Article

Rapid Identification of Candida albicansand Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR

Journal of Clinical Microbiology

July 1998
36(6):1634-41

DOI:10.1128/JCM.36.6.1634-1641.1998

Source
PubMed

Authors:

Cletus Kurtzman

National Center for Agricultural Utilization Research

A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a new Candida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii, C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii, C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5' end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3' end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.

Ultrastructure of fibrillar layer of Candida albicans in serum culture

Article

Jan 2001

Adhesin, hydrolytic enzyme, and germ tube formation are regarded as major virulent factors of Candida albicans. In the pathogenesis of Candida albicans, the first step is adherence to host tissue with adhesin, and then invasion to the tissue by secreting hydrolytic enzymes like protease and phospholipase. The fuzzy coat is of interest as it may play important roles in antigenicity, phagocytosis, and adherence factors that are important in the virulence of C. albicans. The virulent factors could be expressed higher in vivo than in vitro and serum is similar to in vivo condition. Therefore, using transmitting electron microscopy, we studied the effects of serum on fibril expression of C. albicans YWMC132. The fibrillar layer of C. albicans was expressed only in serum. The fibril expression was not inhibited at concentrations of C. albicans until 8×107 cells/ml. However, the thickness of the fibril layer was reduced from 100-130 nm to 20 nm according to increases in cell concentrations from 2×107 cells/ml to 8×107 cells/ml. The fibrillar layer of C. albicans was expressed in serum, early, within 10 min, but the germ tube formation was expressed only after 60 min. When the fibrillar C. albicans was treated with lyticase, protease, or dithiothreitol (DTT), the fibrillar layer disappeared. The protein concentration was higher in the fibrillar cell than in the nonfibrillar cell. These results indicated that the proteinous fibrillar layer of C. albicans YWMC132 was expressed only in serum culture.

Virulence Factors as Promoters of Chronic Vulvovaginal Candidosis: A Review

Article

Full-text available

Dec 2021
MYCOPATHOLOGIA

Introduction The vast majority of the species of the genus Candida spp. is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections. Aim and methodology The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate. Results and Discussion Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here.

Genotyping and Antifungal Susceptibility Profile of Sequential Candida albicans Isolated from the Oral Cavity of HIV-Infected Individuals

Article

Full-text available

Apr 2018

ABSTRACT Objective: The study aimed to evaluate the genotypic profiles of C. albicans (Candida albicans) sequentially isolated throughout the course of HIV infections, and to determine its MIC (minimal inhibitory concentrations) to AMB (amphotericin B), FLC (fluconazole), KTC (ketoconazole), and ITC (itraconazole). Design: samples were collected from the oral cavity of HIV-positive individuals during 4 years, with a sterilized swab. MIC was performed by using the microdilution method AFST/EUCAST. The genetic similarities within and between sequential clones of C. albicans were assessed by DNA fingerprinting using the random amplification of polymorphic DNA technique. Results: A total of 142 oral samples were isolated from 59 HIV-infected individuals who attempted up to five visits each, with or without symptoms of oropharyngeal candidiasis. Profile analysis revealed that yeasts isolated over sequential visits from symptomatic or asymptomatic individuals showed 78% or 87% relatedness, respectively. The degree of similarity among C. albicans was higher for isolates from colonization than for those from infection. Genetically identical C. albicans samples also formed connected subclusters in sequential visits. In regard to susceptibility profile, all isolates were susceptible to AMB, FLC, KTC, and ITC and maintained this pattern all along, no differences in MICs of any given antifungal compound were observed for sequential C. albicans isolates. Conclusions: These data suggest that genotype and susceptibility to antifungal drugs were maintained over time in sequentially isolates of C. albicans colonization and a diverse evolutionary genetic trend in C. albicans sequentially isolated from the oral candidiasis of HIV infected individuals.

Hyphal wall protein 1 gene: A potential marker for the identification of different Candida species and phylogenetic analysis

Article

Full-text available

Dec 2016

Background and Purpose Hyphal wall protein 1 (HWP1) is an important adhesin which usually is expressed on the germ tube and hyphal surface produced by different Candida species. The hyphal wall protein-coding gene (HWP1) was evaluated as a novel identiﬁcation and phylogenetic marker in Candida tropicalis, C. orthopsilosis, C. parapsilosis and C. glabrata. Materials and Methods Initially, four specific primer pairs were designed, and the target was amplified and finally sequenced. A total of 77 Candida isolates from four different species were included in the study. Consensus sequences were used for the evaluation of phylogenetic tree using the CLC Genome Workbench, GENEIOUS, and MEGA softwares and the levels of nucleotide and amino acid polymorphism were assessed. Results According to the results, the specific amplified fragments of HWP1 gene were useful for the differentiation of four species. Intra-species variation was observed only in C. tropicalis with two DNA types. The phylogenetic tree of Candida species based on the HWP1 gene showed consistency in topology with those inferred from other gene sequences. Conclusion We found that HWP1 gene was an excellent marker for the identification of non-albicansCandida species as well as the phylogenetic analysis of the most clinically significant Candida species.

Candida Mastitis in Dairy Cattle with Molecular Detection of Candida albicans

Article

Full-text available

Feb 2016
KAFKAS UNIV VET FAK

This study focused on determining the prevalence of Candida species involved in dairy cattle mastitis with molecular detection of Candida albicans. A total of 150 milk samples were collected from dairy cattle showing clinical mastitis. Isolation and identification of organisms through phenotypical and physiological criteria on different media were performed as well as molecular identification of C. albicans. Fourty one isolates of Candida species were recovered with a prevalence of 27.3%. C. albicans was the dominating species (29.3%). Out of 12 strains phenotypically identified as C. albicans, 8 were confirmed by PCR using species specific primer for the 26S rRNA gene of C. albicans. A specific virulence determinant Phospholipase B1 gene was detected in all molecularly identified C. albicans isolates. This study has clearly shown the prevalence of Candida mastitis and providing a new attractive diagnostic molecular tool for mycotic mastitis caused by C. albicans.

