RESUMO
Resistance to azole antifungals continues to be a significant problem in Candida albicans. One of the major mechanisms of azole resistance in C. albicans has been reported to be mutations in the ERGII gene encoding the azole target enzyme cytochrome P-450-dependent lanosterol 14 alpha- demethylase. This study was carried out to explore the presence of any point mutation in ERGII gene sequence of some C.albicans clinical isolates resistant to fluconazole. Three hundred C. albicans isolates, from various specimens were examined. These isolates were tested for antifungal susceptibility to fluconazole. The minimum inhibitory concentrations [MICs] of resistant C. albicans isolates were done by NCCLs macrobroth dilution method and E test. C. albicans ATCC 90028 was used as a control susceptible strain in all tests. The resistant C. albicans isolates were tested by PCR to amplify the entire ERGII gene. The isolates were further tested for point mutation concerning the ERGII gene by singlestranded conformation polymorphism [SSCP] analysis and DNA sequencing. Sixteen [5.4%] out of 300 of the isolates were resistant to fluconazole by disc diffusion and macrobroth dilution [MICs ranged from 64 - 128 micro g/ml]. E test showed 4 [25%] sensitive isolates [MIC 4-8 micro g/ml] and 12 [75%] resistant [MIC 64 - 256 micro g/ml]. Point mutation [fragment variations] in ERGII gene was detected in 7[43.75%] isolates by SSCP technique. DNA sequencing of the PCR amplified ERGII fragments of four isolates that showed band mutation by SSCP revealed; 25 nucleotide changes, resulting in 4 amino acid substitutions in regions covered by fragments that did and did not show variation by SSCP. Accordingly, the sensitivity of the SSCP technique was [55.5%]. Ten of these changes were also present in strain ATCC90028, other mutations exist in resistant isolates only and these mutations could play a role in drug resistance alone or in association with other mechanisms particularly when they resulted in an amino acid substitution. Although, PCR-SSCP analysis can give false positive results, since not all mutations detected can cause amino acid changes [silent mutations], our results concerning DNA sequencing were confirmative for strains showing positive PCR-SSCP results
RESUMO
Deletions of the AZFc [azoospermic factor c] region of the Y chromosome including DAZ gene are the most common known cause of spermatogenic failure. This study was conducted with the aim of detecting Y chromosome microdeletions involving the DAZ locus in idiopathic male infertility to allow rapid and accurate diagnosis required for proper genetic counseling. The study included 30 male patients with idiopathic azoospermia [24/30] or oligozoospermia [6/30]. A control group consisted of 10 normal fertile males and 5 females. All cases were subjected to detailed history, clinical examination, assessment of testicular volume, semen analysis, serum hormonal profile [FSH, LH, testosterone], testicular biopsy, chromosome analysis, polymerase chain reaction [PCR] amplification of two specific loci of the DAZ gene on the Y chromosome [sY254 and sY255], single strand conformations polymorphism analysis [SSCP]. The result of the study revealed that deletions involving the sY245 and sY255 DAZ loci were found in 4 cases [4/30; 13.3%]; 3 azoospermic patients and one with severe oligozoospermia. All four cases with microdeletions had decreased testicular volume, normal serum LH and T, serum FSH was elevated in 3 of them and normal in one. The two loci were amplified normally in the male control group and failed to amplify in the female control group. SSCP analysis failed to find any point mutations in sY254 and sY255 in patients with absence of microdeletions of DAZ gene. In conclusion, the estimated frequency of microdeletions involving the DAZ locus is 13.3% in azoospermic and severely oligozoospermic Egyptian men with idiopathic infertility. Polymerase chain reaction amplification of the DAZ locus is a rapid and accurate method for the diagnosis of microdeletions of the Y chromosome in patients with idiopathic infertility especially those seeking micromanipulation assisted reproduction