A large batch wâs plElarcd and utilized aU

Rev.

Inst- Med.

tro|.

São paulo 2g(5) t276-2E3, seteñhrò-outubro. lg87

PREDICTIVE VALUE OF SEROTOGIC IESTS

IN

THE DIAGNOSÍS AND

FOLLOI¡V.UP OF PATIENTS W|TH PARAGOCCIDIOIDOMYCOSIS

_tr)

Luz EIeD¡ CIINO & Á[getÐ BESTA,RO

A

serologic study was undertaken

in

a gtoup

of

4A patients

with

_active pe

lacoscidioidomycosis who were treated

in

the same

foÍm

(ketoconazole). _fo¡

identicaJ. periods

of

time (6 months), and followed-up

for

valious periods post. therapy. The tests employed w€re agar gel iÌnrnunodiffusion (.A,GID) and, com: plement fixation

(FC).

AIso studied were S0 seta

from

patients ,with proven

histoplasmosis and pulmonary aspergilloma, B0 patients _{rwith culturaly} proven

tuberculosis as \¡¡¡ell as 92 specimens from healthy individuals, residents

in

thg endemic a.rea

for

pamcoccidioidornlrcosis,

A

single

lot

of

yea,st filtpàte antigen

wâs used throwhout the stud¡r,

ltre

va,lue of each tÆst was measuted acsording

to

GAIEN a¡rd GAMBINO

6.

Both tests v¡ere highly sensi¿ive, gg aÃd. gtVo rcs. tæctively. Regarding their specificity, the AGID was totally specific S¿hile the

CI'

edúbited 96.6Vo and 97qo specificiþ

in front of

tuberculosis patients and healthy individuals respectively and. 82%

in

comperison

ï'ith

patients with other mJ¡coses. The concElt of predlctive value, that is, Ûre certainty one ha.s

in

accepting a positive test ås diagnostic of paracoccidioidomycosis, _{favored tbe}

,q.GID proædure (70OVo) ov4-r the CF

test.

The lattet coutd

sort

out vnllr- 9t7o ceftainty a patient with patacoccidioidomycosis among a group

of

healthy indi. viduals a,nd willf, 97.5%

in

the sase

of

_{TB patients; when the group}

_in

question

was _(877a). composed by individuals with other deep Í\ycoses, sucTì certBinty was lower

T'he above _{results indicâte that both the} .4,GID and the CF iests fumish results

of

high sonfidenc€; one should

not

telay, howevel,

in

the CF alone as a means

to

establish the specific diagnosis

of

pamcoccidioidomycosis.

KEI¿ WORD,S: _{Predictive values; Serologic tests; pamsoccidoidorlycosis; Cros$}

rea.ctions; Systemic lr¡ycoses,

SUMMABY

ParacoccidÍoidomycosis irs

a

polylno4lhio disease and

its

varying clùúcal manifestations

oft€n delay promp¿ consideration by the trea-ting physical of its real etiology.

In

tffs

con_ text serologic _{tests play an important role in}

diagnosist"2,11,u,17,19,25, _{FΡrthermore and due}

to

the ch¡onic nature

of

paracoccidioidoh.vcG sis, such tests are also cruciâl

in

evatuating

the patient's respo¡ìse

to

treatme¡;it 2.as,2,8p4,

(') s¿udy ges€ntod durj¡s the rslt¡M rr¡temãtlotrsl cou(ql¡IuE on psrêcocclauoidomycosis, _{Ileb¡1¡sry t986, M€de¡ü¡,}

eo-loÍrbiB.

cotpo¡ación _!3¡a lnvestlgactones Blológic5s (cIB), qoslitÀt p¿blo l¡obóÃ urtbe, Mectellin, cotombts, south ¡t¡¡rer¡cs.

INIT,ODUGTION

This being so,

it

worrld bs highly deslrâble

to

attempt standatdization

_of

the

various tests currently available and

of

the

reegents erE,

ployed

in

such tests s,7.

cenetally,

in

o¡der

to

achieve standardlza,

tion,

it

is necessårf,r to r¡ndertake the-statist¡ca¡ anâlysis of the most p¡omising _tests 6,2,10.