Photodynamic Therapy in Candida spp. and Epithelial Cell Cultures: An In vitro Study

Article

Dec 2023

Aims: The objective of this study was to determinate the photodynamic effect using methylene blue and diode laser on Candida spp. and epithelial cells in vitro. Study Design: An in vitro study was carried on using cultures of Candida spp. and HEp-2 cells. Place and Duration of Study: This study was developed at the School of Biosciences of the Department of Microbiologic Sciences and at the Laboratory of Pneumology of the Biomedical Research Institute at São Lucas Hospital, both at the Pontifical Catholic University of Rio Grande do Sul, located in Porto Alegre, Brazil, between January and December. Methodology: Cultures of Candida spp. and HEp-2 cells were submitted to aPDT with methylene blue (100 μg/mL) and indium-gallium-aluminum-phosphide (InGaAlP) diode laser at 100 J/cm2, 270 J/cm2 and 450 J/cm2. Results: All of these three doses caused significant inactivation of Candida spp. (P<0.05). At 450 J/cm2, the viability of Candida spp., based on colony forming units (CFUs), was reduced by 72.42%, followed by lesser effects with 270 J/cm2 and 100 J/cm2, 45.87% and 22.83%, respectively. PDT decreased CFUs by 50.44% in C. albicans, while other Candida species showed a 41.18% decline in CFUs (P<0.05). With regard to the average effect of the three doses tested in PDT group, HEp-2 cell viability, based on trypan blue exclusion, declined to 70.81%, which was significantly lower than that observed in the control group (86.21%). Conclusion: Methylene blue plus laser exposure (100 J/cm2, 270 J/cm2 and 450 J/cm2) caused significant inactivation of Candida spp. Photodynamic inactivation of the epithelial cells based on cell viability was 2.24-fold lower than the inactivation of Candida spp., which suggests a safety margin for in vivo application.

Isolation and Characterization of Marine fungi for their Bioactive pigments

Article

Full-text available

Nov 2022

Microorganisms are known resource for the pigment production due to their stability, availability, cost efficacy, downstream processing and yield. The elevated biological activities of natural pigments are incited to evaluate their biological importance against various pathological indices. In this regards, the study aim to evaluate the antibacterial and antioxidant activity of pigments extracted from marine fungi. The marine fungi PC, CL and PP isolated from marine sediments, were identified as Rhodotorula evergladensis, Aspergillus sydowii and Talaromyces coalescens, respectively through ITS sequence similarity analysis. Then, the pigments were extracted from the fungal isolates and their purity was verified using HPLC chromatogram. Further, the pigments from PC and CL found to possess promising antibacterial activity against human pathogens like Escherichia coli, Bacillus cereus, Shigella sp, Salmonella typhi and Klebsiella pneumoniae. But, the pigment from PP did not show such conspicuous antibacterial activity. Further, DPPH and peroxide ion scavenging activity showed 65% and 70% of antioxidant potential for PC and PP pigment extracts respectively. When the extracts were subjected to reducing power assay, CL pigment extract showed 50% reduction to ferrous ions, but PP and PC showed only 30% reducing activity. Thus, the study revealed that the pigments of R. evergladensis (PC)and T. coalescens (PP) are found to have promising antioxidants activity besides antibacterial activity. Among the fungal strains, the pigment from A. sydowii (CL) also exhibited a better metal ion reducing property. Therefore, these fungal pigments can be harnessed in textile, food processing and other beverage producing industries.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis

Article

Full-text available

Jul 2022

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 10¹ copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 10⁴ copies/μL) and internal transcribed spacer (ITS; 6.74 × 10⁵ copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Long-Reads-Based Metagenomics in Clinical Diagnosis With a Special Focus on Fungal Infections

Article

Full-text available

Jan 2022

Identification of the causative infectious agent is essential in the management of infectious diseases, with the ideal diagnostic method being rapid, accurate, and informative, while remaining cost-effective. Traditional diagnostic techniques rely on culturing and cell propagation to isolate and identify the causative pathogen. These techniques are limited by the ability and the time required to grow or propagate an agent in vitro and the facts that identification based on morphological traits are non-specific, insensitive, and reliant on technical expertise. The evolution of next-generation sequencing has revolutionized genomic studies to generate more data at a cheaper cost. These are divided into short- and long-read sequencing technologies, depending on the length of reads generated during sequencing runs. Long-read sequencing also called third-generation sequencing emerged commercially through the instruments released by Pacific Biosciences and Oxford Nanopore Technologies, although relying on different sequencing chemistries, with the first one being more accurate both platforms can generate ultra-long sequence reads. Long-read sequencing is capable of entirely spanning previously established genomic identification regions or potentially small whole genomes, drastically improving the accuracy of the identification of pathogens directly from clinical samples. Long-read sequencing may also provide additional important clinical information, such as antimicrobial resistance profiles and epidemiological data from a single sequencing run. While initial applications of long-read sequencing in clinical diagnosis showed that it could be a promising diagnostic technique, it also has highlighted the need for further optimization. In this review, we show the potential long-read sequencing has in clinical diagnosis of fungal infections and discuss the pros and cons of its implementation.

