This article was downloaded by: [Columbia University]On: 10 December 2014, At: 09:44Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK

Journal of ImmunoassayPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/ljii19

Rapid Identification of CandidaAlbicans by Dot-EnzymeImmunoassayRoland Guinet a , Sylvie Bruneau a & HermanceMarlier aa Centre d'Immunochimie Microbienne, InstitutPasteur , Domaine du Poirier, 69210, Lentilly, FrancePublished online: 23 Oct 2006.

To cite this article: Roland Guinet , Sylvie Bruneau & Hermance Marlier (1991)Rapid Identification of Candida Albicans by Dot-Enzyme Immunoassay, Journal ofImmunoassay, 12:2, 225-231, DOI: 10.1080/01971529108055068

To link to this article: http://dx.doi.org/10.1080/01971529108055068

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all theinformation (the “Content”) contained in the publications on our platform.However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness,or suitability for any purpose of the Content. Any opinions and viewsexpressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of theContent should not be relied upon and should be independently verified withprimary sources of information. Taylor and Francis shall not be liable for anylosses, actions, claims, proceedings, demands, costs, expenses, damages,and other liabilities whatsoever or howsoever caused arising directly orindirectly in connection with, in relation to or arising out of the use of theContent.

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan,sub-licensing, systematic supply, or distribution in any form to anyone isexpressly forbidden. Terms & Conditions of access and use can be found athttp://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

JOURNAL OF IMMUNOASSAY, 12(2), 225-231 (1991)

RAPID IDENTIFICATION OF CANDIDA ALBICANS BY DOT-ENZYM E MMUNOASSAY

Hcland Cuinet , Sylvie Bruneau and Herrnance Marlier C e n t r e d ' lmrnuncchimie Micrcbienne, Insti t u t Pas t eu r ,

Dcmaine du Pcir ier , 69210 Lentil ly, F r a n c e

ABSTRACT

A highly sensit ive and specif ic dc t - enzyme irnrnuncassay f c r t h e rapid ident i f icat icn cf Candida albicans w a s developed using a murine m c n c c l c n a l a n t i b c d y (M ab) , w h i c h a d s c r b e d t c cell s u r f a c e - e x p c s e d determinants . This Mab r e a c t e d with 28 of t h e 28 C. albicans s t r a ins t e s t ed including the serotypes A and B and 2 C. stellatoidea. I t did n e t r e a c t with 32 c the r i s c l a t e s represent ing eight c ther Candida species c e m m o n l y e n c o u n t e r e d in h u m a n m a t e r i a l s . A l l t h e test c c u l d b e pe r fo rmed in four s t eps in less than an hour. The yeas t s w e r e direct ly spo t t ed on a s t r ip cf Immuncdyne membrane. Then t h e s t r ip was incu- ba t ed f c r 5 min. with t h e Mab, f c r 15 min. with a percxyddse-ccnjugate and for 30 min. w i th t h e e n z y m e subs t r a t e and 4-chlcrc 1 naphtcl . This test prcved useful fcr rapid and easy ident i f icat icn cf C. albicans. (KEY WORDS : Candida albicans, Dct-enzyme irnrnuncassay)

INTRODUCTION

Infect ion due t o fungi a r e cf increasing impor t ance in pa t i en t s with

ccmprcmised host de fenses and t h e mcr t a l i t y r a t e asscciated with these

infect ions is very high. C. albicans and C. tropicalis a r e the species

mcst ccmmcnly isclated (1). The rnicrobiclogical diagncsis, based upcn

biochemical tests, is st i l l t i m e consuming (2) in spi te cf t h e develcp-

rnent of miniatur ized k i t s (3, 4) and of a u t c m a t e d system (5, 6). Sl ide

agglutination tests with specie,-specific ant isera were l imited by the

225

Copyright 0 1991 by Marcel Dekker, Inc.

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

226 GUINET, BRUNEAU, AND MARLIER

lack cf specificity cf s e ra s ince C. albicans se rc type A and C. tropi-

calis could not b e distinguished (7, 8). A ccmmcnly used test f c r

C. albicans, t h e "germ tube" test, required th ree hours and cccasicnal

isolates may g ive a f a l s e negat ive test.

Thus t h e laboratcry needs a fast and sensi t ive technique tc rapidly

identify C. albicans. We repor t he re t h e use cf a mcnoclcnal antibody

in a dc t - enzyme irnrnuncassay f c r t h e rapid d e t e r t i c n cf this micro-

crganism.

MATERIALS A N D M ETHOD5

Yeas t s s t ra ins

A t c t a l cf 60 yeas t s cr yeast-l ike fungi belcnging t c t h e gene ra

Candida, .%CChWOmyCeS and Geotrichurn w e r e examined (Table I). M cst

s t ra ins have been isolated f r c m pathclcgicdl p rcduc t s cn Sabcuraud

C h l c r a m p h e n i c c l A g a r ( D i a g n c s t i c s P a s t c u r , M a r n e L a C c q u e t t e ,

France). They w e r e identified by classical c r i t e r i a dcccrding t c van dc r

Walt and Yarrow (2). T h e e the r s t ra ins w e r e stcck cu l tu re s cbtained

f r c m Centraalburedu vcor Schimmelcul tures (CBS, Delf t , The Ne the r -

Idnds), f r c m Naticnal In s t i t u t e cf Heal th (NIH, Bethesda, USA) or f rom

lns t i t u t Pas t eu r (IP, Paris, France). Fo r all studies yeas t s w e r e cul tured

en Sabcuraud Chlcrarnphenicol Agar f c r 24 H a t 30".

