UNIVERSITATEA DE MEDICIN I FARMACIE

IULIU HAIEGANU CLUJ-NAPOCA

CATEDRA DE MICROBIOLOGIE

TESTAREA SENSIBILITII LA ANTIFUNGICE A

SPECIILOR DE CANDIDA
REZUMATUL TEZEI DE DOCTORAT

Conductor tiinific

Doctorand

Prof. Univ. Dr. Lia Monica JUNIE

Cluj-Napoca 2010

Ioana Alina COLOSI

CUPRINSUL TEZEI DE DOCTORAT

INTRODUCERE .......................................................................................................................................... 3
PARTEA I STADIUL ACTUAL AL CUNOATERII................................................................................ 4
Capitolul 1. Levurile din genul Candida......................................................................................................... 5
1.1 Istoric........................................................................................................................................................ 5
1.2 Taxonomie i clasificare............................................................................................................................ 5
1.3 Caractere generale..................................................................................................................................... 9
Capitolul 2. Patogenitate .............................................................................................................................. 11
2.1 Factori de patogenitate ai Candida i factori de aprare ai gazdei............................................................. 12
2.2. Factori de risc asociai infeciei cu Candida............................................................................................ 17
Capitolul 3. Infecii determinate de Candida ................................................................................................ 19
Capitolul 4. Identificarea speciilor de Candida ............................................................................................. 24
4.1 Examenul microscopic direct................................................................................................................... 24
4.2 Teste fiziologice...................................................................................................................................... 25
4.3 Cultivarea pe medii ................................................................................................................................. 26
4.4 Teste biochimice ..................................................................................................................................... 29
4.5 Teste rapide de identificare a unor specii de Candida............................................................................... 30
4.6 Tehnici moleculare.................................................................................................................................. 32
Capitolul 5. Metodele utilizate pentru testarea sensibilitii la antifungice a speciilor de Candida ................. 32
5.1 Metoda microdiluiilor M27-A ............................................................................................................... 34
5.2 Metoda difuzimetric M44-A .................................................................................................................. 38
5.4 Metoda difuzimetric Neo-Sensitabs .................................................................................................... 41
5.3 E-Test (AB Biodisk, Solna, Suedia)..................................................................................................... 42
5.5 Alte metode comercializate, nestandardizate, pentru testarea sensibilitii la antifungice a Candida spp... 47
PARTEA A II-A CERCETRI PERSONALE.......................................................................................... 54
Studiul I: Distribuia i testarea sensibilitii la antifungice a speciilor de Candida din Cluj-Napoca, Romnia
i Grenoble, Frana........................................................................................................................................ 55
Capitolul 6. Ipoteze, scop i obiective .......................................................................................................... 55
6.1 Ipoteza cercetrii ..................................................................................................................................... 55
6.2 Scopul cercetrii...................................................................................................................................... 55
6.3 Obiectivele cercetrii............................................................................................................................... 56
Capitolul 7. Material i metod .................................................................................................................... 56
7.1 Tulpinile studiate .................................................................................................................................... 56
7.2 Testarea sensibilitii la antifungice a tulpinilor de Candida spp. izolate .................................................. 69
7.3 Metodele de analiz statistic utilizate ..................................................................................................... 82
Capitolul 8. Rezultate................................................................................................................................... 84
I. Speciile de Candida izolate........................................................................................................................ 84
8.1 Speciile de Candida izolate din diverse produse patologice ..................................................................... 84
8.2 Speciile de Candida izolate de la pacieni din diferite grupe de risc ......................................................... 98
II. Testarea sensibilitii la antifungice........................................................................................................ 108
8.3 Testarea sensibilitii la Fluconazol i compararea profilului de sensibilitate la Fluconazol a tulpinilor
de Candida izolate din Cluj-Napoca i Grenoble ......................................................................................... 108
8.4 Testarea sensibilitii la Voriconazol i compararea profilului de sensibilitate la Voriconazol a tulpinilor
de Candida izolate din Cluj-Napoca i Grenoble ......................................................................................... 115
8.5 Testarea sensibilitii la Caspofungin i compararea profilului de sensibilitate la Caspofungin a tulpinilor
de Candida izolate din Cluj-Napoca i Grenoble ......................................................................................... 132
8.6 Testarea sensibilitii la Amfotericin B a tulpinilor de Candida spp. .................................................... 140
8.7 Testarea sensibilitii la Itraconazol a tulpinilor de C.krusei................................................................... 143
Capitolul 9. Discuii................................................................................................................................... 144
Capitolul 10. Concluzii .............................................................................................................................. 157
Studiul II: Compararea a dou metode, E-test i metoda difuzimetric Neo-Sensitabs, pentru testarea
sensibilitii la antifungice a speciilor de Candida ....................................................................................... 159
Capitolul 11. Ipoteze, scop i obiective ...................................................................................................... 159
11.1 Ipoteza cercetrii ................................................................................................................................. 160

11.2 Scopul cercetrii.................................................................................................................................. 160

11.3 Obiectivele cercetrii........................................................................................................................... 160
Capitolul 12. Material i metod ................................................................................................................ 161
12.1 Tulpinile studiate................................................................................................................................. 161
12.2 Metodele utilizate pentru testarea sensibilitii la antifungice a tulpinilor de Candida .......................... 161
12.3 Metodele de analiz statistic utilizate ................................................................................................. 168
Capitolul 13. Rezultate............................................................................................................................... 169
13.1 Rezultatele testrii sensibilitii la antifungice a tulpinilor de Candida prin E-test i metoda
difuzimetric Neo-Sensitabs ...................................................................................................................... 169
13.2 Rezultatele testrii sensibilitii la Amfotericin B a tulpinilor de Candida spp. prin E-test i metoda
difuzimetric Neo-Sensitabs ...................................................................................................................... 170
13.3 Rezultatele testrii sensibilitii la Fluconazol a tulpinilor de Candida spp. prin E-test i metoda
difuzimetric Neo-Sensitabs ...................................................................................................................... 172
13.4 Rezultatele testrii sensibilitii la Voriconazol a tulpinilor de Candida spp. prin E-test i metoda
difuzimetric Neo-Sensitabs ...................................................................................................................... 174
13.5 Rezultatele testrii sensibilitii la Caspofungin a tulpinilor de Candida spp. prin E-test i metoda
difuzimetric Neo-Sensitabs ...................................................................................................................... 179
Capitolul 14. Discuii ................................................................................................................................. 184
Capitolul 15. Concluzii .............................................................................................................................. 187
CONCLUZII GENERALE....................................................................................................................... 188
REFERINE............................................................................................................................................. 189

CUVINTE CHEIE
Candida, infecii fungice, factori de risc,
antifungice: Fluconazol, Voriconazol, Caspofungin, Amfotericin B, Itraconazol,
E-test, metoda difuzimetric M44-A, metoda difuzimetric Neo-Sensitabs.

Introducere
Infeciile oportuniste de etiologie micotic se constituie ntr-o nou provocare medical la nceput de secol 21.
Infeciile fungice sunt de obicei oportuniste, asociate cu disfuncii imune i cu prezena unor factori de risc (ex.
utilizarea antibioticelor cu spectru larg). Numrul pacienilor cu disfuncii imune a crescut dramatic datorit pandemiei
SIDA, creterii numrului pacienilor transplantai, chimioterapiei agresive antineoplazice. Fungii sunt de asemenea
recunoscui ca importani ageni etiologici ai infeciilor nosocomiale, determinnd infecii severe la pacienii
imunodeprimai: pacieni cu arsuri extinse, cateterizai, hemodializai, pacieni aflai la vrste extreme.
Se impune necesitatea cunoaterii epidemiologiei (cunoaterea speciilor implicate), patogeniei (ex. izolarea
Candida nu nseamn ntotdeauna infecie), formelor clinice i cunoaterea aspectelor terapeutice a infeciilor micotice.
Pentru aceasta, izolarea i identificarea agentului etiologic precum i determinarea profilului de sensibilitate la
antifungice sunt eseniale.
Ajutorul laboratorului n alegerea terapiei este hotrtor prin precizarea sensibilitii tulpinii de Candida la
antifungicul utilizat. Astfel, determinarea printr-o metod standardizat a sensibilitii la antifungice a Candida spp. este
deosebit de important, mai ales n contextul emergenei speciilor i tulpinilor de Candida rezistente la antifungice.
Scopul cercetrilor prezentate n teza de fa l-a constituit determinarea distribuiei speciilor de Candida
implicate n patologia uman din Cluj-Napoca i a sensibilitii acestor specii la antifungice comparativ cu speciile de
Candida izolate din Grenoble, Frana, precum i evaluarea unei metode difuzimetrice, uor de realizat, fiabile i ieftine
pentru testarea in vitro a sensibilitii la antifungice a speciilor de Candida.

Stadiul actual al cunoaterii

Stadiul actual al cunoaterii a fost structurat n 5 capitole (capitolele 1-5) i prezint o sintez a cunotinelor
privind genul Candida, sintez realizat prin studiul literaturii de specialitate (232 referine).