Difference Between the Profiles Presented by Yeasts that Colonize the Vaginal Mucosa or Cause Primary or Recurrent Candidiasis

Article

Full-text available

Jun 2021
MYCOPATHOLOGIA

Candida yeasts are the most frequent in the vaginal content. This yeast may be a normal microbiota but also causes candidiasis. In symptomatic cases, primary candidiasis (VVC) or recurrence (RVVC) can be considered. This study aims to compare the frequency and in vitro sensitivity profile of Candida species isolated in the vaginal content with the different stages of the presence of yeasts. A total of 258 non-pregnant patients with/without VVC were prospectively screened at a teaching Health Centre of the Faculty of Medicine, in the University of Sao Paulo. The vaginal isolates were identified by traditional and molecular methods. Yeasts were isolated in 160 women. 34% were asymptomatic, 34% with vulvovaginal candidiasis (VVC), and 32% recurrent vulvovaginal candidiasis (RVVC). C. albicans was the most frequent species with 50.1% (82/160), followed by C. parapsilosis 13.7%(22/160), C. glabrata 12.5% (20/160), and C. tropicalis (6.2%). Analysis by the group showed that, in the asymptomatic group, eight yeast species were isolated, C. albicans 44.5% (24/54), C. glabrata 20% (11/54), C. parapsilosis and Rhodotorula rubra being the most frequent. In the VVC group, 11 yeast species were identified. Most isolates were C. albicans 68.5% (37/54), C. tropicalis 7.5% (4/54), and C. parapsilosis 5.5% (3/54). In the RVVC group, ten species were identified, the most frequent being C. albicans 38.5% (20/52), C. parapsilosis 17% (9/52), C. glabrata 4% (8/52), and C. tropicalis 6% (3/52). Less frequent species, such as C. haemulonii and Trichosporon spp, were isolated in the VVC and RVVC groups, C. kefyr was isolated in the three groups studied, and Rhodotorula spp was isolated in the control and RVVC groups. Candida metapsilosis was present in two isolates from the RVVC group. Most isolates were considered sensitive to the tested antifungals. Less sensitivity was seen for caspofungin. In this study, we were able to verify that the most common species of yeasts found in vaginal secretion were isolated in the three groups studied; however, there was the diversity of species in VVC and RVVC. Cryptic species C. haemulonii and were isolated in symptomatic patients. High levels of MICs, some of the antifungals tested, in the control group, draw attention in the group of asymptomatic women. We would like to emphasize that this research aims to assist clinicians and gynecologists, as well as assist in the epidemiological studies of candidiasis, in our country, how to draw attention to the profile of sensitivity/resistance to antifungals.

Hyposalivation, oral health, and Candida colonization in independent dentate elders

Article

Full-text available

Nov 2020
PLOS ONE

Hyposalivation is an important problem in elders and could interfere with several oral functions and microbial ecology. While the number of independent elders who retain more natural teeth increases worldwide, few studies examined hyposalivation in this population. Thus, this study aims to examine relationships between hyposalivation, oral health conditions and oral Candida colonization in independent dentate elders and evaluate factors associated with salivary flow and Candida carriage. We conducted a cross-sectional study in fifty-three dentate elders (≥65 years old with at least 4 pairs of posterior occlusal contacts) with no, or well-controlled, systemic conditions. Participants were interviewed for medical history, subjective dry mouth symptoms, oral hygiene practices and denture information. Unstimulated and stimulated salivary flow rates, objective dry mouth signs, gingival, tongue-coating, and root-caries indices were recorded. Stimulated saliva was cultured on Sabouraud-dextrose agar for Candida counts. Candida species were identified using chromogenic Candida agar and polymerase chain reaction. Statistical significance level was set at p<0 . 05 . The results showed that hyposalivation was associated with higher gingival and tongue-coating indices ( p = 0 . 003 and 0 . 015 , respectively), but not root-caries index. Hyposalivation was also associated with higher prevalence of oral Candida colonization ( p = 0 . 010; adjusted OR = 4.36, 95% confidence interval = 1.29–14.72). These two indices and Candida load were negatively correlated with unstimulated and stimulated salivary flow rates. Interestingly, non- albicans Candida species were more prevalent in denture wearers ( p = 0 . 017 ). Hence, hyposalivation is a risk factor for poorer oral health and oral Candida colonization in independent dentate elders. Because of its potential adverse effects on oral and systemic health, hyposalivation should be carefully monitored in elders.

CONVENTIONAL AND MOLECULAR DETECTION OF CANDIDA ALBICANS AND CANDIDA PARAPASILOSIS ISOLATED FROM BOVINE MASTITIS IN BASRAH-IRAQ

Article

Full-text available

Jan 2019

This study aimed to isolate and identify Candida spp. that cause mycotic mastitis in cows by using molecular techniques for genotype and virulence genes detection. Two hundred and fifty mastitis milk samples from cows in province of Basrah were collected and examined using rapid diagnostic tests and confirmed by polymerase chain reaction (PCR) assay, Candida species was identified in 116/250 (46.4%) of the mastitis milk samples. Based on conventional method and ID-Yst card system Vitek 2, Candida albicans was predominant 60/116 (51.7%), followed Candida parapasilosis 15/116 (12.9%). Concerning the results PCR amplification of 18S rRNA gene for identification of C. albicans and C. parapasilosis, this gene were present 60 samples in C. albicans, while C. parapasilosis was appeared in 15 samples. Secreted aspartyl proteinase 1 like (SAP1) gene was detected in 57/60 (95%) and agglutinin-like sequence (ALS1) gene was detected in 55/60 (91.6%) in C. albicans. While secreted aspartyl proteinase 1 like (SAP1) and agglutinin-like sequence (ALS1) genes were detected in 15/15 (100%) and 14/15 (93.3%) in C. parapasilosis isolates, respectively.