_Ihg

CÁNO, f,. E. & RESTREPO, A. - Èedictive value of se¡orogic tests in the diaglþsis and fotlo\¡¡-u! of pEtionts ìrith

pa¡âcoccidìoidomycoE:s. Rev. IDst. M€d. irolr. São P¿u¡o, 29t 276-2A3, 1987.

tests results obtained when studying series of patients ând

of

control

individuals.

We be-lieve

that

various points should be empha,si-z,ed here as follqws:

1.

The group(s) ur¡del study should be

as homogeneous a,s possible and cons-tituted by p€$ons twith simila¡

charâÆ-teristics.

Ideauy, Û1 the case

of

pa. tients

_-

rwho should have been prc. perly diagnosed

_-

tåey should be

acti-ve ât time

of

filst

testing, be treated

with the

same drug and

for

similar periods

of

time, q'ith bleedings ta,lring place

at

identical pe¡iods during and after therapy. Healthy controls should be chosen among persons living in the

known endemic areas

for

the disease urder

study.

Patients

with

ot¡rer

fi¡n-gal

diseases should

also

be

studied

once their diagnosis has been propedy established.

2.

Identica'l tÆsts should be applied to aü specimeru! and the methods sl¡ould be carefully controlled, avoiding modifica,

tions

to

the teshniques during the pe

Iiod

of

study7 6.

3. It

would be highly desirable

to

use a

single

lot ot

antigen

for all

tests but

if

this

is

not feasÍble, one should em-ploy antigens of comparable potencyT'16.

Ihe

above steps twould, eliminat€ va,riables and would peamit

a

more precise statistical evalu&tion.

IÌ¡ring

the pa.st 6 years rwe have had tl¡e

oppofiunity

to

perform serologic tests

to

a series

of

patients and healthy coDtfol groups who present optimal conditions

for

standardi-zation. The results we obtained form the ba. sis

of tllis

publication.

MATEßIAL ÄND METIIODS

Sera obtained

from

4

grol¡ps

of

percons were studied" as follows:

G¡oup

À:

Composed ttJr 43 pa,tients \trith

laboretory proven parasoccidioidornycosls as

described elsewhet e T'n þE,

Group B: Fo'rmed þy 50 patients vrith othe¡ deep mycoses, lwhose diagnosis had allso been

established b¡¡ laboratory þro¿€dures. There

were 33 cases with histoplasmosis and 17 vrith pulmonary aspergilloma.

Group C: Constituted

by

30 patients with culturally-proven tuberculosis

by M.

tubercu.

losls var, hominis.

Group

D:

Composed

by

92 healthy indivi. duâls,

all

resident

in

endemic a¡eas

for

par€"

coccldioido[rj¡cosis and v¡ho had been previou&

ly

skin-tested with paxacoccidioidin and for¡nd

reactive æ.

Concefnturg group A, tftese patients were

exarniried at various periods.

All

43 v/ere tes-ted

at

time

of

diagnosis, when

their

disease was

active.

They v¡¡ere

all

treated vrith

keto-conazole (200 mg,/dey)

for

a period

of

6

mon-ths;

during

this

time, 42 patients wet€ bled

at

montl¡

3 of

thempy and

4l

at

6

months, Upon determination

of

therapy, 31 patients

were available

for

testi.ng 12 months after fi-nishir¡g drug inta,he, 16 patients

after

24

-and

I

after 36

_-

months.

Patients with paxa.coccidioidom¡¡cosis wer€

classified

as

having pulmonary diseas€ with

dissemination

to

othet organs (29, 67 -ïEo, ot

as

presenting involsement lestricted

to

the

lungs (14, 32-5Vo)

-

Concwrcîf, pulmona"rJ¡

tu-berculosis was found

in

5

patients (11.07¿).

Regarding other parameterc, most patients (42) wele males and

âIl

were adults, ages 35-62 yeals.

Sera were collected before breakfast f¡om

all

individuâls (groups A, B, C, D), prese¡ved

\trith 1:10.000 meÉhiolate

in

bu.ffer solution 8,

and kept

in

the æfdgerato¡ at 4'C

until

time

of

testing; this rwas done before 15 days of collection date.

The serologic t€sts evaluated

ï'ere

com. plement fixation (CF), the 50ø¿ hemo$¡sis

mi-crGtest recoûlmeded

by

the CDC Laboratorl¡

Brar¡ch e, and

tfie

âgar gel immunodiffusion techrique (AGID) suggested by the Panameri.

ca'n Health

Organization serolog¡¡ study grou¡x 8.