Cercetri personale
Prezentarea contribuiilor personale a fost structurat n dou pri distincte, corespunztoare celor dou studii
realizate.
Studiul I: Distribuia i testarea sensibilitii la antifungice a speciilor de Candida din Cluj-Napoca, Romnia i
Grenoble, Frana, cuprinde capitolele 6-10.
Capitolul 6 expune ipotezele, scopul i obiectivele studiului. Scopul cercetrii a fost determinarea distribuiei
speciilor de Candida implicate n patologia uman din Cluj-Napoca i a sensibilitii acestor specii la antifungice
comparativ cu speciile de Candida izolate din Grenoble, Frana.
Capitolul 7 prezint tulpinile de Candida spp. studiate: n perioada iulie 2007- decembrie 2008 s-a realizat
identificarea i testarea sensibilitii la antifungice a speciilor de Candida izolate din diverse produse patologice
provenite de la pacieni spitalizai n Clinica de Boli Infecioase din Cluj-Napoca i de la pacieni spitalizai n Spitalul
Clinic Universitar din Grenoble, Frana. Probele prelucrate n laboratorul Clinicii de Boli Infecioase din Cluj-Napoca
au provenit att de la pacieni internai n aceast clinic ct i de la pacieni internai n alte clinici din Cluj-Napoca,
pacieni internai n secii chirurgicale, de terapie intensiv, pediatrie sau medicale.

Au fost descrise metodele utilizate pentru testarea sensibilitii tulpinilor izolate de Candida spp. la antifungice:
pentru Fluconazol i Voriconazol metoda difuzimetric M44-A i E-test, pentru Caspofungin, Amfotericin B i
Itraconazol E-test.
Metodele de analiz statistic a datelor au fost:
- testul Chi-ptrat, testul exact al lui Fisher;
- testul U al lui Mann-Whitney;
- coeficientul de corelaie (R) al lui Pearson;
Pentru realizarea analizelor statistice i a modelelor de regresie s-a utilizat programul SPSS 13.0.
Capitolul 8 prezint rezultatele obinute n studiul I.
Speciile de Candida izolate
Din Cluj-Napoca s-au identificat 249 tulpini de Candida spp.: C.albicans (45%), C.glabrata (26,1%), C.krusei
(10,04%), C.parapsilosis (5,62%), C.tropicalis (4,82%), C.famata (2,41%), C.guilliermondii (2,01%), C.pelliculosa
(0,8%), C.lipolytica (0,8%), C.kefyr (0,8%), C.sake (0,4%), C.lusitaniae (0,4%), C.inconspicua (0,4%), C.curvata
(0,4%).
Din Grenoble s-au identificat 813 tulpini de Candida spp.: C.albicans (51,91%), C.glabrata (20,66%), C.tropicalis
(6,40%), C.parapsilosis (5,78%), C.krusei (5,41%), C.kefyr (4,43%), C.inconspicua (1,85%), C.lusitaniae (1,72%),
C.guilliermondii (0,98%), C.utilis (0,37%), C.catenulata (0,12%), C.lipolytica (0,12%), C.pulcherima (0,12%).
Din hemoculturi, n Cluj-Napoca, principalele specii izolate a fost C.albicans, C.parapsilosis, C.glabrata,
C.tropicalis,

iar n Grenoble C.albicans, C.glabrata, C.parapsilosis

i C.tropicalis. n Cluj-Napoca, din urini,

principala specie izolat a fost C.glabrata. Din Grenoble, din urini, principala specie izolat a fost C.albicans.
n Cluj-Napoca, de la pacieni internai n secii chirurgicale, pe primul loc ca frecven a izolrii s-a situat
C.glabrata, n secii de boli infecioase dup C.albicans s-a situat C.krusei, iar n secii de pediatrie dup C.albicans s-a
situat C.parapsilosis.
Testarea sensibilitii la antifungice i compararea profilului de sensibilitate la antifungice a tulpinilor de Candida
izolate din Cluj-Napoca i Grenoble
Rezultatele testrii sensibilitii la Fluconazol sunt reprezentate grafic n figurile 83 i 84.
Nu au fost testate la Fluconazol tulpinile de C.krusei (C.krusei fiind rezistent la Fluconazol).
n Cluj-Napoca, toate tulpinile testate de C.kefyr, C.lusitaniae, C.lipolytica, C.guilliermondii, C.inconspicua i
C.curvata au fost sensibile la Fluconazol.
n Grenoble, toate tulpinile de C.tropicalis, C.kefyr, C.guilliermondii, C.utilis, C.lipolytica, C.pelliculosa, C.pulcherima
i C.catenulata au fost sensibile la Fluconazol.
Diferena de sensibilitate la Fluconazol a tulpinilor de Candida spp. izolate din Cluj-Napoca i din Grenoble a
fost semnificativ statistic (p <<0.001, testul chi-ptrat i testul Fisher exact).
Riscul relativ (RR) ca o tulpin de Candida spp. izolat din Cluj-Napoca s fie mai rezistent la Fluconazol dect o
tulpin de Candida spp. izolat din Grenoble a fost 4,038, cu un interval de ncredere 95% de [2,191 7,444].

100%

100%

93.69%

92.31%

91.67%

100%

99.53%

100%

83.33%

100%

97.87%

80%
78.57%

80%

60%

60%

50%

40%

SDD

25%
17.19%

20%

40%

35.71%

16.67%

0%

7.69%

21.43%

16.67%

20%

5.41%

8.33%

8.93%

0,9%

0.24% 0.24%

2.13%

Figura 83. Sensibilitatea la Fluconazol a speciilor de

Candida din Cluj-Napoca

ae
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Figura 84. Sensibilitatea la Fluconazol a speciilor de

Candida din Grenoble

Rezultatele testrii sensibilitii la Voriconazol sunt reprezentate grafic n figurile 90 i 91.

n Cluj-Napoca, toate tulpinile testate de C.famata, C.guilliermondii, C.kefyr, C.lusitaniae, C.lipolytica, C.inconspicua,
C.curvata, i C.pelliculosa au fost sensibile la Voriconazol.
n Grenoble, toate tulpinile de C.parapsilosis, C.tropicalis, C.kefyr, C.inconspicua, C.lusitaniae, C.guilliermondii,
C.utilis, C.lipolytica, C.pelliculosa, C.pulcherima i C.catenulata au fost sensibile la Voriconazol.

94.52%

91.67%
77,5%

80%

100%

99.76%

100%
100%

100%

100%

100%

97.73%

91.67%

88.89%
80%

75%

60%

60%

R
S

R
S

40%
17,5%

20%
5.48%

5%

20%

15%

10%

11.11%

8.33%

5.35%

0.24%

2.27%

2.98%

0%

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Figura 90. Sensibilitatea la Voriconazol a speciilor

de Candida din Cluj-Napoca

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Figura 91. Sensibilitatea la Voriconazol a speciilor

de Candida din Grenoble

Diferena de sensibilitate la Voriconazol a tulpinilor de Candida spp. izolate din Cluj-Napoca i din Grenoble a
fost semnificativ statistic (p <<0.001, testul chi-ptrat i testul Fisher exact).
Riscul relativ (RR) ca o tulpin de Candida spp. izolat din Cluj-Napoca s fie mai rezistent la Voriconazol dect o
tulpin de Candida spp. izolat din Grenoble a fost 6,295, cu un interval de ncredere 95% de [3,033 13,064].
Caspofungin
Toate tulpinile de Candida testate din Cluj-Napoca au fost sensibile la Caspofungin, cu excepia unei tulpini de
C.parapsilosis care a fost non-sensibil la Caspofungin, CMI-ul obinut pentru aceast tulpin a fost de 2 g/ml. Toate
tulpinile de Candida izolate din Grenoble au fost sensibile la Caspofungin.

Diferena de sensibilitate la Caspofungin a tulpinilor de Candida spp. izolate din Cluj-Napoca i din Grenoble fost
nesemnificativ statistic (p = 0.139, testul Fisher exact).
S-a constat c exist o diferen nalt semnificativ statistic (p<<0.001, testul Mann-Whitney) ntre CMI-urile
la Caspofungin obinute pentru tulpinile de Candida spp. izolate din Cluj-Napoca i Grenoble. CMI-urile obinute n
Cluj-Napoca s-au ncadrat n intervalul 0,032 -2 g/ml, cu o median de 0,25 g/ml, iar CMI-urile obinute n Grenoble
la Caspofungin pentru tulpinile testate de Candida spp. s-au ncadrat n intervalul 0,004 - 1g/ml, cu o median de
0,064 g/ml (figura 105).
Rezultatele testrii sensibilitii la Amfotericin B a tulpinilor de Candida spp. (figura 110)
Toate tulpinile de C.albicans, C.inconspicua, C.lusitaniae, C.guilliermondii, C.utilis, C.lipolytica, C.pelliculosa,
C.pulcherima i C.catenulata testate n Grenoble au fost sensibile la Amfotericin B.
Rezultatele testrii sensibilitii la Itraconazol a celor 32 tulpini de C.krusei izolate din Grenoble: 11 tulpini au
fost rezistente (46,87%) i 14 SDD (43,75%).

Figura 105. CMI-urile pentru Caspofungin obinute pentru tulpinile de Candida din Cluj-Napoca i Grenoble

100%

98.20%

100%

98.08%

97.22%

95.74%
86.36%

80%

60%

40%

R
S

20%

0,6%

0%

i
lb
.a

ns
ca
C

b
la
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4.26% 4.54%

1,2% 1.92%

ta
ra
C

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si
ap

sis
C

s
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9,1%
2.78%

ei
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yr
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Figura 110. Sensibilitatea la Amfotericin B a speciilor de Candida izolate din Grenoble.

