A M O D IF IC A T IO N O F T H E IN D IR E C T FL U O R E S C E N C E
T E S T (IF T ) FO R P A R A C O C C ID IO ID O M Y C O S IS
W a lt e r B. Pet an a* a n d Bo d o W a n k e * *
A modification o f the indirect fluorescence test (IF T ) for serological diagnosis o f
Paracoccidioidomycosis is described in which formolized budding forms (yeast cells) o f
the fungus
P a ra co ccidio id e s brasilie n sisare used as antigen. A further modification
introduced is a less eiaborate technical procedure o f the test without lowering the
sensitivity o f the reaction. The test may be considered as adequateiy accurate and easy to
perform in any laboratory with immunofluorescence facilities.
IN T R O D U C T IO N
P a r a c o c c i d i o i d o m y c o s i s is a s e r i o u s c h r o n i c a n d P r o g r e ssiv e d ise a se c a u s e d b y t h e f u n g u s
Paracoccidioides brasiliensis
a n d m a i n l y a t t a c k s t h e lu n g s, m u c o s a o f t h e m o u t h a n d n o s e w i t hf r e q u e n t sp r e a d t o t h e l y m p h n o d e s , a d r e n a l g la n d s a n d o t h e r v isce r a .
T h o u g h th e in fe c tio n is re la tiv e ly easy to diagpose b y h is to lo g ic a l s c ru tin y o f b io p sie d tissues, and in sta in e d p re p a ra tio n s fr o m squashed tissues and o f s p u tu m , c e rta in im m u - no lo gica l tests are a va ila b le to a id th e diagnosis (c o m p le m e n t f ix a tio n , e le c tro im m u n o p h o re s is , im m u n o flu o re s c e n c e , p re c ip itin and in tra d e r- mal te s t). T h e c o m p a ra tiv e s e n s itiv ity o f th e available se ro lo g ica l re a c tio n s f o r p a ra c o c c id io i d o m yco sis has been review ed in d e ta il b y Fava N e tto 1 .
F ra n co
et a í1
describe an in d ir e c t flu o re s c e n ce te s t using as a n tig e n b ro k e n -u p c e llu la r w a lls o f yeast fo rm s o fP. brasiliensis,
w h ic h re m a in in th e s e d im e n t a fte r p re p a ra tio n o f th e p o ly - saccharide a n tig e n f o r th e c o m p le m e n t fix a tio n test. A c c o rd in g to F ra n c oe t al.2
th e results o b ta in e d w it h th e im m u n o flu o re s c e n c e te s tc o m p a re fa v o u ra b ly w it h those o b ta in e d b y the c o m p le m e n t fix a tio n test.
P atients and suspects f o r th e disease are fr e q u e n tly seen in o u r c lin ic and those diagnos- ed f o r th e in fe c tio n are tre a te d in o u r h o s p ita l. T o e x te n d th e scope o f o u r im m u n o flu o re s c e n ce u n it to such cases, a m o d ific a tio n o f th e in d ir e c t flu o re s c e n c e te s t f o r p a ra c o c c id io id o m ycosis was in tro d u c e d . T h e m o d ific a tio n was a im e d f o r a s im p le r w a y in p re p a rin g th e a n tig e n , and a less e ia b o ra te te c h n ic a l p ro c e d u re o f th e te s t, w it h o u t a ffe c tin g th e s e n s itiv ity o f th e re a c tio n .
M A T E R IA L S A N D M E T H O D S
Reagents re q u ire d :
a) p h o sp h a te b u ffe re d saline (PBS) p H 7 .2 b) 2 p e r c e n t. fo r m a lin in p h o s p h a te -b u ffe re d
salin e p H 7 .2
c) 10 per c e n t g ly c e rin e in p h o s p h a te -b u ffe re d salin e p H 8 .0
d) th e a n tig e n
e) a n ti-h u m a n im m u n o g lo b u lin -flu o re s c e in con- ju g a te
f) Evans b lu e 1 per ce n t. aquaeous s o lu tio n
* V is itin g p ro fe s s o r in m e d ic a i p a ra s ito lo g y , D e p a r tm e n t o f P re v e n tiv e M e d ic in e , F a c u lty o f M e d ic in e , Fe d e ra l U n iv e r s ity o f R io d e J a n e iro .
