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FLUCONAZOLE SUSCEPTIBILITY AND ERG11 GENE EXPRESSION IN VAGINAL CANDIDA SPECIES ISOLATED FROM STUDENTS OF A SCHOOL OF NU

CONTRIBUTORS. Pam K.V Akpan J.U., Oduyebo O.O., Smith S.I., Nwokorie F. Ogunsola F.T., Fowora M.A.. INTRODUCTION. Candida spp are ubiquitous organisms inhabits the genital tract as commensals (Mardh et al., 2002; Girardo et al., 2000)But also causes vulvovaginitis.75% of adult women will expe

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FLUCONAZOLE SUSCEPTIBILITY AND ERG11 GENE EXPRESSION IN VAGINAL CANDIDA SPECIES ISOLATED FROM STUDENTS OF A SCHOOL OF NU

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    1. FLUCONAZOLE SUSCEPTIBILITY AND ERG11 GENE EXPRESSION IN VAGINAL CANDIDA SPECIES ISOLATED FROM STUDENTS OF A SCHOOL OF NURSING IN LAGOS NIGERIA Presented by Pam K.V. Post graduate student Department of Medical Microbiology and Parasitology College of Medicine university of Lagos Graduate assistant Department of Microbiology University of Jos

    2. CONTRIBUTORS Pam K.V Akpan J.U., Oduyebo O.O., Smith S.I., Nwokorie F. Ogunsola F.T., Fowora M.A.

    3. INTRODUCTION Candida spp are ubiquitous organisms inhabits the genital tract as commensals (Mardh et al., 2002; Girardo et al., 2000) But also causes vulvovaginitis. 75% of adult women will experience at least one episode of vulvovaginal candidiasis during their lifetime (Chong et al., 2003) approximately 4050% will experience further episodes 5% will develop the recurrent type (RVVC), with at least three symptomatic episodes of vulvovaginitis in 1 year.

    4. Intro fluconazole represents the only systemic therapy that the centers for disease control and prevention (CDC) recommends for the management fluconazole binds to and inhibit the activity of lanosterol 14a-demethylase (erg11p), a key enzyme in the fungal ergosterol biosynthesis pathway (Kelly et al., 1993). resistance to fluconazole has become a problem ; 23% (Cruz, 2003), 5.1%(Xu et al., 2008)

    5. Intro. Mechanisms of resistance over expression of ERG11 (White et al., 2002) amino acid substitutions in the target enzyme Erg11p due to missense mutations in the ERG11 gene (Sanglard and Odds 2002; Perea et al., 2001; White et al., 1998) increased expression of the drug efflux pump genes such as MDR1, CDR1 and CDR2 (Sanglard and Odds 2002; White et al., 1998). Importantly in any one isolate, resistance may be due to a combination of mechanisms (Chau et al., 2004). We need to know the resistance rate of vaginal isolates to fluconazole and the possible mechanisms of resistance.

    6. RATIONAL OF STUDY The only study carried out to examine resistance rate to fluconazole in Nigeria, so far revealed a rate of 9.5%. This study was on oropharyngeal isolates, and did not determine the mechanism, but several mechanisms of resistance to fluconazole have been described in Candida.

    7. OBJECTIVES OF STUDY Evaluate the susceptibility of clinical isolates of Candida to fluconazole. Demonstrate the expression of ERG11 genes in fluconazole resistant isolates.

    8. STUDY DESIGN Clinical isolates were previously obtained from LUTH school of nursing students and stored in glycerol broth at -800 . were sub cultured onto sabouraud dextrose agar Fluconazole susceptibility test was carried out on the isolates. the resistant and susceptible dose dependent species were subjected to polymerase chain reaction using primers that identify ERG11 gene expression.

    9. MATERIALS AND METHODS STUDY POPULATION The isolates were identified using API Candida kit, CHROM Candida agar and germ tube

    10. ANTIFUNGAL DRUG SUSCEPTIBILITY TESTING 2-3 distinct colonies were emulsified in 5mL of sterile saline and inoculated on Mueller-Hinton + Glucose+ Methylene Blue agar plate Fluconazole (25g) was placed on the surface of the inoculated agar plate and plate incubated at 370C for 20 to 24 hours after which zones of inhibition were measured The result was interpreted using Pfaller et al.s (2006) validated CLSI interpretive breakpoints for in vitro susceptibility testing of fluconazole and Candida spp and reported as susceptible S, MIC (zone diameter), 8 g/ml (>19 mm); susceptible dose dependent SDD, 16 to 32 g/ml (15 to 18 mm) and resistant R, 64 g/ml (<14 mm).

    11. AMPLIFICATION OF ERG11 GENES Yeast strains were cultured on Sabouraud Dextrose Agar (SDA) at 370Cfor 24 hours. The ERG11 genes of all fluconazole resistant and susceptible doses dependent isolates were amplified using PCR.