Capitolul 9 interpreteaz i discut rezultatele acestei prime pri a cercetrii, prin raportare la cercetri
asemntoare.

Capitolul 10 prezint concluziile primei pri a cercetrii. Dintre acestea, menionez n continuare urmtoarele:
1. Principalele specii de Candida izolate din Cluj-Napoca i Grenoble au fost Candida albicans i Candida glabrata. n
Cluj-Napoca pe locul 3 ca frecven a izolrii s-a situat Candida krusei (specie rezistent la Fluconazol), n Grenoble
situndu-se pe locul 5, dup Candida tropicalis i Candida parapsilosis.
2. Tulpinile de Candida spp. izolate din Cluj-Napoca au fost mai rezistente la Fluconazol i Voriconazol dect tulpinile
de Candida izolate din Grenoble.
3. Valorile concentraiilor minime inhibitorii la Caspofungin obinute pentru tulpinile de Candida spp. testate la ClujNapoca au fost mai mari dect cele obinute la Grenoble.
4. n iniierea unui tratament antifungic empiric este esenial cunoaterea speciilor de Candida dintr-un anumit areal
geografic, respectiv spital i profilul lor de sensibilitate la antifungice.
5. Diferenele observate ntre Cluj-Napoca i Grenoble subliniaz importana i necesitatea unor protocoale pentru
depistarea i supravegherea pacienilor la risc, pentru stabilirea diagnosticului etiologic micologic, finalizate cu testarea
sensibilitii la antifungice.
Studiul II: Compararea a dou metode, E-test i metoda difuzimetric Neo-Sensitabs, pentru testarea
sensibilitii la antifungice a speciilor de Candida, cuprinde capitolele 11-15.
Capitolul 11 expune ipotezele, scopul i obiectivele. Scopul cercetrii a fost evaluarea unei metode
difuzimetrice, uor de realizat, fiabile, ieftine pentru testarea in vitro a sensibilitii la antifungice a speciilor de
Candida. Obiectivul studiului a fost realizarea unei comparaii ntre dou metode de testare a sensibilitii la antifungice
a speciilor de Candida: E-test (AB Biodisk, Solna, Suedia) i metoda difuzimetric Neo-Sensitabs (Rosco
Diagnostica, Taastrup, Denmark) cu tablete de 9 mm diametru. E-test-ul este o metod de testare a sensibilitii
Candida la antifungice standardizat, uor de realizat dar al crui principal dezavantaj l constituie costul ridicat (10
euro/bandelet), ceea ce l face inaccesibil pentru majoritatea laboratoarelor clinice, impunndu-se astfel gsirea unei
metode care s pstreze avantajele E-test-ului dar care s fie mai ieftin.
Capitolul 12 prezint materialul i metodele utilizate pentru realizarea acestui studiu.
S-a realizat identificarea i testarea sensibilitii la antifungice prin E-test i prin metoda difuzimetric Neo-Sensitabs
a 93 de tulpini de Candida spp. izolate din diverse produse patologice provenite de la pacieni din Spitalul Clinic
Universitar din Grenoble, Frana n perioada februarie aprilie 2009.
S-a determinat procentul concordanelor calitative dintre rezultatele obinute prin E-test i cele obinute prin metoda
difuzimetric Neo-Sensitabs. Au fost identificate urmtoarele discordane: foarte mari - cnd o tulpin a fost rezistent
(R) prin E-test i sensibil (S) cu Neo-Sensitabs, mari - cnd o tulpin a fost S prin E-test i R cu Neo-Sensitabs,
minore cnd o tulpin a fost S sau SDD printr-o metod i SDD sau R prin cealalt.
Analiza statistic a datelor s-a realizat utiliznd modele de regresie liniar i calculul coeficientului de corelaie Pearson
(R) dintre valorile liniarizate prin logaritmare ale CMI (exprimate n g/ml) i diametrele zonelor de inhibiie
(exprimate n milimetri) obinute pentru antifungicele testate, interpretndu-se pentru fiecare regresie n parte
coeficientul de corelaie R al lui Pearson i nivelul su de semnificaie statistic. Pentru realizarea modelelor de regresie
s-a utilizat programul SPSS 13.0.
Capitolul 13 prezint rezultatele obinute n studiul II.

Pentru Amfotericin B procentul de concordan obinut ntre E-test i metoda difuzimetric Neo-Sensitabs
a fost de 90,32%. S-au constatat 6 discordane minore (pentru 5 tulpini de C.glabrata i 1 tulpin de C.albicans) i 3
discordane foarte mari (pentru 2 tulpini de C.glabrata i 1 tulpin de C.krusei). Coeficientul de corelaie R, dintre
Log2CMI Amfotericin B i diametrul zonei de inhibiie a fost -0,622 (figura 121).
Pentru Fluconazol procentul de concordan obinut ntre E-test i metoda difuzimetric Neo-Sensitabs a
fost de 100%. Coeficientul de corelaie R, dintre Log2CMI Fluconazol i diametrul zonei de inhibiie, a fost de -0,654.
Pentru Voriconazol procentul de concordan obinut ntre E-test i metoda difuzimetric Neo-Sensitabs a
fost de 100%. Coeficientul de corelaie R, dintre Log2CMI Voriconazol i diametrul zonei de inhibiie, a fost -0,620.

Figura 121. Amfotericin B reprezentarea grafic

a relaiei dintre Log2CMI (E-test) i diametrul
zonei de inhibiie (Neo-Sensitabs).

Figura 128. Caspofungin reprezentarea grafic

a relaiei dintre Log2CMI (E-test) i diametrul
zonei de inhibiie (Neo-Sensitabs )

Pentru Caspofungin procentul de concordan obinut ntre E-test i metoda difuzimetric Neo-Sensitabs a
fost de 98,92%. S-a constatat 1 discordan mare (pentru 1 tulpin de C.krusei). Coeficientul de corelaie R, dintre
Log2CMI Caspofungin i diametrul zonei de inhibiie, a fost de -0,679 (figura 128).
Valoarea tuturor coeficienilor de corelaie R a prezentat o nalt semnificaie statistic (p<<0,001).
Capitolul 14 - interpreteaz i discut rezultatele celui de-al II-lea studiu din contribuiile personale, prin
raportare la cercetri asemntoare, subliniind punctele forte, dar totodat i limitrile inerente oricrui studiu.
Capitolul 15 prezint concluziile celei de-a doua pri a cercetrii personale. Dintre acestea menionez n
continuare urmtoarele:
1. E-test -ul este o metod de testare a sensibilitii la antifungice a speciilor de Candida standardizat, uor de utilizat
n practic, dar valorile concentraiilor minime inhibitorii (g/ml) sunt uneori dificil de citit mai ales pentru azoli,
datorit fenomenului de cretere rezidual. Un alt dezavantaj al E-test-ului este costul ridicat.
2. Zonele de inhibiie obinute prin metoda difuzimetric Neo-Sensitabs au fost bine delimitate i msurarea
diametrului zonelor de inhibiie s-a realizat cu uurin, fiind unul din principalale avantaje ale metodei. De asemenea,
aceast metod este uor de utilizat n laboratoarele clinice, costul fiind mult mai sczut dect al E-test-ului.
3. S-a obinut o excelent concordan ntre rezultatele E-test-ului i Neo-Sensitabs pentru Fluconazol, Voriconazol i
Caspofungin, un nivel mai sczut de concordan obinndu-se pentru Amfotericin B.

CONCLUZII GENERALE
1. Infeciile fungice rmn afeciuni grave i frecvena lor a crescut considerabil n ultimii ani. Creterea numrului de
pacieni imunodeprimai i explozia interveniilor medico-chirurgicale invazive au contribuit n mare msur la
emergena acestei patologii. n faa acestei modificri epidemiologice posibilitile terapeutice s-au mbuntit,
cuprinznd Amfotericina B, azolii i echinocandinele.
2. n iniierea unui tratament antifungic empiric este esenial cunoaterea speciilor de Candida dintr-un anumit areal
geografic, respectiv spital i profilul lor de sensibilitate la antifungice.
3. Diferenele observate ntre Cluj-Napoca i Grenoble subliniaz importana i necesitatea unor protocoale pentru
depistarea i supravegherea pacienilor la risc, pentru stabilirea diagnosticului etiologic micologic, finalizate cu
testarea sensibilitii la antifungice.
4. Cunoaterea speciilor de Candida implicate n patologie, a distribuiei acestora i a sensibilitii lor la antifungice
este esenial pentru iniierea unei terapii optime ct mai precoce, administrarea corect a antifungicelor dup
rezultatul antifungigramei limitnd tratamentul empiric al infeciilor fungice, prevenind selectarea tulpinilor de
Candida rezistente i eecul terapeutic.
5. Comparaia efectuat ntre metoda difuzimetric Neo-Sensitabs i E-test pentru testarea sensibilitii la
antifungice a Candida spp. este al doilea studiu care evalueaz performanele Neo-Sensitabs pentru Amfotericin
B i Caspofungin comparativ cu metoda E-test i primul studiu care evalueaz performanele Neo-Sensitabs
pentru Fluconazol i Voriconazol comparativ cu metoda E-test.
6. S-a obinut o excelent concordan ntre rezultatele E-test-ului i Neo-Sensitabs pentru Fluconazol, Voriconazol
i Caspofungin.
7. Metoda difuzimetric Neo-Sensitabs pentru testarea sensibilitii la Fluconazol i Voriconazol a Candida spp.
prezint multiple similitudini cu metoda standardizat M44-A, putnd reprezenta o alternativ uor de utilizat n
laboratoarele clinice i mai ieftin dect E-test-ul, existnd posibilitatea unei predicii a CMI n funcie de
diametrul zonei de inhibiie prin realizarea de modele de regresie liniar.

