In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documented UPC2 gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressed ERG11 by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans. Candida albicans is an opportunistic fungal pathogen that causes mucosal, cutaneous, and systemic infections, including oropharyngeal candidiasis (OPC), the most frequent infection in people with AIDS (9, 13). In the United States, Candida is the fourth-most-common organism isolated from nosocomial bloodstream infections and is associated with a mortality rate approaching 40% (24). Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents, treatment failures have occurred that have been associated with the emergence of azole-resistant strains of C. albicans (25a). The azole class of antifungals work by inhibiting the cytochrome P450 enzyme lanosterol demethylase, a critical enzyme in the synthesis of ergosterol which is encoded by the ERG11 gene (14). The efficacy of fluconazole is decreased in clinical isolates of C. albicans by the interplay of several mechanisms of resistance (17,21,23,32). Overexpression of the efflux transporter genes CDR1, CDR2, and MDR1 is a common mechanism of drug resistance in this organism (10,17,26). Point mutations in the ERG11 gene result in reduced binding affinity of azoles to their target without precluding enzymatic function (31). In addition to point mutations, overexpression of ERG11 has also...

Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for

Erythromycin (EM) and clindamycin (CM) susceptibility testing was performed on 222 clinical isolates of group B Streptococcus. A multiplex PCR assay was used to detect the ermB, ermTR, and mefA/E antibiotic resistance genes. These results were compared to the phenotypes as determined by the standard EM/CM double disk diffusion assay.Group B Streptococcus (GBS) is one of the leading causes of neonatal bacterial infection. This type of infection commonly leads to pneumonia, septicemia, or meningitis. Because of the serious nature of neonatal GBS infections, the suggested standard protocol for the obstetrician/gynecologist is that pregnant women should be tested for the presence of GBS at 35 to 37 weeks of gestation (7,15). Once GBS colonization is diagnosed, the typical treatment for these patients is penicillin, to which there is no known resistance. However, there is a significant population of penicillin-allergic patients, a reported 12% of pregnant women (12), for whom the macrolide (erythromycin [EM]) or lincosamide (clindamycin [CM]) class of drugs needs to be administered, in particular, for those patients who are at high risk for anaphylactic shock. Previous reports have cited resistance of GBS to EM and CM of up to 37% and 17%, respectively (7). The resistance is commonly caused by three genes: ermB, ermTR, and mefA/E (1, 9, 10). The ermB and ermTR genes encode 23S rRNA methylases, which alter the binding of the antibiotic target site. The expression of these genes leads to the constitutively expressed and the erythromycin-induced macrolide, lincosamide, and streptogramin B (cMLS and iMLS, respectively) resistance phenotypes (9). The mefA and mefE genes, which are 90% identical, encode 14-and 15-member macrolide efflux pumps and lead to the macrolide only (M) resistance phenotype (1). Because of the presence of ermB, ermTR, mefA/E, and other antibiotic resistance genes on plasmids and/or transposons, these genes can pass from organism to organism, and the monitoring of the antibiotic resistance of GBS should occur regularly (13). We used a multiplex PCR assay to screen for the prevalence of the ermB, ermTR, and mefA/E genes in GBS clinical isolates from 222 patients for whom physicians ordered GBS testing. The samples, representing 20 states in the United States and 60% of which were from Florida, New Jersey, and Texas, were chosen at random. Patient ages ranged from 15 to 82 years, with an average of 31.3 Ϯ 11.8 years. These results were compared to the antibiotic resistance phenotypes as determined by the standard EM/CM double disk diffusion assay (3,11,15)

Mixed vaginitis is due to the simultaneous presence of at least two vaginal pathogens, both contributing to an abnormal vaginal milieu and, hence, symptoms and signs of vaginitis. In mixed vaginitis, both pathogens require specific therapy for complete eradication of concurrent manifestations. In coinfection, although two pathogens are identified, a potential pathogen may be present but may not be a cause of existing vaginal symptoms. Although data remain sparse, mixed vaginitis occurs rarely (<5 %). By contrast, pathogen coinfection occurs frequently in women with vaginitis. Approximately 20 %-30 % of women with bacterial vaginosis (BV) are coinfected with Candida species. Coexistence of BV pathogens and T. vaginalis is even more common, with coinfection rates of 60 %-80 %. Both coinfection and mixed vaginitis have significant clinical and therapeutic implications and are worthy of further investigation.

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.Vulvovaginal candidiasis (VVC) is a common fungal infection that affects healthy women of all ages. At least 75% of women will develop one or more infections once during their lifetime, with 5 to 8% of those individuals developing recurrent infections (5, 7). Current literature examining the species distribution of Candida isolates involved in VVC is limited; however, several important observations have been made. For example, one study shows that Candida albicans accounts for 70 to 90% of all VVC cases, with a recent emergence of nonalbicans species (10). The rise in VVC infections, more specifically in those caused by non-albicans species, could be due to several factors, ranging from an increase in over-the-counter antifungal use to an increase in high-risk patient populations (i.e., diabetics and menopausal women). Candida glabrata is the primary non-albicans species emerging in VVC, accounting for up to 14% of infections in immune-competent women (9, 10).In addition to an increase in non-albicans species overall, it is becoming clear that certain patient populations may experience higher risks of infection from these non-albicans species, often leading to limited treatment options. Interestingly, in a few small studies, C. glabrata was found to be the primary species isolated from diabetic (61.3%) and elderly (51.2%) patients with VVC (2, 4, 6, 11). Often, these non-albicans species are associated with elevated MIC levels for the azoles, the most commonly prescribed class of antifungal drugs. It has been well documented that C. glabrata demonstrates both intrinsically low susceptibility to the azoles and the ability to develop frank resistance (8,12,13,14,15,16). Moreover, a recent increase in the trailing phenotype, with low-level resistance to the azoles, has been observed for the Candida tropicalis isolates (1, 3). This highlights the importance of identifying Candida species within clinical samples in order to provide physicians with information concerning the proper treatment for their patients.