ANTI-PATHOGENIC Candida Spp. Activity DETERMINATION VIA Lactobacillus Spp. ISOLATION AND IDENTIFICATIONS USING CONVENTIONAL AND MOLECULAR METHODS

Article

Full-text available

Jan 2020

Two Hundred and fifty samples of cow's milk from different parts of the province of Basrah were collected from clinical and subclinical mastitis reported using the California mastitis test between March 2018 and September 2019 and examined using conventional PCR assay, Candida species was identified in 116/250 (46.4%). Based on conventional method and ID-Yst card system Vitek 2, Candida albicans was the predominant 60/116 (51.7%), followed by Candida parapsilosis 15/116 (12.9%). Concerning the results of PCR amplification of 18S rRNA gene for identification of C. albicans and C. parapsilosis, this gene was present in 60 samples in C. albicans, and in 15 of C. parapsilosis. Lactobacillus are an industrially important group of probiotic organisms that play an important function in human health through inhibiting dangerous and pathogenic bacteria growth, boosting immune function, and increasing resistance to infection. Ten out of 250(4%) Lactobacillus isolates were obtained from apparently healthy cow milk samples. Lactobacillus isolates were identified according to phenotypic characterization and molecular technique using PCR (16S rRNA) and sequencing, it was seen that L. acidophilus formed 5 isolates (50%), L.amylovorus was three (30%), while 131 L.crisaptus formed only two (20%) only. The results of this study revealed that the BLAST analysis at the NCBI gene bank gave 99.39% homology with L. acidophilus, 99.19% homology with L.crispatus and 97.59% with L. amylovorus. In vitro antimycotic activity of probiotic bacteria (Lactobacillus) against C. albicans and C. parapsilosis using agar well diffusion methods was adapted. The cell-free neutralized supernatant (CFS) of Lactobacilli (10 5 ,10 6 ,10 7) were inhibited the growth of pathogenic C.albicans and C. parapsilosis. It was also noticed that, L. acidophilus showed the strongest antifungal activities against pathogenic C. albicans and C.parapsilosis with different degrees of inhibition zones in comparsion with each of L.crispatus and L. amylovorus, meanwhile, L. amylovorus revealed strongest antifungal activity against pathogenic C.parapsilosis.

ANTI-PATHOGENIC Candida Spp. Activity DETERMINATION VIA Lactobacillus Spp. ISOLATION AND IDENTIFICATIONS USING CONVENTIONAL AND MOLECULAR METHODS

Article

Full-text available

Jan 2020

ANTI-PATHOGENIC Candida Spp. Activity DETERMINATION VIA Lactobacillus Spp. ISOLATION AND IDENTIFICATIONS USING CONVENTIONAL AND MOLECULAR METHODS

Article

Full-text available

Dec 2020

Efficacy of gel-based artificial saliva on Candida colonization and saliva properties in xerostomic post-radiotherapy head and neck cancer patients: a randomized controlled trial

Article

Full-text available

Apr 2021
CLIN ORAL INVEST

Objective To evaluate the efficacy of an edible artificial saliva gel, oral moisturizing jelly (OMJ), and a topical commercial gel (GC dry mouth gel) on Candida colonization and saliva properties.Materials and methodsThis study was a secondary analysis of a single-blinded randomized controlled trial conducted in xerostomic post-radiotherapy head and neck cancer patients. Candida colonization, stimulated salivary flow rate (SSFR), saliva pH, and buffering capacity (BC) were measured at 0, 1, and 2 months after each intervention. Candida colonization was quantified by colony counts and species identified by Candida Chromagar, polymerase chain reaction, and API 20C AUX system. Statistical significance level was 0.05.ResultsA total of 56 participants in OMJ (N = 30) and GC (N = 26) groups completed the study. OMJ significantly increased saliva pH (p = 0.042) and BC (p = 0.013) after 1-month use, while GC only improved saliva pH (p = 0.027). Both interventions tended to increase SSFR but only GC had a significant increase at 2 months (p = 0.015). GC and OMJ significantly decreased the number of Candida species at 1 and 2 months, respectively. Both groups tended to reduce Candida counts but not significant.Conclusions Both OMJ and GC saliva gels could improve saliva pH and decrease the number of Candida species. OMJ is superior to GC in its buffering capacity, while GC may better improve salivary flow rate. Long-term and large-scale study is warranted to test the efficacy of artificial saliva in oral health improvement.Clinical relevanceOMJ and GC gel could decrease the number of Candida species and improve saliva properties in post-radiation xerostomic patients.Trial registration numberClinicaltrials.gov NCT03035825. Date of registration: 25th January 2017.

Isolamento de Candida spp. de estomatite relacionada à prótese no Pará, Brasil.

Article

Full-text available

May 2020

O objetivo deste estudo foi isolar e identificar espécies de Candida da cavidade oral de usuários com estomatite relacionada à dentadura, atendidos na Universidade Federal do Pará (Cidade de Belém, Pará, Brasil). Foram incluídos 36 usuários de protese com estomatite relacionada à prótese, e foram observadas estomatite tipo I (50%), tipo II (33%) e tipo III (17%). Candida spp. foram isolados em 89% dos casos e incluíram cinco espécies diferentes de Candida. C. albicans, frequentemente recuperada (78% dos casos), seguida por C. famata e C. tropicalis. Observamos uma associação significativa entre o isolamento da espécie Candidas e a condição insatisfatória da prótese (p = 0,0017). Nossos resultados demonstraram a alta frequência de isolamento de espécies de Candida em usuários de próteses com estomatite relacionada à prótese e mostraram a relação entre essas espécies e má manutenção da prótese.

Conventional and Molecular Detection of Candida albicans and Candida parapasilosis Isolated from Bovine Mastitis in Basrah, Iraq

Article

Full-text available

Dec 2019

Conventional and molecular detection of C. albicans and C. parapsilosis isolated from bovine mastitis in Basrah-Iraq.

Article

Full-text available

Dec 2019

This study proved the detection of the important two species of Candida in milk samples collected from cows with mastitis by conventional and molecular methods.

Molecular Identification of a Possible Causative Agent of Stomach Distension Syndrome in Rainbow Trout Onchorhynchus mykiss

Article

Full-text available

Oct 2017

A rainbow trout Onchorhynchus mykiss farm located in Gangwon province, South Korea, experienced approximately 10% mortality in June 2017. Most diseased fish had a markedly distended, gas-filled stomach, and exhibited abnormal behavior at the water surface. In this study, we attempted to identify the cause of stomach distension syndrome in those rainbow trout. The stomach of most of the affected fish were full of unidentified gases and some exudate, and yeast was isolated from the stomach mucosa. Pure cultures of yeast were identified using a multilocus sequence typing scheme based on 18S rRNA, internal transcribed spacers, large subunit rRNA, and the gene encoding the largest subunit of RNA polymerase II (RPB1). The RPB1 gene sequences were compared with those of related species available in a database. The yeast was identified as Scheffersomyces coipomoensis (Candida coipomoensis) based on sequence analyses. This is the first study to reveal that S. coipomoensis is a potential causative agent of stomach distension syndrome in farmed rainbow trout. Our results will be helpful for future related studies, and indicate that farmers and stakeholders should observe this emerging disease closely.

