran á 3 0 3 ,2 4 0 3 0 - 2 1 0 , N iteró i, RJ, Brasil. R eceb id o p ara p u b licação em 3 0 / 0 8 / 9 4 .
compo nents that are most frequently damaged in leptospirosis. Our aim w as to answ er certain key questio ns relating to the human disease, w hich co ntinues to be characterized by high mortality rates due to the frequent o ccurrence o f hemorrhagic diathesis12 23 24 25.
MATERIALS AND M ETHODS
The study co v ered hem ato lo gical,
histopathological and immuno -histochemical alteratio ns related to the hem o rrhagic syndrome in Guinea pigs infected w ith a virulent strain o f L e p t o s p ir a in t e r r o g an s serovar co penhageni. Seventy animals, divided into seven groups o f ten animals each, w ere killed after blo o d co llectio n. One group (gro up 0 - control) w as intra-peritoneally ino culated with lm l o f sterile Fletcher medium, w hile the six o ther groups (gro ups 1 - 6 ) w ere inoculated w ith lm l o f leptospira culture, i.e. Fletcher medium co ntaining betw een 107 and 108 microorganisms per ml and sequentially killed from the first (gro up 1) to the sixth day (gro up 6) after inoculation.
Silva ]JP, So uz a Netto BA, Lilem baum W, A lv im M EAM , O liveira AV. The hem o rrhagic sy ndro m e o f leptospirosis: an expei'imental study in G uinea pigs. Revista da So ciedade Brasileira de M edicina Tro pical 2 8 :1 6 9 - 1 7 7 , jul- set, 1995.
In o u r h isto p atho lo g ical analy sis the fo llo w ing techniq ues w ere used : hem ato xy lin- e o s in , p h o s p h o tu n g s tic a l h e m a to x y lin , M asso n’s trichro m e, G o m o ri’s reticulin, PAS, Jo n e s’ silv er and W arthin-Starry im p regnatio n stain (M anual o f H isto lo g ic and Sp ecial Staining Techniq u es, 2nd Ed. 1964 - M cGraw Hill Bo o k Co m p any, Inc. NY).
Fo r the d etectio n o f L. ict ero haem o rrhagiae
antig en using the av id in-bio tin-p ero xid ase m etho d , w e em p lo y ed the Vectastain A BC Pero xid ase (Rab b it Ig G ) kit fro m V ecto r Labo rato ries Inc. - USA (ref. n2 PK-4001).
The statistical sig nificance o f o ur results w as assessed by “t” testing at p< 0.05.
RESULTS
G ro u p s 1 an d 2 d isp lay e d slig h t alteratio n s in their h em ato lo g ical and anato m o p atho lo g ical p aram eters, m o st no tably w ith an increase in p ro thro m bin tim e and w ith cap illary co n g estio n in lung , heart and gastro cnem ius m uscle.
In g ro up 3, PT co ntinued to b e g reater than in the co ntro l g ro up , and w as asso ciated w ith an increase in PTT and a red uctio n in the p latelet co u nt and fibrino g en co ncentratio n. Cap illary co ng estio n w as accentuated in lung, heart, liver, kid ney and gastro cnem ius m uscle, and hem o rrhag e fo ci w ere also fo und , chiefly in lung, liv er and g astro cnem ius m uscle. Intrav ascular co ag u latio n fo ci w ere d etected in liver, g astro cnem ius m uscle and kid ney, and scattered fo ci o f co ag u latio n necro sis w ere o bserv ed in liver. Im m uno p ero xid ase assays w ere slig htly p o sitiv e in liver, heart, kid ney (v ascu lar lu m en and interstitium ) and g astro cnem ius m uscle, w ith p arasite antig en ap p earing m ainly in a fibrillar fo rm.