Bibliografie selectiv
1. White TC. Mechanism of resistance to antifungal agents. In Murray PR, editor. Manual of Clinical Microbiology. 8th
ed. Washington D.C (USA): ASM PRESS; 2003:p.1869-79.
Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs and mode of transmission. Clin Infect Dis
1996;22(suppl.2):89-94.
2. Espinel-Ingroff A, Barchiesi F, Hazen KC, Martinez-Suarez JV, Scalise G. Standardisation of susceptibility testing
and clinical relevance. Med Mycol 1998;36:68-78.
3. Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs and mode of transmission. Clin Infect Dis
1996;22(suppl.2):89-94.
4. Mares M, Bazgan O. Diagnosticul de laborator al infeciilor produse de fungi. n Buiuc D, Negu M. Tratat de
microbiologie clinic. Ed.Medical, Bucureti; 2008. p.953-6.
5. Espinel-Ingroff A, Canton A, Gibbs D, Wang A. Correlation of Neo-Sensitabs tablet diffusion assay results on three
different agar media with broth microdilution M27-A2 and disk diffusion M44-A results for testing susceptibilities of
Candida spp. and Cryptococcus neoformans to Amphotericin B, Caspofungin, Fluconazole, Itraconazole and
Voriconazole. J Clin Microbiol 2007;45(3):858-64.
6. Espinel-Ingroff A, Canton E. Antifungal susceptibility testing of yeasts. In Antimicrobial susceptibility testing
protocols. Edited by Schwalbe R, Steele-Moore L, Goodwin AC. CRC Press, Taylor & Francis Group 2007.
7. Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr, Filler SG, Fisher JF, Kullberg
BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD; Infectious Diseases Society of America. Clinical

practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin
Infect Dis 2009;48(5):503-35.
8. Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. In vitro susceptibilities of Candida spp. to
caspofungin: four years of global surveillance. J Clin Microbiol 2006;44(3):760-3.
9. Thompson GR, Wiederhold NP, Vallor AC, Villareal NC, Lewis JS, Patterson TF. Development of caspofungin
resistance following prolonged therapy for invasive candidiasis secondary to Candida glabrata infection. Antimicrob
Agents Chemother 2008;52(10):3783-85.
10. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol
Reviews 2007;20(1):13363.
11. Ostrosky-Zeichner L, Rex JH, Pappas PG, Hamill RJ, Larsen RA, Horowitz HW, Powderly WG, Hyslop N,
Kauffman CA, Cleary J, Mangino JE, Lee J. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in
the United States. Antimicrob Agents Chemother 2003;47(10):3149-54.
12. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Meis JF, Gould IM, Fu W, Colombo AL, Rodriguez-Noriega E;
Global Antifungal Surveillance Study. Results from the ARTEMIS DISK Global Antifungal Surveillance study, 1997 to
2005: an 8.5-year analysis of susceptibilities of Candida species and other yeast species to fluconazole and voriconazole
determined by CLSI standardized disk diffusion testing. J Clin Microbiol 2007;45(6):1735-45.

Dr. Ioana Alina COLOSI

Asistent universitar
Universitatea de Medicin i Farmacie Iuliu Haieganu Cluj-Napoca, Romnia
Catedra de Microbiologie
Str. Louis Pasteur nr. 6, cod 400349, Cluj-Napoca, Romnia
Email: ioana.colosi@gmail.com

CURRICULUM VITAE
1. Nume: OPRI, cstorit COLOSI
2. Prenume: IOANA ALINA
3. Data i locul naterii: 27august 1974, Cluj-Napoca
4. Cetenie: romn
5. Stare civil: cstorit
6. Studii:
Liceul
Teoretic U.M.F. Iuliu
Instituia
Haieganu,
Zalu
Cluj-Napoca
Perioada:
Grade sau
diplome obinute

sept.1988iun.1992
Diploma de
bacalaureat

7. Titlul tiinific: MSc, doctorand

8. Experiena profesional:
Perioada:
ian.1999feb.2000
Cluj-Napoca
Locul:
Spitalul
Instituia:
Clinic de
Aduli

oct.1992sept.1998

U.M.F. Iuliu Haieganu

Cluj-Napoca
Catedra de Informatic
Medical i Biostatistic
nov.2007- iun. 2009

Doctor-medic,
specializarea
Medicin
General

mar.2001mar.2003
Cluj-Napoca
Spitalul Clinic de
Aduli

Master Metodologia
Cercetrii n tiinele
Vieii

U.M.F. Iuliu
Haieganu
Cluj-Napoca
Catedra de
Microbiologie
nov.2005Doctorand
domeniul
Medicin

ian.2003oct.2004dec.2007
prezent
Cluj-Napoca
Cluj-Napoca
Spitalul
U.M.F. Iuliu
Municipal
Haieganu,
Cluj/U.M.F.
Cluj-Napoca,
Iuliu
Catedra de
Haieganu
Microbiologie
Medic stagiar
Medic rezident
Medic rezident
Asistent
Funcia:
Medicin de
Medicin de
universitar
Familie
Laborator
9. Locul de munc actual i funcia: Universitatea de Medicin i Farmacie Iuliu Haieganu Cluj- Napoca,
Asistent universitar
10. Vechime la locul de munc actual: 5 ani
11. Membru al asociaiilor profesionale:
Colegiul Medicilor din Romnia
Societatea Romn de Micologie Medical i Micotoxicologie
Societatea Romn de Parazitologie
12. Limbi strine cunoscute: engleza, franceza
13. Specializri i calificri:
Medic specialist Medicin de Familie
Medic specialist Medicin de Laborator
Master UMF Cluj-Napoca, Metodologia Cercetrii n tiinele Vieii
doctorand UMF Cluj-Napoca, domeniul Medicin

14. Cursuri postuniversitare:

- 2 martie 10 aprilie 2009 curs de Mycologie Mdicale, Institutul Pasteur, Paris, Frana.
- 2 februarie 27 februarie 2009 - Stagiu n Laboratoire de Parasitologie-Mycologie a CHU de Grenoble, Frana,
bursa ERASMUS
- 15 mai - 15 august 2008 Stagiu n Laboratoire de Parasitologie-Mycologie a CHU de Grenoble, Frana, bursa
ERASMUS
- Noiembrie 2007 - Curs Methods of studying biofilms and hipermutability organizat n cadrul al 43-lea
Simpozion ESCMID, 2007 Palma de Mallorca, Spania.
- 2003 Curs de pregtire postuniversitar organizat de UMF Cluj-Napoca- Nouti n pneumologie- Boli
caracterizate prin limitarea fluxului aerian
- 2002 Curs de pregtire postuniversitar organizat de UMF Cluj-Napoca- Tuberculoza- Problem de sntate
major n Romnia
- 2001 Pregtire pedagogic, curs postuniversitar organizat de UMF Cluj-Napoca
- 2000 Curs de pregtire postuniversitar organizat de Catedra de Pediatrie III din UMF Cluj- Napoca- Urgene
pediatrice
- 2000 Curs de pregtire postuniversitar organizat de Colegiul Medicilor Cluj i Clinica de Pediatrie II Cluj
Concepte i soluii de tratament i profilaxie a alergiilor la nou nscut i sugar
ACTIVITATE TIINIFIC:
Lucrri publicate in extenso
1. Colosi I, Faure O, Lebeau B, Colosi H, Junie M, Pelloux H. Comparison of two methods, E-test and NeoSensitabs tablet diffusion assay, for testing susceptibility of 93 Candida strains to amphotericin B, fluconazole,
voriconazole, and caspofungin. Sc Parasit 2009:1-2:112-22.
2. Colosi I, Costache C, Junie M. Patogenia infeciilor cu Candida: factorii de patogenitate i factorii de risc ai
infeciilor cu fungi din genul Candida. Clujul Medical 2009;82(1):30-4.
3. Colosi I, Costache C, Junie M. Rezistena biofilmelor de Candida la antifungice. Fungi &Mycotoxins
2009;1(3):242-9.
4. Costache C, Colosi IA, Colosi HA. Immunochromatography versus microscopy for the identification of Giardia
lamblia and Cryptosporidium parvum in human feces. Sc Parasit 2009:1-2:26-31.
5. Colosi I. Methods of studying biofilms and hipermutability. Sc Parasit 2008;1:22-4.
6. Christofidou M, Spiliopoulou A, Vamvacopoulou S, Stamoulis V, Dimitracopoulos G, Anastassiou E, Junie LM,
Costache C, Colosi I, Ciobanca PT. Species distribution and antifungal susceptibility of Candida isolates collected
from hospitalized patients in Romania and Greece. Sc Parasit, 2008;1:61-7.
7. Costache C, Colosi I, Junie M. Specii de Candida izolate din infeciile micotice. Sc Parasit 2008;1:68-73.
8. Costache C, Colosi I. Metoda difuzimetric n testarea sensibilitii la antifungice. Sc Parasit 2008;1:74-8.
9. Costache C, Junie M, Colosi I, Minica F. Incidena parazitozelor la pacienii din Spitalul Clinic Judeean de
Urgen i Clinica de Boli Infecioase. Sc Parasitol 2007;8(1):39-44.
Lucrri publicate n rezumat
1. Colosi I, Faure O, Bourdon C, Dessaigne B, Lebeau B, Pelloux H. Comparison of two methods, Disk diffusion
and E-test, for testing susceptibility of 102 filamentous fungi to amphotericin B, itraconazole, voriconazole,
caspofungin, and posaconazole. Posterpresentation at the 4th Trends in Medical Mycology (TIMM-4), 18-21
October 2009, Athens, Greece. Mycosis 2009;52(S1):30.
2. Colosi I, Costache C, Junie M, Gocan G. Serologic profile for toxoplasmic infection in pregnant women in Cluj
county. Posterpresentation at the X-th European Multicolloquium of Parasitology, Paris, August 24-28, 2008,
Abstract book p.183.
3. Costache C, Muresan L, Colosi I. Intestinal parasites in children hospitalized for different pathologies.
Posterpresentation at the X-th European Multicolloquium of Parasitology, Paris, August 24-28, 2008. Abstract book
p.131.