Comparison between molecular methods (PCR vs LAMP) to detect Candida albicans in bronchoalveolar lavage samples of suspected tuberculosis patients

Article

Full-text available

Jan 2018

With the increase of patients suffering from immune deficiency infections also increased pulmonary fungi even in people with defective immune system can cause fatal and lethal candidiasis. The timely diagnosis of pulmonary candidiasis is one of the problems that has been detected. Polymerase chain reaction (PCR) test and Loop mediated isothermal amplification (LAMP) method optimized on the basis of α INT1 gene and then sensitivity and specificity were evaluated. LAMP is a novel nucleic acid amplification technique with high specificity and sensitivity which has been done under isothermal condition. Samples were the bronchoalveolar lavage suspected of tuberculosis (TB) reviews for TB disease negative have been reported. DNA extraction carried out by standard phenol/chloroform method on samples and PCR test and LAMP was done. PCR and LAMP testing was performed on samples and products of 441 bp were amplified and observed with agarose gel electrophoresis. At the end of the LAMP reaction, SYBR Green was used for identifying negative and positive results. Among the 60 quantities sera, only 7 cases were PCR positive but 8 cases were LAMP positive. In comparison, between LAMP and PCR, the LAMP technique in spite of its simplicity, high sensitivity and specificity, could be an appropriate replacement for PCR.

Erratum to: Application of high-resolution melting analysis for differentiation of spoilage yeasts

Article

Dec 2017

In the article by Erdem et al. published in Journal of Microbiology 2016; 54, 618–625, the figure 1 should be corrected as below.

Impact Evaluation of Gender and Age on Percentage Distribution of Candidiasis Across Settings in Port Harcourt Metropolis in Nigeria

Article

Full-text available

Jan 2017

Umasoye Aaron

Gender and age could be important factors determining prevalence distribution of candidiasis in community and hospital settings. The ability of an individual to defend itself against invasion by pathogens (bacteria, fungi, viruses, etc.) to a large extent determines his/her vulnerability to Candida infections. Candida infections occurring either in the community or hospital setup , can be clinically observed as localized infections of the mouth, throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, or gastro-intestinal tract or become systemic. Therefore, the identification of Candida species is very important in the diagnostic laboratory, because such identification shows prognostic and therapeutic significance, allowing early and correct antifungal therapy. However, this study tries to assess the possibilities involved in the clinical distribution of Candida species along Gender and age divides. Also, it seeks to discuss the possible ways at which incidence of Candida infections can be halted. Furthermore, samples used in this study were obtained from routine laboratory sample at the University of Port Harcourt teaching hospital and processed. The inocula were prepared by growing the various Candida isolates on separate Sab-oraud dextrose agar plates for purity which is then used for normal saline microsco-py, germ tube test and carbohydrate assimilation tests to confirm Candida specie. Therefore, this study showed an indifferent distribution of Candida species among the various age groups for community settings while hospital settings occurrence of Candida isolates among age group 21-30 (18.6%) was the highest followed by 31– 40 (17.9%). The result generally showed a decreasing trend as you move left or right of peak group 21 – 30 (the Candida species highest percentage occurring group). On the other hand, it was observed that age group 40 – 50 (15.8%) had the highest frequency followed by age group < 10 (13.6%) had within the community setting. Nevertheless , from this present study, we can advance that; emphasis should be placed on public education and advocacy to enhance behavioral and attitudinal change. Similarly, the adoption of sustained and consistent good hygienic practices as a routine day to day life style should form part of the advocacy.

The Distribution of Candida Species from Clinical Specimens in Different Areas of Ondo State

Article

Full-text available

May 2017

A total of 400 various clinical samples which included HVS, urine, stool and oral swabs were collected from patients in the four geographical locations of Ondo State, Akure, Ondo, Ikare and Okitipupa. Candida species were isolate from 63 out of the 400 samples after culturing. Of these total numbers of positive isolates, C. albicans, C. Krusei, C. glabrata, C. parapsilosis accounted for 73.2%, 12.3%, and 4.6% respectively. The results revealed a statistically significant relationship (P<0.5) between the candida infection and the debilitated state of individuals. There was no significant relationship (P>0.5) between the gender and the age. The incidence of the Candida albicans are often present in the absence of vaginitis, therefore isolation in healthy individuals does not necessarily imply infection.

GENOTYPIC IDENTIFICATION OF CANDIDA SPP. ISOLATED FROM ONYCHOCANDIDIASIS PATIENTS BY PHENOTYPIC METHODS, PCR AND RAPD-PCR

Article

Jan 2014

Imran

A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient

Article

Full-text available

Mar 2015

Background and Purpose Invasive candidiasis (IC) is a significant cause of morbidity and mortality in patients with hematologic disorders and bone marrow transplant recipients. Rapid, specific and sensitive test for the timely accuracy in immunocompromised patients to reduce mortality rates and prevent IC progress is necessary. We established a real-time PCR assay on blood for the diagnosis and differentiation of the causative Candida species. Materials and Methods Whole blood samples were collected twice, from 72 patients for Real Time PCR and blood culture assays. The primers and hybridization probes were designed to potentiate the specific sequence of 18S rRNA genes using Light Cycler system and Fluorescence Resonance Energy Transfer (FERT). The patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC based on the revised European Organization for Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria. Results From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML) (27.8%) and acute lymphoblastic leukemia (ALL) (26.4%). Out of 72 patients, 11 patients (15.3%) had positive real time PCR /probe results. Based on the melting temperature (Tm) analysis, 5 (45.4%) C. krusei, 3 (27.2%) C. tropicalis, 2 (18.1%) C. parapsilosis and 1 C. albicans (9%) were identified. According to the revised EORTC / MSG, 1 patient (9%) and 10 patients (91%) were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC

Application of high-resolution melting analysis for differentiation of spoilage yeasts

Article

Sep 2016

A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination.