In g ro up 4, PT and PTT v alues rem ained hig h, w ith m arked p latelet d ep letio n but no rm aliz ed fib rino g en co ncentratio ns. H isto p atho lo g ical alteratio ns w ere aggravated b y an in tensificatio n o f the co ng estio n, hem o rrhag e and intrav ascular co ag u latio n alread y o b serv ed in heart, lung, liver, kid ney and g astro cnem iu s m u scle. C o ag ulatio n n ecro sis ap p eared to b e m o re intense, sp read ing o u t in a m u ltifo cal fo rm . Im m uno p ero xid ase assay s co ntinued to b e p o sitiv e in heart, liver, kid ney and g astro cnem ius m uscle.
In hem ato lo g ical term s, gro up 5 w as sim ilar to g ro up 4, w ith m arked p latelet d ep letio n, increased PT and PTT, and red uced fibrino g en co ncentratio ns. The histo p atho lo g ical p icture, m eanw hile, w as further ag g rav ated b y increased co n g estio n , hem o rrhag e and intrav ascular co ag ulatio n in all the tissues exam ined . C o ag ulatio n n ecro sis also increased , ap p earing in a m ultifo cal fo rm in liver. Im m u no p ero xid ase results w ere intensified in liver, kid ney , heart and gastro cnem ius m uscle, w ith p arasite antig en ap p earing in filam entary and granular forms.
In gro up 6, the PT, PTT and fibrino g en v alues returned to no rm al, w hile the p latelet co unt rem ained lo w . The histo p atho lo g ical p ro file w as m o d ified , w ith a red uctio n in intrav ascular co ag u latio n; co n g estio n and hem o rrhage, ho w ev er, rem ained intense in all the tissues exam ined . The im m uno p ero xid ase assays w ere intensely p o sitiv e, w ith the sam e ap p earance as in g ro up 5.
Silva J/P, So uz a Netto BA, Lilem baum W, Alvitn M EAM , O liveira AV. The hem o rrhagic sy ndro m e o f leptospirosis: an experim ental study in G uinea pigs. Revista da So ciedade Brasileira de M edicina Tro pical 2 8 :1 6 9 - 1 7 7 , jul- set, 1995.
D ISC U SSIO N
O ur system atic o bserv atio n o f g uinea pigs sequentialy killed fro m the first to the sixth day after ino culatio n w ith a v irulent strain o f lep to sp ira, p ro v id ed us w ith a clear p icture o f the kinetics o f this d isease.
O n the first tw o d ays (g ro up s 1 and 2), the anim als did no t exhib it a sig nificant clinical p icture, a find ing that w as co m p atib le w ith o ur o bserv atio ns o f o nly slight v isceral im p airm ent in the fo rm o f v ascular co ng estio n, m ainly in lung, heart and gastro cnem ius m uscle.
S ilv a JJP , S o u z a N et t o BA , L ile m b au m W, A lv itn M EA M , O liv eira A V . T he h e m o r r h a g ic s y n d r o m e o f lep t o s p iro s is: a n e x p e r im e n t al s t u d y in G u in e a p ig s . R e v ist a d a S o c ie d a d e B r as ile ir a d e M e d ic in a T r o p ic al 2 8 :1 6 9 - 1 7 7 , ju l- s e t , 1 9 9 5 .
interaction w ith cellular surface proteins prior to attack.
The third day after ino culatio n (group 3) represents a dividing line betw een the first stage (invo lv ing mild and relatively no n sp ecific manifestatio ns) and the seco nd stage
(characterized by impo rtant clinical,
pathological and hematolo gical alterations). The p latelet co unt beg ins to d ecline, acco m p an ied by an in c rease in PT and PTT and a reductio n in fibrinogen levels. A lthough clinically w ithout hemorrhage, in histo patho lo gical tests the animals exhibited accentuated vascular congestio n, extravasated red blo o d cells and slight depo sits o f fibrin in capillary blo o d vessels in liver, kidney and muscle.
A m o n g th e s e h i s to p a th o l o g i c a l
p heno m ena, co ng estio n w as the most
constant, mainly in liver, kidney and lung. We believe that this co ngestio n preced es and is asso ciated w ith the vascular damage that leads to extravasation o f red blo o d cells to the interstitium15. Other authors, such as Higgins and Cousineau16 and more recently Venugopal
and Ratnam 30, hav e em p hasized this
histo patho logical pattern, linking it to the hemorrhagic diathesis that is characteristic o f serious forms o f human lep to spiro sis10 23.
