Comparative Methods of Speciation of Candida Isolated from Patients with Vulvovaginitis in Ekpoma and Environs, Nigeria. N. J. Inyang1, I. B. Enweani2, F. I. Esumeh3, E. Agwu4, J. A. Olaboye1, J. O. Ikheloa1 INTRODUCTION Candida albicans is the most incriminating pathogen.Candidiasis can either be as a result of primary or secondary infection involving a member of the genus Candida... The frequency of infections caused by Candida has been increasing worldwide due to a multiplicity of predisposing factors such as AIDS, diabetes, leukemia and cancer, which facilitates the conversion of the commensal form to the parasitic existence.The incidence of nosocomial candidemia has risen sharply in recent years, particularly in critical care units.There has beenan important shift in the type of Candida infections—away from Candida albicans—to more treatment-resistant, non-albicans varieties. As the pathogenicity and antifungal susceptibility of C. albicans often vary among strains, identification of the disease-causing strains is crucial for diagnosis, clinical treatment and epidemiological surveillance. Study was undertaken to compare the effectiveness of various methods of Candida identification isolated from high vaginal swabs (HVS) of patients suffering from vulvovaginalcandidosis in Ekpoma  Fig.1: DNA Amplified Products of C. albicans suspected to be Candida dubliniensis Lane 1= molecular markers, 2= negative control, 3= C. dubliniensis Known Isolate, lane 4-8 C. albicans strains. MATERIALS & METHODS: A total number of 696 High Vaginal Swabs were collected. These were processed for Candida speciation employing different techniques(Germ tube production,Chlamydospore formation, Ability of yeast to grow at 450C differentiated C. albicans from C. Dubleniensis and sugar fermentation tests) to ascertain the effectiveness of the methods was undertaken for the isolates. ChromagarCandida was used to presumptively identify the yeast species. Phenotyping was carried out using ID32C for carbohydrate assimilation. read visually, with mini API(R) instrument or with ATBTM (BioMereux(R) Paris, France). Identification was then obtained using the identification software. Three sets of primers were designed for this study, C. dubliniesis Specific primers, a universal primer sets that could amplify 610 bp from any fungi template and a 25SrRNA C.albicans specific primer set was also deigned. DNA amplification was done using PCR thermalCycle program while Analyses of PCR products were done by Agarose gel electrophoresis of Yeast Amplified products. Genotypic identification was employed  Fig.2: DNA Amplified Products of Candida albicans, lane 1= C. dubliniensisstandard stain, lane 2-9 C. albicans isolate, lane 10 = molecular marker   Fig 3: Southern blot of EcoR1 digested DNA of C. albicans strains hybridize with Ca3 probe lane 1-3 High vaginal swab, lane 4and5 wound swab, lane 6 mid stream urine, lane 7 catheter urine lane 8 semen and lane 9 sputum isolated DNAs DISCUSSION: Colour on Chromagar medium, sugar assimilation pattern on ID32C and the ability of all the isolate to copy at 610bp using universal fungal primer, showed a statistically significant difference at P<0.05. Using the universal fungal primers 612 isolates were confirmed as yeast, with all the amplified product copying at 610bp (Figs. 1& 2). Fig. 2 showed the genomic DNA of different strains of Candida albicans isolated from different specimens. Result obtained using the Candida dubliniensisspecific primers revealed that some suspected C. dubliniensis strains were C. albicans, with none yielding additional 288bp band unique to C. dubliniensis (Fig. 3). Fig.3 showed the numerous bands generated by cutting genomic DNA of selected C. albicans isolates with EcoR1. Hybridization with Ca3 probe revealed some strain similarities and differences among C. albicans isolates. For instance , isolate 3 from HVS, 4 and 5 from wound swab and 6 from urine showed similar finger print patterns with additional and unique fragments at 4.5bp and 2.2bp (lanes 3-6, Fig.3). Isolates 7 (Urine) and 8 (semen) have similar finger print patterns (lanes 7 and 8, Fig.3), different from isolates on lanes 1, 2, and 9 with separate patterns   This showed the effect of diagnostic tools in Candida characterisation. Candida albicans was seen in co-infections with C. tropicalis, C. glabrata and S.cerevesiae and in triple infections with C. tropicalis, C. glabrata and C. norvegensis. All strains formerly identified as C. stellatoidea with some techniques were found to be non- sucrose fermenting variant of C. albicans with genotypic identification. RESULTS: Germ tube production, chlamydospore formation and sugar fermentation test grouped the isolates into C. albicans 205 (81.8%), C. stellatoidea 11(4.3%) and other Candida species 35 (13.9%). Further phenotypic identification using ChromagarCandida ID32C, growth at 450C, and PCR analysis identified six species which included C. albicans 216 (26.1%) C. glabrata, 11 (4.4%).C. tropicalis 9 (3.6 %) C. parapsilosis 5 (20%) C. norevegensis 4 (1.6%), S. cerevesiae 3 (1.2%), others 3 (1.2%). In a single co-infection, C.albicans and C. tropicalis; C. albicans and C. glabrata; C. tropicalis and C.glabrata were isolated. In triple co-infection, C. albicans,C.glabrata and C. tropicalisas well as C. albicans, C. glabrata and C.norevegensiswere recovered from HVS samples 1Irrua Specialist/Teaching Hosp., Irrua, NIGERIA, 2Nnamdi Azikiwe Univ., Nnewi Campus, NIGERIA, 3Ambrose Ali Univ., Ekpoma, NIGERIA, 4Kampala Intl. Univ., Western Campus, UGANDA