Both tÆsts employed .a yeast

filtrate

ánti-gen obtained according

to

techniques alleady

descdbed 23, from _{P. b¡adliensls} isoþte _8339.

c'aNo' _{pâEcoccidioidomycos:s.} L' E & RESTREPo _Rev. A - _IDst. Pre'lictivs Yslue of serotoglc tests tn the diasÐosis sn¿r folloey..up _{of lauênts with}

Med. tlop. São p¿uro, 29: }?6-283. I9B?.

For

comparison putf)oses,

_a

_single

_¡ot

_of Histoplasl¡La capsulatum _whole yeast oells a.n

tigen \Ã'as prel)âted ¡5 _{and used in the CI' tests.} CF results werc considered positive _when

the titer

was

>

1:8; IDGA results qrere DG Êitive when band 1 either alone

or

accompa. nied by bands 2 and g, q'as detected 1?,1s.

St¿tistical analS¡sis eacompassed thè deter_ mination

of

sensitivity, especificitr anal pæatic.

tive

velue

as

defined

by

GALEN and. GAM-g1¡9

_{o, and}

_ITOZINN

_and

_{TASCIIDIIÂN r0} (Table

_1).

_{Tlre Student}

_t

_{test was used to de_}

temine

statistical significance.

?ÅBLE 1

D€f¡nitioÌì of teIÃls (+)

ooncePt

30 TB patients GsVo) as well as B of the

nor-mal

controls (3.37¿), _{tumed out positive.}

When the

IL

capsulatum whole yeast cells

antigen was used

in

tfìe C¡' test (Fig. 1), at¡ histopla.smosis sel6r reâcted; a high proportion G7 or 86Eo) of the para,coccidloidomlrcosis ca, ses also tecogn¿ed ttre heteÌo,logous antigen. S€ven serum samples

from

aspergilloma pa.

tients

(

_{þqo), one}

_of

_{the TB patients} (8.32¿) and

I

from _{the healthy control group} (9.8%) werc also positive.

ïhe

sensitivity of botll serologia tests was

determined

in

_trmtients

_witf¡

para4occid.ioido-myoGsis, a¿ every one of the study perioals (Fig.

2).

At

time

of

diagnosis both AGID ar¡d CF proved highlt sensitive (Bg _{ând g0Z¿)}

æsDecti-vely.

lherapy caused.

a

_{certain loss}

oi

sen-sitivity

with

â

trend towards

loweÌ

figures

ráith time

of

thempy.

At

the end

of

J yeâ.ß

alter

cessation

of

treatment, AEE, of.

lhe

ca,-ses could

still

be detected by AGID and 85Zo

by the CF: IIowever, ¿here _{were no statistical}

differences betrween the two tests.

The sf,ecifìcity was determined

in

the

con-trol

gÎoups

(8,

C and

D).

.q,s eai] be seen

in

Fig._3 _{the AGID test was totally specific,} ,ì¡,iû¡ no false positive reactions being recorded for

pati€nts

with

other myoosis, tuberculosis or healtl¡Jr controlË. .A,s

it

_lærtains the _{CF test} (Fig. _{3), only}

₃

_{false positive reætions (g7Eo} specificity) were recorded

fot

the healtùJr

con-trols, one

for

the TB patients (96.62¿) and

I

for

the group of patients with histoptasmosis

and aspergilloma (gZZo

_{specificity).}

_Ihe

dif-ference between _{healthy controls, and pâtients} which other mj¡co,ses was statisticaltlr sigt¡i. Sensitiyttt percentage _ot

dlse8se i¡dtir4aluah

qriüh positivo test

æsults

Speclftcitj' percenktge _of non-diseased individuåls

Ì\¡ith negative ¿est results.

k€dicttye percen¿Bge _of

v¿Iue of s _{lositive reactols} positive test _{having disease}

D€ftnftioE Equation employed to meâsure vhtue of test

(+) TaI€n from cât€n ânat Gambioq ^ ,.rìar r<ozjnn ar; T,P. '

T.P.+IlN...

T.N. r.'

---_x100

T.N. + P.

T,P. _--X¡OO

T.P. +FP. *i.*

T¿schdliÂû ro.