A no ther sig nificant find ing w as the detectio n (via immuno peroxidase testing) o f lep to spira antigens w ith a filamentary o r granular aspect in liver sinusoid s, kidney interstitium and tubules, heart tissue and gastrocnemius muscle. This finding o pens new
p ersp ectiv es fo r und erstand ing the
physio patho genic pathw ays o f the disease, an area that is still far from being clarified1611. The p red o minance o f these antigens in capillaries reinfo rces the id ea that they play an important
ro le in the causatio n o f hemo rrhagic
pheno mena.
In summary, the platelet depletion, the red uced fibrino gen lev els and the increases in PT and PTT, all o f w hich are asso ciated w ith a clinical picture co mpatible w ith obstruction o f m icro circulatio n28, lend w eight to the hypo thesis that DIC is pro bably respo nsible fo r
the hem o rrhagic p heno m ena w hich
increasingly o ccur as the d isease runs its course.
O n the o ther hand , Laing20 d escribes acu te p latelet d ep letio n asso ciated w ith
m icro angio p athy and slight alteratio ns to co agulatio n facto rs, a co nd itio n that he id entifies as thro m bo tic thro m bo cyto p enic purpura.
The essential fact is that all these pheno mena lead to thrombo cyto penia, w hich
appears to b e a co nstant feature o f
leptospirosis and w hich plays a central ro le in the causation o f hemorrhagic syndrome141621.
We have cho sen to fo cus on this syndrome no t o nly because o f its intensity and co nstancy in the human d isease, but principally because o f the po ssibilities o f its reversal through therapy.
From an anato mo patho lo gical po int o f view , a significant finding in our study w as the o ccurrence o f coagulatio n necrosis, mainly o f liver tissue. In our opinion, the intensification o f this necro sis from the 3rd day (gro up 3) co nforms w ith the to xic-ano xic picture that is also characteristic o f serio us forms o f the human disease24 26. The co agulatio n necrosis o bserved in liver (w hich is different from the d escrip tio ns in the literature o f iso lated necro sis) destroys tissue to a variable extent, keeping a clo se relationship w ith the presence o f fibrin thrombi, and o f fibrillar and granular- formed parasite antigen.
This type o f necrosis, w hich has no t been previo usly o bserv ed in lep to sp iro sis, exacerbates the endothelial damage, possibly playing a seco nd ary ro le in relation to the direct actio n o f the parasite o r its to xic products. The latter, besid es causing vascular obstruction, co uld trigger the DIC pro cess9 17 22.
The animals’ morbidity on the 4th day (group 4 ) w as patently mo re ad vanced than in previous groups, w ith more serio us clinical pathological findings, mainly in the form o f increased hemorrhagic p heno m ena in lung,
kid ney and gastro cnem ius muscle,
accompanied by intravascular coagulatio n in lung, liver, kid ney and gastrocnemius muscle.
Co agulatio n necro sis w as intensified ,
Silv a J/P , S o u z a N et to BA , L ile m b au m W, A lv im M EA M , O liv e ir a A V . T he h e m o r r h a g ic s y n d r o m e o f le p t o s p iro s is; a n e x p e r im e n t a l s t u d y in G u in e a p ig s . R e v ist a d a S o c ie d a d e B r as ile ir a d e M e d ic in a T r o p ic al 2 8 :1 6 9 - 1 7 7 , ju l- s e t , 1 9 9 5 .
D esp ite the ap p arently p arad o xical behavio r o f the fibrinogen, the o verall pattern o f alterations strengthens the hypothesis o f DIG. The increase in, and subsequent normalization o f fibrino gen levels may be due to a co mpensating mechanism that allow s synthesis o f fibrinogen, thereby offsetting its destruction; alternatively, it may be due to the mobilizatio n o f a tissue p o o l Lu respo nse to the severe systemic attacks27.