D o t enzyme irnmuncassay

All t h e s t eps w e r e perf orrned at rocm temperature .

S t ep I : The blastospores or t h e a r th rc spc res w e r e remcved

f r c m t h e su r face of t h e aga r p l a t e and direct ly spc t t ed on Imrnuncdyne

membrane (Pall Industries, St-Germain-en-Layc, France). Ten srrains

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

RAPID IDENTIFICATION OF CANDIDA ALBICANS 227

Figure 1

24 H cld cu l tu re s frcrn : Candida albicans (Ca), C. guilliermondii (Cg), C. krusei (Ck), C. parapSi1osi.s (Cp), C. pseudotropicalis (Cps), C. stellatoidea (Cst), C. tropicalis (C t) and Saccharomyces cerevisiae (Sc) w e r e direct ly spc t t ed en Immuncdyne membrane. A f t e r incubaticn with t h e Mab, t h e percxydase ccn juga te and substrate , spc t s f r c m C. albicans w e r e s t r c n g l y b l u e c c l c r e d . A l l c t h e r s p c t s w e r e n e t d e tee ted .

w e r e spot ted cn a s t r ip cf 5 x 100 mm. Then the s t r ips were scaked in

Tris buffered saline pH 7.5 (TBS : Tris 20 r n M , NaCl 500 rnM) ccntai-

ning 3 % (V/V) gelat in (Bicrad Labcratcr ies , Richmrnd, USA) fcr 1 min,

t c block t h e remaining sites.

S t e p 2 : Incubaticn fcr 5 rnin with t h e Mab N A B 806,

Cherniccn, El Segundc, Cal i fcrnia , USA) di luted 1 : 200 in TBS ccntai-

ning 0.1 % Tween 20 (TBST, Merck, Darrnstadt, FRC) fc l lcwed by twc!

washing in TBST fcr I rnin. each.

S t e p 3 : Incubaticn f c r 15 min with percxydase ccnjugated

rabbi t anti-rncuse Ig (Ref. P 161, Dakcpet ts , Copenhagen, Denmark)

diluted 1 : 200 in TBST.

S tep 4 : Afte r t h ree washing in TBST f c r I r n i n . and cne in

TBS f c r I min. t he b l c t s were revealed fcr 30 rnin in sclution cf TRS

containing 0.33 % H202 (Merrk, Darmstadt , FRG) and 0.05 % 4-chlcrc-1

naphtcl (Sigma Ch imie SA, L a Verpilliere, France).

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

228 GUINET, BRUNEAU, AND MARLIER

Table 1 Results of yeasts identification in Dct-enzyme immuncassay

Tested strains

C. albicans 792 NIH, 311 NIH, 1431, 1422, 1543, 1189, 1513, 184, 34, HM, 1565, 1434, 1583, 5346, 1285, DUR, 1468, 1485, 214, 243, 740, GIN, BAY, 1309, MOR, BEK

C. albicans received as C. stellatoidea CBS 1905, CRS 2990

C. glabrata IP 810, 1P 811, 5090, 2036

C. guillierrnondii IP 624, 206

C. krusei 573 CBS, 208 1P

C. rnacedoniensis CBS 600

C. parapsilosis IP 45, IP 83, 2147

C. pseudotropicalis CBS 607, IP 42, 1020

C. tropicalis CBS 94, IP 43, 104, 5025, 9763, 4620, 5476, 1079, 1559, 4881, 4439, 1325, 1831

S. cerevisiae 1P 605, IP 635, 1248

G. candidurn 1065

Pcsit ive Negative Number results results

26 26 0

2 2

4 0 4

2 0 2

2 0 2

1 0 1

3 0 3

3 0 3

13 0 13

3 0 3

I 0 1

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

229 RAPID IDENTIFICATION OF CANDIDA ALBICANS

RESULTS-

T c evaluate the reactivity cf t h e Mab with C. albicans we tested a

panel cf fungi frequently encountered in human materials. A strcng blue

cclcur ccrrespcnded t c a pcsitive reacticn easy to read (Figure I ) . Al l

t h e 28 C. albicans strains were detected including the t w c serctypes A

and B and the sucrcse negative variant C. stellatoidea. Ncne cf the

cther 32 yeast strains reacted with the Mab even C. tropicalis which

shared many prcperties with C. QlbiCanS (Table 1). All the tes t cculd be

p e r f u m e d i n less than an hour.