Epidemiology of nosocomial candidiasis: The importance of specific identification

Article

Jan 2003

Nosocomial infections with Candida species are recognized as a significant cause of morbidity and mortality in both seriously ill immunocompetent and immunocompromised patients. Infections with Candida albicans and non-albicans Candida species have become a significant cause of infection in humans. Several of the more commonly Candida spp isolates are less susceptible to the antifungal drugs currentlly applied in clinical treatment, a factor that means significant difficulties for effective treatment. The modern mycology laboratory has an important role to play in several aspects relating to these organisms, including therapy, detection, identification and epidemiological analysis. In this study, we have provided an initial comparison of differences in species distribution among Candida isolates from four general hospitals of São Paulo,SP. Overall, 40 isolates of C. albicans, C. parapsilosis and C. tropicalis were isolated respectively in 35%, 50% and 15%, revealed a tendency of higher frequency of non-albicans species. The species distribution in patients with candidemia showed that the most commonly species were C. parapsilosis (45,4%), followed by C. albicans (36,4%) and C. tropicalis (18,2%); thus, we have an increase of non-albicans species. The three different species were include in 6, 3, and 4 different biotypes, respectively C. albicans, C. parapsilosis e C. tropicalis. This study emphasizes the importance of periodic evaluation of Candida species distribution especially in centers caring for patients at risk.

A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient

Article

Full-text available

Mar 2015
Curr Top Med Mycol

Tahereh Shokohi

Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1–D2 region sufficient for identification of clinical fungi?

Article

Aug 2015
MYCOSES

Although conventional identification of pathogenic fungi is based on the combination of tests evaluating their morphological and biochemical characteristics, they can fail to identify the less common species or the differentiation of closely related species. In addition these tests are time consuming, labour-intensive and require experienced personnel. We evaluated the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeast and 160 moulds in our clinical mycology laboratory. While the yeast isolates were identified at species level with 100% homology, 102 (63.75%) clinically important mould isolates were identified at species level, 56 (35%) isolates at genus level against fungal sequences existing in DNA databases and two (1.25%) isolates could not be identified. Consequently, Whatman FTA filter matrix technology was a useful method for extraction of fungal DNA; extremely rapid, practical and successful. Sequence analysis strategy of D1-D2 region of the large ribosomal subunit gene was found considerably sufficient in identification to genus level for the most clinical fungi. However, the identification to species level and especially discrimination of closely related species may require additional analysis. © 2015 Blackwell Verlag GmbH.

Advances in yeast systematics and phylogeny and their use as predictors of biotechnologically important metabolic pathways

Article

Full-text available

Jun 2015
FEMS YEAST RES

Detection, identification and classification of yeasts have undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences is leading to a major revision of yeast systematics that will result in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed, as will use of phylogeny for prediction of biotechnological applications. Published by Oxford University Press on behalf of FEMS 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Bloodstream infection by candida in patients with hematologic neoplasia: polyphasic taxonomy and antifungal susceptibility / Infecção sanguínea por candida em pacientes com neoplasia hematológica: taxonomia polifásica e suscetibilidade antifúngica

Article

Feb 2022

Bloodstream infection (BSI) by species of Candida has been identified as an important cause of death in patients with neutropenia who undergo chemotherapy for the treatment of hematologic malignancies This study aimed to verify the occurrence of bloodstream infections by Candida species in patients admitted to the haematology-oncology service of a public hospital specialized in the treatment of cancer in Northeast Brazil. A total of 105 clinical samples from 62 patients with haematological malignancies were analyzed at the Laboratory of Medical Mycology at the Federal University of Pernambuco. Only 7 of 105 individuals were in the ICU environment. The mycological diagnosis was performed through automation (BACTEC 9120 / PHOENIX™), proteomic identification (MALDI-TOF MS) and molecular analysis (PCR). The antifungal susceptibility test followed the bloodstream infection recommendations of Clinical Laboratory Standards Institute. Among the samples studied, nine strains (8,57%) were of the genus Candida, being six C. tropicalis and three C. albicans. The isolates were completely susceptible to the antifungal agents tested. Deaths occurred in 66,6% of the cases. Patients with hematologic malignancies hospitalized in intensive care and the state of septic shock present a higher risk of occurrence of BSI by Candida and death by this opportunistic pathogen.

İnvazivve İnvaziv Olmayan Örneklerden İzole Edilen Candida albicans Suşlarının Genotip Dağılımı Genotype Distribution OfCandida albicans Strains Isolated From Invasive And Noninvasive Samples

Article

Feb 2007

KOYUNCU Esra;DOLAPÇI

Ketorolac-fluconazole: A New Combination Reverting Resistance in Candida albicans from Acute Myeloid Leukemia Patients on Induction Chemotherapy: In vitro Study

Article

Full-text available

Jun 2021

Background and Objectives Candida albicans is a significant source of morbidity and mortality for patients with acute myeloid leukemia (AML). Prolonged use of fluconazole as empirical antifungal prophylaxis in AML patients leads to overexpression of efflux pump genes that resulted in the emergence of azole-resistant species. Consequently, the introduction of a new strategy to improve the management of C. albicans infections is an urgent need. Nonsteroidal anti-inflammatory drug (NSAID) ketorolac is associated with a reduction in cancer relapses. The present study was performed to investigate the use of ketorolac-fluconazole combination to reverse fluconazole resistance in C. albicans isolated from AML patients on induction chemotherapy. Patients and Methods One hundred and seventy AML patients were evaluated. Fifty C. albicans were isolated and subjected to disc diffusion assay and broth microdilution for fluconazole alone and combined with different concentrations of ketorolac. Efflux pump gene (CDR1, CDR2, and MDR1) expressions were quantified by real-time PCR. Results The tested ketorolac acted synergistically with fluconazole against resistant C. albicans with the minimum inhibitory concentration (MIC) of fluconazole decreased from >160 μg/mL to 0.3–1.25 μg/mL in (93.8%) of resistant isolates with fractional inhibitory concentration index (FICI) value of 0.25. The majority of the resistant isolates overexpressed CDR1 (71.1%) and MDR1 (60%). Conclusion Ketorolac-fluconazole in vitro combination would be a promising strategy for further clinical in vivo trials to overcome fluconazole resistance in AML patients on induction chemotherapy.