4. Costache C, Colosi I, Junie M, Colosi H, Bobo C. Antifungal susceptibility testing by disk diffusion on three
different media. Posterpresentation at the 7-th European Congress of Chemotherapy and Infection October 19-22,
2005 Florence, Italy. Abstract book p.109.
5. Junie M, Costache C, Colosi I. The molecular basis of resistance phenomenon to antibiotics of Pseudomonas
aeruginosa strains isolated from patients with urinary tract infections. Al VI-lea Congres Naional de Farmacologie,
Terapeutic i Toxicologie Clinic, iunie 2005, Cluj-Napoca. Volum de rezumate p.478-84.
6. Junie M, ulescu D, Matinca D, Ferke A, Rdulescu A, Colosi I. Pseudomonas aeruginosa strains isolated
from urine samples in Hospitals from Romania and Greece. Posterpresentation at the 11th International Congress on
Infectious Diseases, Cancun, Mexic, March 3-7 2004.
7. Junie M, Ferke A, Tatulescu D, Radulescu A, Colosi I. Resistance pattern of Enterobacter species isolated from
urine and peritoneal liquid. 11th International Congress of Infectious Diseases Cancun, Mexic, March 4-7,
2004;Abstract book p.4.
8. Junie M, Tatulescu D, Costache C, Radulescu A, Colosi I. Identification, clinical and epidemiologic aspects in
candidiasis. Clinical Microbiology and Infection, 13th European Congress of Clinical Microbiology and Infectious
Diseases (ECCMID), Glasgow, UK, May 10-13, 2003;Vol. 9(1):218, (abs).
9. Junie M, Ferke A, Vancea D, ulescu D, Colosi I. Resistance to beta-lactam antibiotics and to cephalosporins
of strains isolated from urine. 13th European Congress of Clinical Microbiology and Infectious Diseases
(ECCMID), Glasgow, UK, May 10-13, 2003; Abstract book p.272.
10. Junie M, Ferke A, Vancea D, Colosi I. Therapeutically aspects in urinary tract infections: the resistance pattern
of Gram-negative bacilli to aminoglycosides, quinolones and other antibiotics. 13th European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID), Glasgow, UK, May 10-13, 2003; Abstract book p. 349.
ACTIVITATE DIDACTIC
Colaborator la cursurile postuniversitare ale Catedrei de Microbiologie: Micologie medical i Parazitologie
medical.
Lucrari practice de Microbiologie cu studenii Facultii de Medicin General anul II, III, VI, secia romn i linia
englez, studenii Facultii de Medicin Dentar anul II, secia romn i linia englez, studenii Facultii de
Farmacie anul II secia romn.
Experiena acumulat n programe/proiecte naionale/internaionale:
Programul/Proiectul
CEEX 78 Constituirea unei reele naionale privind studiul unor zoonoze
parazitare i implicaiilor lor n sigurana alimentelor.
CEEX 99 TRICHID Evaluarea i optimizarea interdisciplinar a metodelor de
screening, diagnostic i tratament n trichineloza i echinococoza chistic uman
i animal n centrul i nord-vestul Romniei.
CEEX - 151 Constituirea unei reele naionale de cercetare a dermatofitozelor la
om i animale.
CEEX- 98/01.08.2006 Studiul biologic, biochimic, corologic i bioproductiv al
unor specii spontane din genul epilobium (fam. Onagraceae) din Transilvania, n
scopul obinerii unor extracte farmaceutice utilizabile n tratarea adenomului de
prostat.
CNMP-PN2-contract nr. 51013 TOXANOM
Optimizarea supravegherii infeciei cu Toxoplasma gondii la unele specii de
animale i la om, ca problem de sntate public n centrul i vestul Romniei.

Funcia
Membru

Perioada:
de la - pn la
2006-2008

Membru

2006-2008

Membru

2006-2008

Membru

Membru

2006-2008

2007-2010

Iuliu Haieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Faculty of Medicine, Department of Microbiology

THE SUSCEPTIBILITY TESTING OF CANDIDA SPECIES

TO ANTIFUNGAL AGENTS

PhD THESIS ABSTRACT

Scientific Supervisor
Prof. Lia Monica JUNIE, MD, PhD

PhD Student
Ioana Alina COLOSI, MD, MSc

Cluj-Napoca 2010

Keywords
Candida, fungal infections, risk factors,
Antifungal drugs: fluconazole, voriconazole, caspofungin, amphotericin B, itraconazole,
E-test, standardized disk diffusion method M44-A, disk diffusion method Neo-Sensitabs.

Introduction
Opportunistic fungal infections represent a new medical challenge at the beginning of the 21st century.
Fungal infections are usually associated with immune dysfunctions and the presence of risk factors (eg, use of
broad spectrum antibiotics). The number of patients with immune disorders has increased dramatically due to AIDS
pandemic, increasing number of transplant patients, aggressive cancer chemotherapy. Fungi are also known as
important etiologic agents of nosocomial infections, causing severe infections in immunosuppressed patients:
neutropenic patients, patients with extensive burns, patients with indwelling catheters, patients at extreme ages.
There is a need of knowledge regarding epidemiology (species involved), pathogenesis (e.g. Candida
isolation does not always mean infection), clinical forms and knowledge of therapeutic aspects of fungal infections.
Therefore, isolation, identification of etiologic agent, and determining the antifungal susceptibility profile of
Candida are essential.
Laboratory assistance for the choice of therapy is crucial by specifying the susceptibility of Candida strains
to antifungal drugs. Thus, a standardized method to determine the antifungal susceptibility of Candida spp. is very
important, especially in the context of the emergence of Candida strains and species resistant to antifungal drugs.
The aim of research presented in the thesis was to determine the distribution of Candida species involved in
human pathology in Cluj-Napoca, their antifungal susceptibility compared to Candida species isolated in Grenoble,
France, and the evaluation of a tablet diffusion assay, as a reliable and affordable in vitro testing method for
antifungal susceptibility of Candida species.
Literature review
The literature review was structured in 5 chapters (Chapters 1-5) and outlines a synthesis of current
knowledge regarding Candida species, synthesis achieved by studying national and international literature (232
references, up to January 2010).
Personal contributions
Original research was structured into two distinct parts, corresponding to the two studies.

The first study: Distribution and antifungal susceptibility testing of Candida species in Cluj-Napoca, Romania and
Grenoble, France, includes chapters 6-10.
Chapter 6 points out the key assumptions, study aim, and objectives. Research aim was to determine the
distribution of Candida species involved in human pathology in Cluj-Napoca and the antifungal susceptibility of
these species compared to Candida species isolated in Grenoble, France.