DISCUSSION

A tc ta l cf 60 yeasts or yeast-like strains belonging t c the genera

Candida, Saccharomyces and Geotrichum were examined. They repre-

sented the 8 Candida species the rncst cften isclated frcm human

clinical materials and kncwn t c be virulent species. G. candidum and

S. cerevisiae w e r e f r e q u e n t l y e n c c u n t e r e d i n c l in ica l m a t e r i a l a s

ccmmensal.

Using the dct-enzyme immunoassay we cbtained 100 % specificity

and 100 % sensitivity fcr the identificaticn cf C. albicans. Ncne cf the

c t h e r C. albicans i d e n t i f i c a t i c n m e t h c d a c t u a l l y used (biochemical ,

serclegical or mcrphclcgical) showed such a specificity and sensitivity.

Particularly, nc false pcsitive results were cbserved with C. tropicalis.

C. albicans and C. tropicalis a r e 2 related species shewing identical

DNA C + C % contents (91, and mcre than 80 % of cross-reacting anti-

gens in quantitative immuncelectrcphoresis (10).

This tes t is rapid t c perfcrm : 60 min., versus 3 h for the germ-

tube test, the fastest identification methcd currently available. An

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

230 GUINET, BRUNEAU, AND MARLIER

i m p o r t a n t point is t he yeas t f ixat icn on the lmmuncdyne s t r ip which is

quick and s t rcng f c r all s t ra ins studied. The p rc t ccc l cculd b e shortened

by using an e n z y m e labeled Mab.

This test is easy t c perform and resul ts a r e r ead by visual cbserva-

t icn, which el iminates the need for an ELlSA p l a t e reader or a f lucres-

c e n t rnicrcscope. The develcpment of rapid, sensit ive techniques such a s

this for t he de t ec t ion of C. albicans infect icns will resul t in ear l ier

t r e a t m e n t . In additicn this Dct-ELISA test has the potent ia l cf being

used f c r d i r e c t C. albicans de tec t ion i n cl inical swabs.

K EF ER E N C ES

1.

2.

3.

4.

5.

6.

7.

Odds, F.C. Ecclclgy and epidemiclcgy cf Candida. In: Odds, F.C., ed. Candida and Candidcsis. Leicester: Leicester University Press , I9 79: 50-75.

Van de r Walt, J.P. and Yarrcw, D. Methcds f c r t h e isolation, main- tenance, classification and ident i f icat icn cf yeasts. In: Kreger-Van Rij, N.J.W., ed. The yeasts. Amsterdam: Elsevier Sc ience Publishers, 1984: 45- 104.

Land, G.A., Harriscn, B.A., Hulme, K.L., Cocper , R.H. and Byrd, J.C. Evaluation cf t h e new API 20C str ip for y e a s t ident i f icat icn against a ccnvent icnal methcd. J. Clin. Micrcbicl. 1979,lO: 3 5 7 - 64.

Salkin, I.F., Land, G.A., Hurd, N.J., Goldscn, P.G. and M c Ginnis, M.R. Evaluation cf yeas t ident i f icat ion and Uni-Yeast-Tek yeas t i d e n t i f i c a t i c n s y s t e m s . J. C l in . i 3 i c r c b i c l . 1 9 8 5 2 5 : 624-2 7.

Land, G., Stc t l e r , R., Land, K. and Ztanck, J. Upda te and evaluat icn cf t h e au tcmic rcb ic yeas t ident i f icat icn system. J. Clin. Micrcbicl . 1984;20: 649-52.

Salkin, I.F., Schadcw, K.H., Bankaitis, L.E., M c Ginnis, M.R. and Kemrna, M.E. Evaluaticn of Abbc t t Quantum 11 yeas t ident i f icat icn system. J. Clin. Micrcbicl. 1985;22 442-44.

Taguchi, M., Tsubigi, M. and Tsuchiya, T. Rapid ident i f icat icn of yeas t s by serological methcds. A combined serclcgical and biclcgical methcd. Sabcuraudia 1979 ; lZ 185-91.

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014

RAPID IDENTIFICATION OF CANDIDA ALBICANS 231

8. Shincda, T., Kaufman, L . and Padhye, A.H. Compara t ive evaluat icn cf t h e i a t r c n sercicgical Candida check k i t and t h e API 20C k i t i c r t h e i d e n t i f i c a t i c n cf m e d i c a l l y i m p c r t a n t Candida s p e c i e s . 3. Clin. Micrcbicl. 1981;Ik 513-18.

9. Nakase, T. and Kcrnagata , K. Signif icance cf DNA base ccrnpcsit icn in the classi f icat icn cf yeas t genus Candida. J. Gen. Appl. illicrc- bicl. 1971;l 2 259-79.

10. Gabriel-Bruneau, S.M. and Guinet, K.M .F. Antigenic re la t icnships amcng scrne Candida species studied by c,rcssed-line immuncelectrc- phcresis : taxcncmic significance. Int. J. Syst. Dactericl . 1984;34: 227-36.

Dow

nloa

ded

by [

Col

umbi

a U

nive

rsity

] at

09:

44 1

0 D

ecem

ber

2014