Evalution The Antifungal Activity of Lactobacillus Against Some isolates of Candida spp Isolated From Bovine Mycotic Mastitis

Method

Full-text available

Dec 2020

mastitis, molecular methods, enzymatic activity

Evalution The Antifungal Activity of Lactobacillus Against Some isolates of Candida spp Isolated From Bovine Mycotic Mastitis

Thesis

Full-text available

Dec 2002

Lactobacillus isolated from milk and milk-based as well as non-milk based fermented foods have been conferred the GRAS (generally recognized as safe) status and have widely been used in food and medicine, because of their probiotic attributes. Lactobacillus species have the ability to produce several antimicrobial substance including hydrogen peroxide, acetic acid, lactic acid, bacteriocins such as small heat stable lantibiotic (SHSL), non- lanthionine containing membrane - active peptides (MAP), large heat- labile proteins (LHLP). Because of the ability to produce various antimicrobial agent, these probiotic could be candidates for the control and treatment of different infections. Firstly Candida spp isolated from milk samples of cows with mastitis then diagnosed conventionally and conventionally PCR. API zym test was used to evaluate the enzymes of the most common species of candida isolated from bovine mastitis which are Candida albicans and Candida parapsilosis. Lactobacillus spp were isolated from milk of apperantly healthy cows and diagnosed by conventional PCR. The antifungal activity was assessed by using two methods, well diffusion method and gene expression by Real time qPCR. Two hundred and fifty of cow milk from different areas of Basrah province had been collected from clinical and subclinical mastitic cows reported by using California mastitis test during the period (March 2018 up to September 2019). Results of the current study showed that 116 (46.4%) of Candida isolates out of 250 milk samples were obtained from cows with mastitis based on cultural, morphological and commercially available ID –YST card system (Vitek 2 system Bio Mèrieux, France for pathogenic yeast). The highest percentage among isolated Candida spp was belong C.albicans which was 60/116 (51.7%) followed by C. parapsilosis was 15/116 (12.9%), C. famata 10/116 (8.6%), C. krusei 10/116 (8.6%), C. lusitaniae 7/116 (6%), C. spherica II 5/116 (4.3%), C. glabrata 4/116 (3.4%), C. inoconspica/ Lambica 3/116 (2.5%), and the lowest percentage was for C. tropicalis 2/116 (1.7%). A total of 90 (77.5%) isolates of Candida spp out of 116 isolates obtained by Candida chromogenic agar, the majority of the common Candida spp identified were C. albicans 53/90 (58.8%), C. parapsilosis 12/90 (13.3%), C. famata 8/90 (8.8%), C. krusei 7/90 (7.7%), C. lusitaniae 6/90(6.6%), C. glabrata 2/90 (2.2%), and C.tropicals 2/90 (2.2%). Some randomly choosed isolates of C. albicans and C. parapsilosis were used for detection of their enzymatic activities by API ZYM test. In case of C. albicans (n=30) the isolates produced n-acetylo-β-glucosyloaminidase strongly when the percentage was 30/30 (100%), esterase and valine arylamidase (C4) 29/30 (96.6%), leucine arylamidase 28/30 (93.3%), esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase 25/30 (83.3%), α-glucosidase 23(76.6%), β-glucosidase 22 (73.3%), α-mannosidase 4 (13.3%), with a lowest activity of cystine arylamidase as 1(3.3%). Regarding C. parapsilosis (n=10) the isolates showed the highest activity of leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase which was 10/10 (100%) for each, followed by esterase lipase (C8) 9/10 (90%), valine arylamidase and alkaline phosphatase 8/10 (80%) for both and esterase (C4) 7/10 (70%). Molecular identification of Candida spp in this study, done by conventional PCR by partial amplification gene encoding for large subunit of 18S rRNA gene by specific primer sequences. The yield of the detection C. albicans and C. parapsilosis, was 60/60 (100%) and 15/15 (100%), respectively. The result concordant with those of Vitek 2 system and Candida chromogenic agar Fifty five isolates out of 60 (91.6%) belong C. albicans were positive for ALS1 gene, Fourteen isolates out of 15 (93.3%) belong C. parapsilosis were positive for ALS1 gene.While 57/60 isolates belong C.albicans were positive for SAP1 gene as (95%). Fifteen isolates (100%) belong C.parapsilosis were III positive for SAPP1 gene. Fifty-eight isolates (98.3%) belong C.albicans were positive for CPH1 gene, whereas 14/15 (93.3%) isolates belong C.parapsilosis were positive for CPH1 gene. Molecular evalution of enzymatic activity was done on C.albicans and C.parapsilosis which were positive by API ZYM test, Conventional PCR was done for partial amplification of sterol esterase, alkaline phosphates and alpha glucosidase genes by specific primer sequences. The results of the PCR amplification of these genes for C.albicans and C.parapsilosis were, in case of sterol esterase it was present in C. albicans as 28/30 (93.3%) while in C.parapsilosis as 8/10 (80% ). Concerning alkaline phosphatase gene this was present in C. albicans as 27/30 (90%) while in C. parapsilosis as 10/10 (100%). Regarding alpha glucosidase gene this was present in C. albicans as 29/30 (96.6 %) while in C.parapsilosis as 10/10 ( 100% ). Ten Lactobacilli out of 250 (4%) were isolated from appearently healthy cow's milk, were identified on the base of morphology Gram positive, catalase negative, long rods shaped bacilli, arranging singly or in chains and non-spore forming. By molecular technique using PCR (16S rRNA) and sequencing, it was seen that L. acidophilus formed 5 isolates (50%), L.amylovorus was 3 (30%), while L.crisaptus formed only 2 (20%) only. The results of this study revealed that the BLAST analysis at the NCBI gene bank gave 99.39% homology with L. acidophilus, 99.19% homology with L.crispatus and 97.59% with L. amylovorus. Antifungal profile of Candida isolates from bovine milk with mastitis in the current study and according to antibiotic disc diffusion method were applied to each of fluconazole, nystatine, ketoconazole and amphotericin B on all isolated C.albicans (60) and C.parapsilosis (15). In C. albicans the resistance proportion for amphotericine B is 60 (100%) and nystatine 55 (91.6%), while those sensitive to fluconazole were 45 (75%) and ketoconazole 56 (93.3%). IV C.parapsilosis isolates demonstrated resistance to each of amphotericine B, nystatine as 15 (100%) and fluconazole 8 (53.3%), meanwhile sensitivity proportion to ketoconazole is 10 (66.6%). The cell-free neutralized supernatant (CFS) of Lactobacilli (105,106,107) were inhibited the growth of pathogenic C.albicans and C.parapsilosis by well diffusion method. It was also noticed that, L. acidophilus showed the strongest antifungal activities against pathogenic C. albicans and C.parapsilosis with different degrees of inhibition zones in comparsion with each of L.crispatus and L. amylovorus, meanwhile L. amylovorus revealed strongest antifungal activity against pathogenic C.parapsilosis. Zone of inhibition of probiotic (Lactobacillus) against C. albicans was compared among 105, 106 and 107 concentrations. Regarding L. acidophilus, the best zone of inhibition was obtained at 106 concentration. L.crispatus, the best zone of inhibition was obtained at 107 concentration. while the best inhibition zone of L. amylovorus was obtained at 106 concentration. In case of C.parapsilosis, zone of inhibition of probiotic was compared among 105,106 and 107 concentrations. Regarding L. acidophilus, the best zone of inhibition was obtained at 107 concentrations. In case of L. crispatus, the best zone of inhibition was obtained at 106 concentration. The best zone of inhibition was obtained at the concentration of 106 of L. amylovorus . Finally in this experiment which was done to evaluate the expression of ALS1, SAP1, CPH1 genes of Candida albicans when it was grown with three species of Lactobacillus and incubated for 24 hr and 48 hr respectively, in comparison with controls. The highest down regulation was seen in ALS1 gene belongs C. albicans when incubated with L. acidophilus for 24 hr, when the mean was (0.035), the same result with a mean of (0.024) where obtained when incubated for 48 hr with the same species. V It was seen that the highest down regulation value of the SAP1 gene of a mean (0.218) belongs C.albicans was obtained with L. acidophilus when incubated for 24 hr, in case of down regulation for the same gene was occurred after 48 hr which is the highest one a mean of (0.023) with L.acidophilus. Regarding the best down regulation of CPH1 gene of C.albicans was obtained when incubated with L.acidophilus after 24 hr when a mean was (0.228), while the highest down regulation of this gene after 48 hr was obtained with L.amylovorus which was a mean of (0.145). The experiment which was done to evaluate the expression of ALS1, SAPP1, CPH1 genes of Candida parapsilosis when it was grown with three species of Lactobacillus and incubated for 24 hr and 48 hr respectively. It was seen that the highest down regulation value of the ALS1 gene of a mean (0.562) belongs C.parapsilosis was obtained with L. acidophilus when incubated for 24 hr, in case of down regulation for the same gene was occurred after 48 hr which is the highest one a mean of (0.509) with L.acidophilus. The highest down regulation was seen in SAPP1 belongs C. parapsilosis when incubated with L. acidophilus for 24 hr, when the mean was (0.807), the same result with a mean of (0.280) where obtained when incubated for 48 hr with the same species. Regarding the best down regulation of CPH1 gene of C.parapsilosis was obtained when incubated with L.acidophilus after 24 hr when a mean was (0.320), while the highest down regulation of this gene after 48 hr was obtained with L. acidophilus which was a mean of (0.602).