    12. METHOD.... PCR DNA was extracted from yeast isolates using a previously described method by Kitamura et al., in 1971. In this study we modified their procedure. PCR was carried out by using primers that span the entire ERG11 open reading frame: 5-GTT GAA ACT GTC ATT GAT GG (forward) and 5-TCA GAA CAC TGA ATC GAA AG (reverse). The PCR was performed in a 25 well thermocycler. The system was programmed to 3 min of denaturation at 92oC and this was followed by 30 cycles, each consisting of 1 min of denaturation at 92oC, 1 min of annealing at 43o ,1 min of extension at 72oC.

    13. ERG11 gene. At the final cycle, an additional 10 min of incubation at 72oC was performed for complete extension. a negative control was also included containing the reaction buffer, dNTP, Taq polymerase without the target DNA. The PCR product was detected by electrophoresis on a 2% agarose gel using X1 Trisacetate EDTA buffer (40 mM Tris acetate, 1mM EDTA (pH 8.4)) the gel was stained with 1-2 drops of ethidium bromide (ethidium bromide intercalates DNA present and fluoresces under UV light) and photographed under UV light (Clinx Science Instruments Genosens 1500)

    14. RESULTS

    15. Table 1: In vitro susceptibilities of Candida spp. to fluconazole as determined by CLSI disk diffusion method Species Number of isolates (%) tested S SDD R C. albicans 14(50%) 12(85.7%) 1(7.1%) 1(7.1 %) C. tropicalis 6(21.4%) 3(50%) 3(50%) - C. glabrata 2(7.14%) 1(50%) 1(50%) - C. kefyr 2(7.14%) 1(50%) 1(50%) - C. famata 2(7.14%) 1(50%) - 1(50%) C. guilliermondii 1(3.57%) - 1(100%) - C. parapsilosis 1(3.57%) - 1(100%) - Total 28(100%) 18(64.3%) 8(28.6%) 2(7.14%) S, susceptible; SDD, susceptible dose dependent; R, resistant.

    16. PCR products lanes 11, 12, 13 with marked amplification other lanes have faint amplification at about 92bp.

    17. PCR products lanes 11, 12, 13 with marked amplification other lanes have faint amplification at about 92bp.

    18. DISCUSSION Most of the major Candida spp were represented including C. albicans (50%), C. tropicalis (21.4%), C. glabrata (7.14%), C. famata (7.14%), C. kefyr (7.14%), C. guilliermondii (3.57%) and C. parapsilosis (3.57%). In epidemiological studies and for clinical therapy it is important to distinguish the different Candida spp involved in an infection. For antifungal resistant species C. krusei, glabrata

    19. C albicans Most of the C. albicans (67%) were sensitive to fluconazole, 22.2% were susceptible dose dependent in which increase in the dosage of fluconazole when administered clinically could yield good therapeutic results in-vivo (Andes, 2003; Rex et al., 2001). Only one isolate of C. albicans showed in vitro resistance.

    20. Non-albicans most surveillance studies conducted on Candida spp have reported susceptible dose dependence in non- albicans Candida spp, in this study 57.1% of them were susceptible dose dependent C. tropicalis, C. glabrata, C. famata, C. kefyr , C. gulliermondi, C. parapsilopsis all which have been documented to cause vulvovaginal candidiasis. Use a dose higher of fluconazole up to 400mg in some cases (normally 150mg) Probably a result induced resistance because studies have associated susceptible dose dependence with previous exposure of isolates but 1(one) isolate of C. famata was resistant to fluconazole- boric acid, other azoles.

    21. An overall resistance rate of 7.14% (two isolates) was noted, similar to that reported by Enwuru et al. (2008) where they had a fluconazole resistance rate of 9.5% in Candida isolates Although their isolates were obtained from HIV patients with oropharyngeal Candidiasis.

    22. Lanosterol demethylase is encoded by the gene ERG11 (also known as CYP51) In this study there was an over expression of the ERG11 gene in three (3) of the isolates (fig 1) that were susceptible dose dependent (S-DD). The isolates that over expressed ERG11 gene were susceptible dose dependent, probably confirming need to increase dose of drug to overcome the excessively produced enzymes, which inhibit fluconazole action. Other co-existing mechanisms may be in place

    23. There was no over expression of the ERG11 gene in the two isolates that were resistant (R) to fluconazole. Their mechanisms of resistance could be amino acid substitutions in the target enzyme Erg11p due to missense mutations in the ERG11 gene (Sanglard and Odds 2002; Perea et al., 2001; White et al., 1998) increased expression of the drug efflux pump genes such as MDR1, CDR1 and CDR2 (Sanglard and Odds 2002; White et al., 1998). Routine detection of resistance to fluconazole is clearly desirable, because early recognition of resistance in clinical isolates contribute to successful clinical and microbiologic outcomes (Tan and Patterson 2005; Rex and Pfaller, 2002).

    24. CONCLUSIONS AND RECOMMENDATIONS 7.14% of vaginal Candida isolates from school of nursing students in LUTH Lagos were resistant to fluconazole while 28.6% were susceptible dose dependent to fluconazole. We therefore, highlight the need for routine antifungal susceptibility testing on vaginal candida Over expression of ERG11 genes existed in 3(30%) of the fluconazole resistant and dose dependent isolates. A study of the resistance mechanisms of fluconazole is a potentially valuable tool to track the emergence and spread of fluconazole-resistant C. species

    25. THANK YOU

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