Disease plesent: . _{T. p. = t¡ue postLrvo ¡eac¿or}

'. I¡. N. : tâlse negårive reâcto¡

DiseÀse absent: .¡* _{..** T.} N, ₌ true negåflv€ teâcrot

F. p.: Jalse positrvo rcaøor

RESIJLTS

I'igute 1 sr¡mmarizes the reactivitJr

of

the

two tests employed.

_ft

can be obs€rved that

in

the

_{AGID positive æsults were obtained}

only

with

patients \4'Íth paracoccidioidomyco. sis (group A'),38 (BB.4Vo)

_of

_{whom p¡oduced} precipitin lines ,with

p.

_{bmsitiensls antigen.} Patients

q'ith

other mycoses, tubercì¡losis and healttry conttols \áere nûn-rea.ctive.

Conce¡ning

cF

test (Fig.

_t),4i

_{@Bqù of.}

group .4. patients also reacted _{to p. br¿silietrds}

yeast antigen; hete, however, sela from other

groups also proved reactive. Eight

of

the BB

patients with histoplasmosis (2S.SEø),

I

of

_tlfte

17

with

aspergiloms (S.BVù aDd. one

of

tho

fisant(p=<0.01).

ConceTning _{the ptedictive value}

_of

_{a Dosi.} tive AGID test (Fig. 4), suoh a value was tìr07o both

in

telation to the healthy controls and to the patients wíth either TB

or

r¡ycoses diffÊ

rent to _{paracocqidioidosrycosis. As}

_for

_{ttre CF}

test its prediotive value in relation to tlre

heal-thy

control was high (qgEò: sucr¡ a value di_ minlshed dudng and

after

treatme¡t

but, Ít

is stilt of 73Eo

^11æ 86 months of therapy ces.

sation.

In

relation

_to

the group

_of

patients

\pith

other mycoses,

the

prcdictive value of the CF test at dlagaosis wa.s lowe¡ _{<gtEo, tf¡an}

that

for

_{healthy controts (gT/o) and}

_TB

_Da'

tierúf <97.57or. AfteÌ therapy, t,Le value decrãa

sed steadely

to

rcach SOlo at 86 monfirs Dost

CANO, 1,, E, & REST.REPO. .{. _{-¡P¡edictive value} of serologic t€€ts in tho di8g[osls a¡d fol¡ow-up ot p¿tieÈts vltD

p€¡8¿occtaltotaloE¡¡cosis.

' Bee, IDsÉ. Mert- tÉp. São Pd¡lo, â. nÈW, 198î .

Groups

m

Porococcidioidomycosis

I7--

H¡stoplosmosis

I

Pulmonory Aspergillomo Ef:f:tl _TB

If

Heolthy controls

Fig. 1 _- Semrogic test¡¡g with P. Dmsulensls arld lI. cslFul¡tum ÂDttgenE in patients with systemic Eycosês, tubercutosis

and nolmal pe¡soDs.

Agor gel

irnmunodiff usion (AGID)

w¡th porûcocc¡d¡oidin

Itre

results

of

t̡e prcsent study confim lrevious indications conceming

the

adequate sensitiviw of both tåe .6.GID and the CF tests

for

the

dt8€nosis

of

pamÆoccidioido4lrcosis 1,34;6).1,,12,t314,17'23,27. _{Concernlng tl¡e specifici.} W

of

such tests, our results offer statisticaüJr

supported datå

for

the AGID, former¡J¡ shown to be highly specific 3¡,8,ú,13.2¡.

In

ttris ægar4

the

cF

test atthough l¡ighly spec;fic (g'IEo) in front of healthy controls, tsas shoen to be less

preûise nrhen cornpBred

to

patlents

_{wÍth his}

toplasmosis and aspergiUom¿

(82øo).

îress tEo mycotic dlso¡ders are collrmon

in

the en demic ar€as

for

pamcoccldioidomõ¡cosis 5r6gt

and,

itr

consequence, one may

rim

lnto

düfi-culties !Íhen &tt€mpting

a

defiDite d¡agnoslg

l¡r a pafticular pattent,

Se8rching for Ê mef,hod to cledry the abo

vo problem, we have utillzed a ststigtical

coD-cept, tbat of tha p¡edlctive _ìsatue

of

a positlve

DISCUSSION

Complemenl Fixofion (CF)

with porococcidio¡d¡n

tÈ5tJ _ì

vr¡lh _{U:ggplglgrul!} whole

teost cells

test ånd applied

It

to

paracoccidioidoÍSrcosis.