* * A ssista n t p ro fe s s o r in c h arg e o f m y c o lo g y la b o r a to r y , D e p a r t m e n t o f P re v e n tiv e M e d ic in e , F a c u lty o f M e d ic in e , F e d e ra l U n iv e r s ity o f R io d e J a n e iro .
A dd re ss f o r re p r in ts : D r. B o d o W a n k e , D e p a rt a m e n t o d e M e d ic in a P re v e n tiv a , C a ix a P ostal 1 8 5 9 , P avilhão C arlo s C hagas, R u a L a u ra d e A r a ú jo , 3 6 , R io d e J a n e iro , Brasil.
3 2 4 Rev. Soc. Bras. M e d . T ro p . V o l. X N ? 6
Fig. 1 a n d 2. — P h o to m ic r o g ra p h s h o w in g th e a p p e a ra n c e o f y e a s t fo rm s o f P. b r a s i l i e n s i s a f t e r e x p o s u re t o sera fr o m p a tie n ts w it h a c tiv e f o r m o f P a ra c o c c id io id o m y c o s is . N o t e th e w e ll d e fin e d flu o re s c e n c e a r o u n d
N O V - D E Z /7 6 R ev. Soc. Bras. M ed . T ro p . 3 2 5
1 — T h e a n tig e n : th is is p re p a red fr o m 3 0 days o ld c u ltu re s o f
P. brasiliensis
g ro w n on n u tr ie n t agar a t 3 7 ° C . T h e c u ltu re s are o v e rla y - ed w it h 2 p e r c e n tf o r m a lin in PBS, to c o ve r th e f u n g u s c o l o n i e s , a n d th e t u b e s are le f t o n th e bench f o r 2 4 h o u rs to k ill th e b u d d in g fo rm s (yeast celts). A f t e r 2 4 h o u rs th e m o u ld is scraped c a re fu lly fr o m th e agar surface, th e suspension is tra n s fe rre d in to a s u ita b le glass Co n t a i n e r i n t o w h ic h som e glass beads have been added, and th e e m u ls io n is a g ita te d b y hand o r in a m e ch a n ica l shaker f o r 10 m in u te s to sepparate th e ye a st celts. T h e e m u ls io n is filte r e d tw ic e th ro u g h six layers o f surgical gauze and th e f ilt r a t e is w ashed fiv e tim e s w ith PBS b y s p in n in g in a c e n trifu g e a t 3 ,0 0 0 rp m , fo r th re e m in u te s b e tw een each w ash. A ft e r th e last spin th e s u p e rn a ta n t flu id is d isca rd e d and the s e d im e n t fr o m each o f th e tu b e s is p o o le d . A d ro p o f th e c o n c e n tra te is e x a m in e d u n d e r a co ve rslip w it h th e m ic ro s c o p e f o r d e n s ity o f th e yeast ce lls a n d th e presence o f p a rtic le s o f i m p u r i t y . T h e a n tig e n c o n c e n tra te is tb e n d i l u t -ed w it h PBS to give b e tw e e n 3 0 — 5 0 ye a st cells per m ic ro s c o p e fie ld (x 4 5 o b je c tiv e and x 10 eyepiece) a n d is s to re d in a re frig e ra to r a t 4 ° C . D u rin g th e storage th e yeast cells te n d to fo r m sm all c lu m p s ; these can be b ro k e n -u p b y a d d in g a fe w glass beads in to th e b o ttle and a g ita tin g fo r tw o m in u te s , a fte r w h ic h th e cells are s u ffic ie n tly dispersed and re a d y f o r d is tr ib u tio n on th e te s t slide.2 — T h e im m u n o g lo b u lin -flu o re s c e in c o n ju - gate: a n ti-h u m a n IgG was used o b ta in e d fr o m W ellcom e D ia g n o s tic Reagents, E ngla nd. P rio r use th e ly o p h ilis e d c o n ju g a te was d ilu te d w ith PBC to 1 :4 0 ; as c o u n te rs ta in 1 0
/