Chapter 7 presents the studied Candida spp. strains between July 2007-December 2008. We identified and
performed the antifungal susceptibility testing of Candida species isolated from different pathological products of
patients hospitalized in the Infectious Diseases Clinic from Cluj-Napoca and of patients hospitalized in the
University Hospital of Grenoble, France. Samples processed in the laboratory of Infectious Diseases Clinic of ClujNapoca came from both patients hospitalized in the clinic and patients hospitalized in other clinics of Cluj-Napoca,
patients hospitalized in surgical wards, intensive care units, pediatric units and medical departments.
The methods used for susceptibility testing of isolates of Candida spp. to antifungal agents were also
described: for fluconazole and voriconazole - disk diffusion method M44-A and E-test, for caspofungin,
amphotericin B, and itraconazole - E-test.
The following methods of data analysis were used:
- Chi-square test, Fisher's exact test;
- Mann-Whitney U test
- Pearsons correlation coefficient (R values).
To perform statistical analysis and regression models we used SPSS 13.0.
Chapter 8 describes the results obtained in the first study.
Isolated Candida species
In Cluj-Napoca we identified 249 strains of Candida spp.: C.albicans (45%), C.glabrata (26,1%), C.krusei
(10,04%), C.parapsilosis (5,62%), C.tropicalis (4,82%), C.famata (2,41%), C.guilliermondii (2,01%), C.pelliculosa
(0,8%), C.lipolytica (0,8%), C.kefyr (0,8%), C.sake (0,4%), C.lusitaniae (0,4%), C.inconspicua (0,4%), C.curvata
(0,4%).
In Grenoble we identified 813 strains of Candida spp.: C.albicans (51,91%), C.glabrata (20,66%), C.tropicalis
(6,40%), C.parapsilosis (5,78%), C.krusei (5,41%), C.kefyr (4,43%), C.inconspicua (1,85%), C.lusitaniae (1,72%),
C.guilliermondii (0,98%), C.utilis (0,37%), C.catenulata (0,12%), C.lipolytica (0,12%), C.pulcherima (0,12%).
The main Candida species isolated from blood cultures in Cluj-Napoca were C.albicans, C.parapsilosis,
C.glabrata, C.tropicalis, and in Grenoble the main candida species were: C.albicans, C.glabrata, C.parapsilosis,
and C.tropicalis.
The main Candida species isolated from urine in Cluj-Napoca was C.glabrata, and in Grenoble - C.albicans.
In Cluj-Napoca, the main Candida species isolated from patients hospitalized in surgical wards was C.glabrata,
from patients hospitalised in departments of infectious diseases - C.albicans followed by C.krusei, and from patients
hospitalized in pediatric department - C.albicans followed by C. parapsilosis.
Antifungal susceptibility testing and comparing the antifungal susceptibility profile of Candida strains isolated in
Cluj-Napoca and Grenoble
Fluconazole susceptibility testing results are plotted in figures 1 and 2. C.krusei strains were not tested against
fluconazole (C.krusei is resistant to fluconazole).
In Cluj-Napoca, all tested strains of C.kefyr, C.lusitaniae, C.lipolytica, C.guilliermondii, C.curvata, and
C.inconspicua were susceptible to fluconazole.
In Grenoble, all tested strains of C.tropicalis, C.kefyr, C.guilliermondii, C.utilis, C.lipolytica, C.pelliculosa,
C.pulcherima, and C.catenulata were susceptible to fluconazole.

The susceptibility difference to fluconazole of Candida spp. strains isolated from Cluj-Napoca and Grenoble was
statistically significant (p <<0.001, chi-square and Fisher exact test). The relative risk (RR) of a Candida spp. strain
isolated from Cluj-Napoca to be more resistant to fluconazole than a Candida spp. strain isolated from Grenoble
was 4.038, with a 95% confidence interval [2.191 - 7.444].
100%

100%

93.69%

92.31%

91.67%

100%

99.53%

100%

83.33%

100%

97.87%

80%

78.57%

80%

60%

60%

50%

40%

SDD

25%
17.19%

20%

40%

35.71%

16.67%

5.41%

7.69%

8.33%

21.43%

16.67%

20%

0%

8.93%
0.24% 0.24%

0,9%

2.13%

Figure 1. The susceptibility to fluconazole

of Candida strains isolated from Cluj-Napoca.

ae
ni

ita

pi

.in
co

C.
lus

ns

sil

C.
ke

os

cu

fy
r

is

is

SDD

a
.s

ra
p

al

m
.fa

.p
a

at
a

ca
pi

pic

o
.tr

C.
tro

br

is

os

an

sil

.p

ap
ar

ke

C.
gla

b
la
.g

a
at

ic

ca

lis

alb

i
lb
.a

ta
ra

0%

ns

C.

74.40%

57.81%

Figure 2. The susceptibility to fluconazole

of Candida strains isolated from Grenoble.

Voriconazole susceptibility testing results are plotted in figures 3 and 4.

In Cluj-Napoca, all tested strains of C.kefyr, C.lusitaniae, C.guilliermondii, C.lipolytica, C.famata,
C.inconspicua, C.curvata, and C.pelliculosa were susceptible to voriconazole.
In Grenoble, all strains of C.parapsilosis, C.tropicalis, C.kefyr, C.inconspicua, C.lusitaniae, C.guilliermondii,
C.utilis, C.lipolytica, C.pelliculosa, C.pulcherima, and C.catenulata were susceptible to voriconazole.

94.52%

91.67%

100%

100%

97.73%

91.67%

88.89%
80%

77,5%

80%

100%

99.76%

100%

100%
100%

75%
60%

60%

R
S

40%
17,5%

20%
5.48%

20%

5%

15%

10%

11.11%

8.33%

5.35% 2.98%

0.24%

2.27%

.k
e

fy
r

ei
.k
ru
s
C

sil
o
ap
.p
ar

.tr
o

pi
ca

sis

lis

ra
ta
.g
la
b
C

ca
.a
lb
i
C

pi
ca
l
.tr
o

.s
a

is

ke

.p
a

ra
p

sil
o

sis

ei
.k
ru
s
C
C

.g
la
b

ra
ta

s
ca
n
.a
lb
i

ns

0%

0%

SDD

40%

SDD

Figure 3. The susceptibility to voriconazole

of Candida strains isolated from Cluj-Napoca.

Figure 4. The susceptibility to voriconazole

of Candida strains isolated from Grenoble.

The susceptibility difference to voriconazole of Candida spp. strains isolated from Cluj-Napoca and Grenoble
was statistically significant (p <<0.001, chi-square and Fisher exact test). The relative risk (RR) for a strain of
Candida spp. isolated from Cluj-Napoca to be more resistant to voriconazole than a strain of Candida spp. isolated
from Grenoble was 6.295, with a 95% confidence interval [3.033-13.064].

Caspofungin susceptibility testing and comparing the pattern of susceptibility to caspofungin between
Candida strains isolated in Cluj-Napoca and Grenoble
In Cluj-Napoca, all tested Candida strains were susceptible to caspofungin, except for 1 strain of
C.parapsilosis which was non-susceptible to caspofungin. The MIC obtained for this strain was 2 mg/ml.
In Grenoble, all tested Candida strains were susceptible to caspofungin.
The susceptibility difference to caspofungin of Candida spp. strains isolated from Cluj-Napoca and Grenoble
was statistically insignificant (p = 0.139, Fisher exact test).
A highly significant difference (p <<0.001, Mann-Whitney test) was found between the MICs obtained at
caspofungin for Candida spp. strains isolated from Cluj-Napoca and Grenoble. In Cluj-Napoca, the MICs obtained
at caspofungin for the tested Candida spp. strains were within the range 0.032 -2 mg/ml, with a median of 0.25
mg/ml. In Grenoble, the MICs obtained to caspofungin ranged between 0.004 - 1g/ml, with a median of 0.064 mg /
ml (figure 5).

Figure 5. The MICs to caspofungin obtained for Candida spp. strains in Cluj-Napoca and Grenoble.

Amphotericin B susceptibility testing results for Candida spp. strains are plotted in figure 6.
100%

98.20%

100%

98.08%

97.22%

95.74%
86.36%

80%

60%

40%

R
S

20%

0,6%

0%

ic
lb
.a

s
an
C

ab
gl

4.26% 4.54%

1,2% 1.92%

ta
ra
C

op
.tr

ica

lis

.p

o
sil
ap
ar

sis
C

s
ru
.k

9,1%
2.78%

ei
C

yr
ef
.k

Figure 6. The susceptibility to amphotericin B of Candida strains isolated from Grenoble.

All strains of C.albicans, C.inconspicua, C.lusitaniae, C.guilliermondii, C.utilis, C.lipolytica, C.pelliculosa,
C.catenulata, and C.pulcherima tested in Grenoble were susceptible to amphotericin B.

Itraconazole susceptibility testing results of the 32 C.krusei strains tested in Grenoble: 11 strains were
resistant (46.87%), 7 strains were susceptible (9.38%) and 14 strains were SDD (43.75%).
Chapter 9 interprets and discusses the results of the first study by comparison with similar studies.
Chapter 10 presents the main conclusions for the first part of personal contributions. In this abstract, I will only
mention some of these conclusions:
1. The main species of Candida isolates in Cluj-Napoca and Grenoble were Candida albicans and Candida
glabrata. In Cluj-Napoca, Candida krusei (a fluconazole resistant specie) ranged 3rd in frequency of isolation. In
Grenoble, C.krusei ranged 5th, after Candida tropicalis and Candida parapsilosis.
2. Candida spp. strains isolated from Cluj-Napoca were more resistant to fluconazole and voriconazole than
Candida strains isolated from Grenoble.
3. Minimum inhibitory concentration values obtained to caspofungin for the strains of Candida spp. tested in ClujNapoca were higher than those obtained in Grenoble.
4. The knowledge of Candida species involved in fungal infections for a given geographical area, or hospital, and
their susceptibility profile to antifungal drugs are essential for the initiation of empirical antifungal treatment.
5. The differences observed between Cluj-Napoca and Grenoble underline the importance and need for screening
and surveillance protocols for patients at risk for fungal infections, for an accurate mycological diagnosis,
completed with antifungal susceptibility testing.