ï1lis

concept advanced

by

GALEN and

cAM-BINO ó, _furrishes

_a

_{more precise inforD¿tio¡} concerning

the

possibility

_tbât

a¡¡ individual who has rea€ted

in

t,lle test,

truly

has pam,

coccldioÍdom!¡cosis.

Ibe

results iDdicate tbat

s

serum reactive

in

the AGID fest should come

f¡om a patient who has

_-

ot

has had

_-

paÊ

mcoccldioidomycosis,

The

certainty

comes

ffom

the 1007¿ prcdictive values exhibited by this technique eitheÌ at the tllne

of

diagnosis

or

et

var¡ous pelioals dur¡¡g and after thera.

py.

In

tl¡e case

of

the CF test, a posiuve

te

silrt

in

arl und¡agûosed pati€Dt inaUcates tbøt

in

comparison witf¡ hestt\y inauviduals

in

the

eDdehlc aleas,

tl¡is

patient has 937¿

possibl-lilies

of

llaving paÍacoccitlioidorqycosis; this is also a meaningful

flgure.

tlowever, a¡nong â

polrulation Of patients \tr¡tlx suspected ll]ycoses,

tåe

Ofs

pÉdict¡ve value.

is

lower @!qo) etrtd althóugh

not

yet

€xtrcmety ùnþofta.nt, there

t,

CATYO, L. E. & R;ESTREPO, A. _- P¡€dictive v¡ilue of seÌoloAic tests ir the aüâglosis a¡d fottow-up of pati€nts wtth

pamcoccidioialoevcosjs. _{Rev. IDst. Med. trop. São Psr¡lo, z9t} 276-283, 19A7.

i.

I

;

_'õ

ô-àe

a¡ararrrìotl

L _{Diogno.i. ond rtreropy J}

¡'ig 2 _- Sensit¡vity oI s€¡ologic tests with P. brasiließis súige¡s t¡ patieDts with paÈcoccictioiatomycosis þeto¡e, duliûB s,nd afteÈ slecific the¡e?y.

illness,

.As

it

pertains tuberculosis, the

pre-dictive va.lue was lrigh (925/or, tnùcatì¡lg

*øt

the t€st is capable of discrird¡ating wilh high efficienclr bef,ween this drsotder and paracocci. dioidorqycosisr.

ff

treatment

for

tho mycoses

is

offered, the prcdictive velue deüeases anal after 36 months

of

therapy cessation, the pos-sibiuties

of

colTectly deternjning tlre etiolosr

of

the paat. illness

is

only 5080.

fn

some diagnostic centers,

it

has b€€[¡ costumary to perform botl¡ the AGID and

tlþ

CF tests because the

fonrer,

¿lthough much

simpler

to

perform, does not vaqr enterisivelJr

duaing the follow-up studies, while Ure latterr

procedu¡e lenals itseLf to adequ¿te quantitifica"

túon.

Ibe

rcsults we p¡esent hero indicate th¿t

it

is

stiu

sowrd

to

proceed atong such lùres. Cross reactivibr amoDg varior¡s n¡rcotio antigens has been exteÐsivet!' docum¡9nted ü¡ tåe past 35.13,15¡BÆ,28,

with

_tlre

llore

_sensitivê

teshniques caflryiag the lerger burden of noû.

L

_{Perio¿. of Observot¡on ( Months)}

J

I

Post - Th eropy

AGID

spesificibt,ó,21. Cleârly there is need to studJ¡

the antigenio make up of P.

brrs

ieDsis and of

other mycotic _{agents causing deeÞseated}

Í\y-cosis

in

otder to isolate specific antigenio

moi-ties. fn

this

folm.,

it

B'ould be possiblo

to

prcduce specific ant¡gens whic.l¡ would ft,srilt

tn

t€sts ü¡ith higlrer predictlve values a,ûd

j¡

a

more prccise diàgnosis

for

the patient

l¡

need

of

ploE¡tt, specific theraly s,?8.

RÐST'MEN

Valor pledisffvo de

las

_Inuebas serofodcss Irara el dlagnosdco y seguimtento de Ia

Ila¡æorcialtoidomicosls.