j mg o f Evans blue was added t o each one m l o f th e d ilu te d conjugate.3 — T h e sera: in th e te s t tr ia l 4 2 sera w ere used o b ta in e d fr o m 2 6 p a tie n ts w it h va rio u s stages o f th e in fe c tio n . S om e o f th e sera w ere ta ke n b e fo re th e tr e a tm e n t, in th e course o f th e tr e a tm e n t and o n c o m p le tio n o f th e th e ra p y . A n equal n u m b e r o f sera fr o m n o rm a l persons w ere used as Co n t r o l s . F o r th e te s t th e se r a w ere d ilu te d to a c o n c e n tra tio n o f 1 :1 0 ; 1 :2 0 ; 1:40; 1 :8 0 ; and 1 :1 6 0 . Sera w h ic h w ere re a ctive in th e last d ilu t io n w ere d ilu te d f u r th e r t o th e end titr e .
4 — T h e te s t: a d ro p o f a n tig e n is p laced in th e c e n tre o f each o f 10 A R A L D IT E rings m a rke d on a m ic ro s c o p e slid e and th e slides are d rie d on a h o t p la te a t 4 5 ° C fo r m in u te s . T h is tr e a tm e n t fix e s th e yeast cells s u ff ic ie n tly to th e glass su rfa ce w it h o u t th e d anger o f th e m
b e in g re m o ve d d u rin g w ashing in PBS. O n each o f fiv e a n tig e n fie ld s a d ro p o f th e d ilu te d serum is p laced, th e f ir s t fie ld h o ld in g th e lo w e s t and th e last th e h ig h e st serum d ilu tio n , and th e slides are in c u b a te d in a h u m id cham - ber a t ro o m te m p e ra tu re f o r 4 5 m in u te s . A fte r in c u b a tio n th e sera are rem oved fr o m th e slides w it h a gentle je t o f PBS and are w ashed in PBS f o r fiv e m in u te s w it h a g e n tle a g ita tio n on a ro ta tin g ta b le . A f t e r w a sh in g each slid e is d ip p e d in to d is tille d w a te r to rem ove th e PBS a n d th e slides are d rie d in an u p r ig h t p o s itio n w ith th e h e lp o f an h a ir d ry e r. A ft e r d ry in g each o f th e a n tig e n fie ld s is covered w it h a d ro p o f im m u n o g lo b u lin -c o n ju g a te and th e slides are in c u b a te d f o r fu r th e r 4 5 m in u te s (h u m id ch a m b e r, ro o m te m p e ra tu re ). A t th e end o f th e second in c u b a tio n p e rio d th e im m u n o g lo b u lin is rem oved w it h a je t o f PBS and th e slides are w ashed f o r fiv e m in u te s in PBS ( w ith s lig h t a g ita tio n ). A f t e r th e w ash each slid e is shaken v ig o ro u s ly to rem ove excess PBS and th e m o is tu re u n d e m e a th th e slides is
wiped
sw ay w ith h y g ie n ic tissue. T h e te s t fie ld s are sand- w ic h e d b e tw e e n 10 per c e n t.g ly c e rin e in PBS (p H 8 .0 ) and a c o v e rs lip re a d y fo r e x a m in a tio n . T he p re p a ra tio n s w ere e x a m in e d b y in c id e n t lig h t ilu m in a tio n w it h th e V IC K E R S PHO- T O P L A N m ic ro s c o p e u sin g 2 0 0 w a tt hig h pressure m e rc u ry v a p o u r la m p and BG 12 as e x c ite r f ilt e r w it h O G 1 as a b a rrie r filte r .R E S U L T S
W ith re a ctive sera th e re was a w e ll pro- n o u n c e d degree o f greenish flu o re sce n ce m a in ly a ro u n d th e c e ll w a ll. S om e o f th e sm a lle r cells show ed a d is tin c t o ve ra ll flu o re sce n ce w h ils t in th e larger cells th e flu o re sce n ce was c o n fin e d to th e ce ll w a ll o n ly , w it h a redd ish -g re y sta ing insid e th e ce ll (F ig . 1 and 2 ). W ith th e n o n -re a c tiv e (c o n tro l sera) th e yeast cells appeared re d d ish -g re y w it h a fa in t w h itis h o u tlin e a ro u n d th e ce ll w a ll (F ig . 3 ). T h e re was n o d i f f i c u l t y in d iffe r e n tia tin g th e re active sam ples fr o m th e n o n -re a ctive .