The second study, Comparison of two methods, E-test and Neo-Sensitabs tablet diffusion assay, for
susceptibility testing of Candida strains to amphotericin B, fluconazole, voriconazole, and caspofungin, includes
chapters 11 through 15.
Chapter 11 points out the assumptions, study aim, and objective. Research aim was to evaluate a tablet diffusion
method, easy to perform, reliable, affordable to test in vitro antifungal susceptibility of Candida species. The
objective was to perform a comparison between two methods of antifungal susceptibility testing of Candida species
to antifungal drugs: E-test (AB Biodisk, Solna, Sweden) and Neo-Sensitabs tablet diffusion assay (Rosco,
Denmark). E-test is a standardized method for testing antifungal susceptibility of Candida, easy to perform, but
whose main disadvantage is the high cost, making it inaccessible for most clinical laboratories, so it is important to
find a cheaper alternative with similar advantages.
Chapter 12 presents the material and methods used to achieve the above goals. Ninety-three strains of Candida
isolated from clinical samples from Grenoble (France) Hospital inpatients were identified and evaluated by E-test
and Neo-Sensitabs tablet diffusion assay between February and April 2009.
We determined categorical agreement levels between E-test MICs and tablet end-points, using the
following disagreement parameters: very major discrepancies - R to E-test and S to tablet; major discrepancies - S
to E-test and R to tablet; minor discrepancies - shifts between S and SDD or SDD and R.
Statistical analysis was performed using linear regression analysis and Pearsons correlation coefficients (R values)
between the log transforms of MICs and the inhibition zone diameters of the antifungal agents. Regression models
were obtained using SPSS 13.0 for Windows.

Chapter 13 describes the results obtained in the second study.

For amphotericin B the categorical agreement between E-test and Neo-Sensitabs assay was 90.32%. Six minor
disagreements (5 strains of C.glabrata and 1 strain of C.albicans), and 3 very major discrepancies (2 strains of
C.glabrata and 1 strain of C.krusei) were observed. The correlation coefficient R between Log2 MICs and the
inhibition zone diameters was -0.622 (figure 7).

Figure 7. Amphotericin B graphical representation

of the linear relation between Log2 MIC (E-test)
and inhibition zone diameters (Neo-Sensitabs).

Figure 8. Caspofungin graphical representation

of the linear relation between Log2 MIC (E-test)
and inhibition zone diameters (Neo-Sensitabs).

For fluconazole, categorical agreement found between E-test and Neo-Sensitabs was 100%. The
correlation coefficient R between Log2 MICs and the inhibition zone diameters was -0.654.
For voriconazole, the percentage of categorical agreement between E-test and Neo-Sensitabs was 100%.
The correlation coefficient R between MICs and inhibition zone diameters was -0.62.
For caspofungin, the percentage of categorical agreement between E-test and Neo-Sensitabs was 98.92%.
One major discrepancy was found (1 strain of C.krusei S by E test and R by the tablet method). One strain of
Candida glabrata was R (non-susceptible) by both methods, with a MIC> 32 mg/ml respectively, an inhibition zone
diameter of 0 mm. The correlation coefficient R between MICs and inhibition zone diameters was -0.679 (figure 8).
In all above cases the computed R-values exhibited a high statistical significance (p<<0.001).
Chapter 14 interprets and discusses the results of the second part of personal research, by comparison with similar
studies. Advantages as well as inherent limitations of our study were highlighted and discussed.
Chapter 15 presents the main conclusions for the second part of personal contributions, of which here I will only

mention the following three:

1. E-test is a standardized, easy to use method for testing susceptibility of Candida to antifungal drugs. MIC
values (g/ml) are sometimes difficult to read (due to the phenomenon of residual growth observed for azoles) and
the method is expensive.
2. The diameters of the inhibition zones obtained using Neo-Sensitabs are well defined and easy to read, proving
this method to be easy to use. Also, the cost of Neo-Sensitabs is lower than for E-tests.

3. Categorical agreement obtained between Neo-Sensitabs and E-test for voriconazole, fluconazole, and
caspofungin was excellent, a lower categorical agreement with E-test was found for amphotericin B.

GENERAL CONCLUSIONS
1. Fungal infections remain a serious threat and their frequency has increased considerably in recent years.
Increasing number of immunocompromised patients and invasive medical or surgical interventions have contributed
to the emergence of this pathology. Faced with these epidemiological changes, therapeutic possibilities were
improved, including amphotericin B, azoles and echinocandins.
2. The knowledge of Candida species involved in fungal infections in a given geographical area, or hospital and
their susceptibility profile to antifungal drugs are essential for the initiation of empirical antifungal treatment.
3. The differences observed between Cluj-Napoca and Grenoble underline the importance and need for screening
and surveillance protocols in patients at risk for fungal infections, for an accurate mycological diagnosis, completed
with antifungal susceptibility testing.
4. The knowledge of Candida species involved in pathology, their distribution and their susceptibility to antifungal
drugs is essential in order to initiate an early and efficient therapy .
5. Comparison between Neo-Sensitabs tablet diffusion assay and E-test for antifungal susceptibility testing of
Candida strains is, to our knowledge, the second study which evaluates the performance of caspofungin and
amphotericin B Neo-Sensitabs tablet method versus E-test, and it is the first study to evaluate the performance of
fluconazole and voriconazole Neo-Sensitabs tablet method versus E-test.
6. Categorical agreement obtained between Neo-Sensitabs and E-test for voriconazole, fluconazole, and
caspofungin was excellent, a lower categorical agreement with E-test was found for amphotericin B.
7. Neo-Sensitabs tablet diffusion assay for testing susceptibility of Candida strains to fluconazole and voriconazole
presents many similaryties with standard method M44-A and may represent a cost-effective and simple alternative
to E-tests in clinical laboratories.
8. Based on the diameter of the inhibition zone, there is a possibility of MIC prediction by carrying out linear
regression models.

Selective bibliography
1. White TC. Mechanism of resistance to antifungal agents. In Murray PR, editor. Manual of Clinical
Microbiology. 8th ed. Washington D.C (USA): ASM PRESS; 2003:p.1869-79.
Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs and mode of transmission. Clin Infect Dis
1996;22(suppl.2):89-94.
2. Espinel-Ingroff A, Barchiesi F, Hazen KC, Martinez-Suarez JV, Scalise G. Standardisation of susceptibility
testing and clinical relevance. Med Mycol 1998;36:68-78.
3. Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs and mode of transmission. Clin Infect Dis
1996;22(suppl.2):89-94.
4. Mares M, Bazgan O. Diagnosticul de laborator al infeciilor produse de fungi. n Buiuc D, Negu M. Tratat de
microbiologie clinic. Ed.Medical, Bucureti; 2008. p.953-6.
5. Espinel-Ingroff A, Canton A, Gibbs D, Wang A. Correlation of Neo-Sensitabs tablet diffusion assay results on
three different agar media with broth microdilution M27-A2 and disk diffusion M44-A results for testing
susceptibilities of Candida spp. and Cryptococcus neoformans to Amphotericin B, Caspofungin, Fluconazole,
Itraconazole and Voriconazole. J Clin Microbiol 2007;45(3):858-64.

6. Espinel-Ingroff A, Canton E. Antifungal susceptibility testing of yeasts. In Antimicrobial susceptibility testing

protocols. Edited by Schwalbe R, Steele-Moore L, Goodwin AC. CRC Press, Taylor & Francis Group 2007.
7. Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr, Filler SG, Fisher JF,
Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD; Infectious Diseases Society of
America. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases
Society of America. Clin Infect Dis 2009;48(5):503-35.
8. Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. In vitro susceptibilities of Candida spp.
to caspofungin: four years of global surveillance. J Clin Microbiol 2006;44(3):760-3.
9. Thompson GR, Wiederhold NP, Vallor AC, Villareal NC, Lewis JS, Patterson TF. Development of caspofungin
resistance following prolonged therapy for invasive candidiasis secondary to Candida glabrata infection. Antimicrob
Agents Chemother 2008;52(10):3783-85.
10. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin
Microbiol Reviews 2007;20(1):13363.
11. Ostrosky-Zeichner L, Rex JH, Pappas PG, Hamill RJ, Larsen RA, Horowitz HW, Powderly WG, Hyslop N,
Kauffman CA, Cleary J, Mangino JE, Lee J. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates
in the United States. Antimicrob Agents Chemother 2003;47(10):3149-54.
12. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Meis JF, Gould IM, Fu W, Colombo AL, Rodriguez-Noriega
E; Global Antifungal Surveillance Study. Results from the ARTEMIS DISK Global Antifungal Surveillance study,
1997 to 2005: an 8.5-year analysis of susceptibilities of Candida species and other yeast species to fluconazole and
voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol 2007;45(6):1735-45.