Con

el

objeto de determinar

el

vdlor

p¡a

dictivo ale las pn¡ebas serológicas (F¡jacióD do

Complemento, trE,

e

fnmunodlfusió¡

e[

cel

de

Agar, IDGA), utilizaalas ¡egulaFmente €r¡ nuestro laboratorir _¡)ara

el

diagnóstico

y

el

SANO, !. E. & R.ESTR¡IPO, A. _- Preillctive value ot s€rologio teÁhs in the drãgaostB a¡d follow-üp of pstr€nts sith

Bev. LEst, lvled. trop. Sio Pr{o, Æt È18.2æ, 19A7.

.;

à!

Groups

Fig. _{¡ -} S!êcificity or se¡orogic t€sts reith P. brl¡ rersû ÀntigeDs in fhlee ditte¡ent controt smups_

Grougs

æl

Heolthy cmtrols

rEsr

_____J

Ø

Polienls with hisloplqsmos¡s

ond osperg¡llomo

I

Poti€nls with TB

60

.:

= fL

^40

036

L _During

_{f."ot."nt J}

Eig. 4 _- Pr€diclive calue ol ð positùe serologic test tn p¿tients with para{occldioidoll¡¡¡coâis _{atrd ià t¡æe} d¡tfeft'at

conü¡oI Bror¡ls, acco¡ating to tine of t¡rerapy.

AGID

L

_Pêriod

_of

_{Obs€rvol¡on (}

_Uont¡slJ

.'.'.

Heollhy conlrols

r¡rl

TB pol¡€nts

-

Pqtionts other

mycoseS

CF

72

cÂNO, _{pâmcoccidioidonycos:s.} I t & RESTREPO, _{nev. IDst. Med. trop.} A - Pr€dlctive lalue of se¡ologic tests iE the altBglosis å¡at fotloF-r4r of Þsti€ûts wità Sio Psuto, nr y¡6-Z&3,79A7,

anaJizaron los datos Þrovenientes de 43

pacien-tes son

la

enfermedad. Tales pacient€s fueron

tratados en forma identica (Ketoconazol, 200 mg/dia por 6 meses); además ambas pruebâs serológicas utilizaron un mismo lote de antig+ no, eliminando asl posibles variables. FU€ra de los sueros provenientes de los 43 pacientes con paracoccidioidomicosis (¡{), _se estudiaron 50 suercs de pacientes con otrâs micosis

pro-fundas (B)

y

92 sueros de personas sar¡as (C). Empleamos las fórmula,s dadas por GALEN y

GAMBINO ó

_y

_{hallamos que la sensibiltdad de} ambas pruebas serológicas era comparable, sin

djferencia estadísticamente significativa entre

ell8s (FC

₌

E7,IæA

₌

88AVo). Dicha seû sibilidad tend.Ía

a

disminuir con el

tratamien-io,

siendo más marc¿da su baja du-rante las

obseúsaciones post-terâpia. .Al a'nalizar

la

eg

pocifibialad se enconttó que

la

IDGA era

to-talmente esp€cífica,

no

encontrandqse falsos

positivos en las poblaciones controles estudia,

dâs

(B

y C).

La

F'C mostró

w

S6.7Eo de especificidqd frente

a la

poblaciód

C

y

un 82Eo trenl,ø a la B, siendo estedistisarnente sig-nificativa

la

diferencia entre ambas. Basados en los datos ariterior€s, se buscó et valof

pte

dictivo, hallaridose que

la

IDGA d¿ seguddâd

completa para el diagnóstiso en cualquiera de los perfodos evaluados

no

asi

la

Fc,

la

que mostró disminución de tales va.lotes durante la te¡apia y la post-terapia. La cl¡Iva æspèctiva mostró como a medida que pasaba eI tiempo,

disminuÍa

la

posibiildad de diferenciar

la

pa.

ra,coccidioldomicosis (.4.) _de otra,s micosis (B), manteniendose, sinembargo,

Ia

diferencia con los controles normales (C).

AqINOTÍLEDGEVIEl{T

T'lre authors Ð<press their appreciatton to

the patients for theit constånt cooperation and

to

Dr. William Mejfa' \¡¡ho kind¡y checked the

statistical analysis.

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R€cebido para pl'lbllcadãô ú.