D IS C U S S IO N
3 2 6 Rev. Soc. Bras. M ed . T ro p . V o l. X N ? 6
*
Fig. 3 — A p p e a ra n c e o t th e y e a s t cells a f t e r e x p o s u re t o sera f r o m n o rm a l person s. T h e ce lls a re o n ly d im ly o u tlln e d w it h a f a in t w h itis h lin e a r o u n d th e ce ll w a ll.
te s t was negative. O n one o f these p a tie n ts th e te s t was re peated som e m o n th s la te r and was fo u n d to be p o s itiv e in a tit r e o f 1 :10. J u d g in g fr o m th e c lin ic a i c o n d itio n o f th is p a tie n t th e p o sitive te s t suggested a relapse o f th e in fe c tio n th o u g h th is was n o t possible t o c o n fir m b y a m y c o lo g ic a l s c ru tin y .
I t is w e ll k n o w n th a t tr e a tm e n t f o r Para c o c c id io id o m y c o s is m a y e x te n d o ve r a lo n g p e rio d and th a t relapses a fte r a p p a re n tly suc- cessful th e ra p y q u ite o fte n . A se ro lo g ica l te s t th e re fo re is an u seful aid in th e assessment o f a c tiv ity o f th e in fe c tio n and on th e e ffe c ts o f th e ra p y . T h e m o d ifie d ve rsio n o f th e re a c tio n
described here appears to be a d e q u a te ly sensiti- ve, and re la tiv e ly s im p le to p e rfo rm , in a n y la b o ra to ry w it h th e f a c ilit y f o r im m u n o flu o rescence.
A C K N O W L E D G E M E N T S
We are g ra te íü l to P ro fe sso r J. R odrig u e s C oura, M .D ., P rofessor o f T ro p ic a l M e d ic in e , D ire c to r o f D e p a rtm e n t o f P re ve n tive M e d icin e , F a c u lty o f M e d ic in e , Federal U n iv e rs ity o f R io de J a n e iro , f o r his in te re s t and h e lp fu l sugges- tio n s.
RESUMO
Descreve-se uma modificação da reação de imunofluorescencia indireta para o
diagnóstico sorológico da paracoccidioidomicose na qual células formolizadas da fase
leveduriforme do
P a ra cco cid io id e s brasilie n sisforam empregadas como antígeno. Outra
modificação foi a de simplificar a técnica da reação sem prejuízo da sua sensibilidade. O
teste pode ser considerado como suficientemente sensível e de fácil execução em qualquer
laboratório equipado para reações de imunofluorescencia.
R E F E R E N C E S
1. F A V A N E T T O , C. — Im u n o lo g ia da Para c o c c id io id o m ic o s e .
Rev. Inst. Med. trop.
São Paulo.
7 5 :42-53, 1976.2. F R A N C O , M .F . de, F A V A N E T T O , C. & C H A M M A , L.G . — Reação de im u n o flu o