Dr. Ioana Alina COLOSI

Teaching Assistant
University of Medicine and Pharmacy Iuliu Haieganu Cluj-Napoca, Romania
Microbiology Department
Str. Louis Pasteur nr. 6, zip code 400349, Cluj-Napoca, Romania
Email: ioana.colosi@gmail.com

CURRICULUM VITAE
1. Last name: OPRI, spoused COLOSI
2. First name: IOANA ALINA
3. Place of birth: August 27th, 1974, Cluj-Napoca
4. Citizenship: Romanian
5. Marital status: married
6. Studies:
High School
U.M.F. Iuliu
Institution
Haieganu,
Zalu
Cluj-Napoca
Period:
Diploma

Sept.1988-June
1992
High school
Diploma

U.M.F. Iuliu Haieganu

Cluj-Napoca
Medical Informatics and
Biostatistics Department

Oct.1992Sept.1998
M.D. Licensure
Diploma,
profile: Medicine,
specialty: General
Medicine

7. Scientific title: MSc, PhD student

8. Professional experience:
Period:
Jan.1999Feb.2000
Cluj-Napoca
Place:
Adults
Institution:
Clinical
Hospital

Mar.2001Mar.2003
Cluj-Napoca
Adults Clinical
Hospital

Nov.2007- June 2009

Master in Research
Methodology for Life
Sciences

U.M.F. Iuliu
Haieganu
Cluj-Napoca
Microbiology
Department
Nov.2005PhD student

Jan.2003Oct.2004Dec.2007
present
Cluj-Napoca
Cluj-Napoca
Municipal
Iuliu Haieganu
Hospital
U.M.F. ,
Cluj/Iuliu
Cluj-Napoca,
Haieganu
Microbiology
U.M.F.
Department
Intern
General
Laboratory
Teaching Assistant
Function:
physician
Practitioner
Medicine
Resident
Resident
physician
physician
9. Current work and function: University of Medicine and Pharmacy Iuliu Haieganu Cluj- Napoca, Teaching
Assistent
10. Seniority at current workplace: five years
11. Membership in professional and scientific societies:
Romanian College of Physicians
Romanian Society of Medical Micology and Micotoxicology
Romanian Society of Parasitology
12. Language skills: English - very good, French - good
13. Specializations and qualifications:
General Practitioner Specialist

Laboratory Medicine Specialist

Master in Research Methodology for Life Sciences
PhD student, UMF Cluj-Napoca, Domain Medicine
14. Postgraduate courses:
- March 2 - April 10, 2009 Course Mycologie Mdicale, Pasteur Institut, Paris, France
- February 2 - February 27, 2009 - training stage in Laboratoire de Parasitologie-Mycologie CHU de Grenoble,
France, ERASMUS program
- May 15 - August 15, 2008 - training stage in Laboratoire de Parasitologie-Mycologie CHU de Grenoble, France,
ERASMUS program
- November 2007 - Course Methods of studying biofilms and hipermutability, ESCMID, 2007 Palma de
Mallorca, Spain.
- 2003 Postgraduate course at the University of Cluj-Napoca, Romania: Updates in pneumology
- 2002 Postgraduate course at the University of Cluj-Napoca, Romania: Tuberculosis - a major public health
problem in Romania
- 2001 Postgraduate course at the University of Cluj-Napoca, Romania: Training course in teaching methods
- 2000 Postgraduate course at the University of Cluj-Napoca, Romania: Pediatric emergencies
- 2000 Postgraduate course at the University of Cluj-Napoca, Romania: Allergies in new born babies and small
children-concepts and solutions for treatment and prophylaxis
SCIENTIFIC ACTIVITY:
Papers published in full text
1. Colosi I, Faure O, Lebeau B, Colosi H, Junie M, Pelloux H. Comparison of two methods, E-test and NeoSensitabs tablet diffusion assay, for testing susceptibility of 93 Candida strains to amphotericin B, fluconazole,
voriconazole, and caspofungin. Sc Parasit 2009:1-2:112-22.
2. Colosi I, Costache C, Junie M. The pathogenicity of Candida infections: pathogenic factors and risk factors of
infections with Candida species. Clujul Medical 2009;82(1):30-4.
3. Colosi I, Costache C, Junie M. Candida biofilm resistance to antifungal drugs. Fungi &Mycotoxins
2009;1(3):242-9.
4. Costache C, Colosi IA, Colosi HA. Immunochromatography versus microscopy for the identification of Giardia
lamblia and Cryptosporidium parvum in human feces. Sc Parasit 2009:1-2:26-31.
5. Colosi I. Methods of studying biofilms and hipermutability. Sc Parasit 2008;1:22-4.
6. Christofidou M, Spiliopoulou A, Vamvacopoulou S, Stamoulis V, Dimitracopoulos G, Anastassiou E, Junie LM,
Costache C, Colosi I, Ciobanca PT. Species distribution and antifungal susceptibility of Candida isolates collected
from hospitalized patients in Romania and Greece. Sc Parasit, 2008;1:61-7.
7. Costache C, Colosi I, Junie M. Candida species isolated from fungal infections. Sc Parasit 2008;1:68-73.
8. Costache C, Colosi I. Disk diffusion method for antifungal susceptibility testing. Sc Parasit 2008;1:74-8.
9. Costache C, Junie M, Colosi I, Minica F. Incidence of parasitosis in patients from the Emergency County
Hospital and Infectious Diseases Clinic from Cluj-Napoca. Sc Parasitol 2007;8(1):39-44.
Papers published as abstracts
1. Colosi I, Faure O, Bourdon C, Dessaigne B, Lebeau B, Pelloux H. Comparison of two methods, Disk diffusion
and E-test, for testing susceptibility of 102 filamentous fungi to amphotericin B, itraconazole, voriconazole,
caspofungin, and posaconazole. Poster presentation at the 4th Trends in Medical Mycology (TIMM-4), 18-21
October 2009, Athens, Greece. Mycosis 2009;52(S1):30.
2. Colosi I, Costache C, Junie M, Gocan G. Serologic profile for toxoplasmic infection in pregnant women in Cluj
county. Poster presentation at the X-th European Multicolloquium of Parasitology, Paris, August 24-28, 2008,
Abstract book p.183.
3. Costache C, Muresan L, Colosi I. Intestinal parasites in children hospitalized for different pathologies. Poster
presentation at the X-th European Multicolloquium of Parasitology, Paris, August 24-28, 2008. Abstract book
p.131.

4. Costache C, Colosi I, Junie M, Colosi H, Bobo C. Antifungal susceptibility testing by disk diffusion on three
different media. Poster presentation at the 7-th European Congress of Chemotherapy and Infection October 19-22,
2005 Florence, Italy. Abstract book p.109.
5. Junie M, Costache C, Colosi I. The molecular basis of resistance phenomenon to antibiotics of Pseudomonas
aeruginosa strains isolated from patients with urinary tract infections. The VI-th National Congress of
Pharmacology, Therapeutics and Toxicology, June 2005, Cluj-Napoca. Abstract book p.478-84.
6. Junie M, ulescu D, Matinca D, Ferke A, Rdulescu A, Colosi I. Pseudomonas aeruginosa strains isolated
from urine samples in Hospitals from Romania and Greece. Poster presentation at the 11th International Congress
on Infectious Diseases, Cancun, Mexic, March 3-7 2004.
7. Junie M, Ferke A, Tatulescu D, Radulescu A, Colosi I. Resistance pattern of Enterobacter species isolated from
urine and peritoneal liquid. 11th International Congress of Infectious Diseases Cancun, Mexic, March 4-7,
2004;Abstract book p.4.
8. Junie M, Tatulescu D, Costache C, Radulescu A, Colosi I. Identification, clinical and epidemiologic aspects in
candidiasis. Clinical Microbiology and Infection, 13th European Congress of Clinical Microbiology and Infectious
Diseases (ECCMID), Glasgow, UK, May 10-13, 2003;Vol. 9(1):218, (abs).
9. Junie M, Ferke A, Vancea D, ulescu D, Colosi I. Resistance to beta-lactam antibiotics and to cephalosporins
of strains isolated from urine. 13th European Congress of Clinical Microbiology and Infectious Diseases
(ECCMID), Glasgow, UK, May 10-13, 2003; Abstract book p.272.
10. Junie M, Ferke A, Vancea D, Colosi I. Therapeutically aspects in urinary tract infections: the resistance pattern
of Gram-negative bacilli to aminoglycosides, quinolones and other antibiotics. 13th European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID), Glasgow, UK, May 10-13, 2003; Abstract book p. 349.
TEACHING ACTIVITY
Collaborator in postgraduate courses of the Department of Microbiology: Medical Mycology and Medical
Parasitology.
Practical activities of Microbiology with students of the Faculty of Medicine IInd and IIIrd year, Romanian section
and English study-line, students of the Faculty of Dentistry IInd year, Romanian section and English study-line,
students of the Faculty of Pharmacy IInd year, Romanian section.
Experience in national programs / projects:
Program/Project
CEEX 78 Formation of a national network for study of parasitic zoonoses and
their implications for food safety.
CEEX 99 TRICHID Interdisciplinary evaluation and optimization of
screening methods, diagnosis and treatment of human and animal trichinellosis
and cystic echinococcosis in central and northwestern Romania.
CEEX - 151 Formation of a national networks for research of dermatophytosis
in humans and animals.
CEEX- 98/01.08.2006 Biological, biochemical, and bio productiv study of a
wild species of the genus Epilobium (fam. Onagraceae) in Transylvania, in
order to obtain pharmaceutical extracts used in treating prostate adenoma.
CNMP-PN2-contract nr. 51013 TOXANOM
Optimizing the surveillance of Toxoplasma gondii infection in some species of
animals and humans, as public health problem in central and western Romania.

Function
Member

Period:
2006-2008

Member

2006-2008

Member

2006-2008

Member

Member

2006-2008

2007-2010