The 5th and 6th days (Gro ups 5 and 6) w ere characterized by the acute o nset o f the -d isease, w ith the guinea pigs displaying serious anato mo patho lo gical symptoms, such as tubular necro sis, intersticial nep hritis, miocarditis, miositis, co agulation necro sis and intense hemorrhage in virtually all organs and tissues. A no table finding w as the aggravation o f co agulatio n necro sis in liver, w ith multifocal manifestations in abo ut 80% o f the animals. The o v erall clinical p icture co uld be
attributable to an o bstructio n o f
microcirculation, w ith all the accompanying symptoms o bserv ed in W eil’s syndrome10 2425.
In gro up 5, p latelet d ep letio n w as accentuated , acco m p anied by increases in PT and PTT and red uctions in fibrinogen levels, a pattern fully co mpatible w ith DIC. O n the sixth day (gro up 6), the platelet depletio n persisted, but PT and PTT returned to normal, and fibrinogen co ncentratio ns increased. This pattern is co nsistent w ith the reduction in histo patho lo gical evid ence o f intravascular coagulation.
Our analysis o f this co m p lex arra_ / lesio ns reveals a clinical picture in w hich
hem o rrhagic p heno m ena no t o nly
predo minate, but also seem to be largely respo nsible fo r the o nset o f acute systemic impairment. A ttacks o n endo thelia (probably o f a to xic-ano xic nature) are pro vo ked by the parasite o r its products, acting either directly o n ho st cells o r indirectly via immunological pathw ays. In o ur view , any o f these alternative mechanisms co uld be respo nsible fo r the triggering o f the DIC pro cess, w ho se central ro le has been clearly demo nstrated in this study.
RESUMO
O s a u t o r e s e s t u d am a s ín d r o m e h e m o r r á g ic a d a le p t o s p ir o s e e m c o b a i o s in o c u la d o s c o m am o s t r a v ir u le n t a d e Lep to sp ira interro g an s s o r o v a r
c o p e n h ag e n i. S ã o a b o r d a d o s a s p e c t o s h e m a t o ló g ic o s , h ís t o p a t o lô g ic o s e i m u n o -h is t o q u ím ic o s . A u m e n t o d o t e m p o d e p r o t r o m b i n a ( P T ) e d o t e m p o p a r c i a l t r o m b o p las t in a ( P T T ) , b e m c o m o r e d u ç ã o d o n ú m e r o d e p l a q u e t a s e d o f i b r i n o g ê n i o a c o m p a n h a r a m - s e d e c o n g e s t ã o v as c u lar , h e m o r r ag ias e c o a g u l a ç ã o in t r av as c u lar n o fíg a d o , rim , c o r a ç ão , m ú s c u lo e s q u e lé t ic o e p u lm ã o . O an t íg e t z o d e Leptospira f o i d e t e c t a d o p r i n c i p a l m e n t e e m t e c id o h e p á t ic o , r e n a l e c a r d í a c o . S ão d is c u t id o s p o s s ív e is m e c a n i s m o s p a t o g ê n i c o s r e s p o n s á v e i s p e l a s í n d r o m e h e m o r r á g i c a , c o m ê n f a s e n a a g r e s s ã o t ó x ic o -a n ó x i c -a d o e n d o t ê lio v -as c u l-ar , q u e p r o p i c i -a r i -a -a i n s t a l a ç ã o d e c o a g u l a ç ã o i n t r a v a s c u l a r d i s s e m i n a d a ( C IV D ) o b s e r v ad a , s o b r e t u d o , n o s c o b a io s d o s g r u p o s 4 , 5 e 6.
P a l a v r a s - c h a v e s : L e p t o s p i r o s e , S í n d r o m e h e m o r r á g i c a , P l a q u e t o p e n i a . C o a g u l a ç ã o i n t r a v a s c u l a r d i s s e m i n a d a, C I V D , Im u n o p e r o x id as e .
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