Detection, Quantification, and Identification of Yeast in Winemaking

Chapter

Sep 2019

Yeast have a fundamental role in winemaking. They carry out alcoholic fermentation and they contribute to the quality of the wine, although they can also cause spoilage during grape must transformation and in the final product. To detect and identify wine yeast and control their activities, a plethora of different methods can be utilized. As reported in the present chapter, these methods have different degrees of complexity and vary in terms of cost, rapidity and sensitivity. Those based on yeast isolation, namely culture-dependent methods, are widely utilized to define the composition of the microflora associated with wine-related environments and for yeast identification at the strain level, besides providing a means for ex-situ preservation of wine yeast biodiversity. Culture-independent methods bypass microorganisms cultivation, thus avoiding any bias introduced by their isolation and uncovering cell populations undetected by culture-dependent methods. These methods can be utilized to evaluate the impact of all of the components of the wine microbiota on the quality of the final product, to implement a quality control system based on real-time detection and quantification of specific targets, such as the inoculated starter (s) or the spoilage yeast, or to provide further insights into the composition of the microbial communities involved in the grape must transformation.

Oral Candida colonization in xerostomic post-radiotherapy head and neck cancer patients

Article

Jul 2019

Objectives: To evaluate 1) oral colonization of Candida species, especially for non-albicans Candida species (NACS), in xerostomic post-radiotherapy head and neck cancer patients and 2) risk factors affecting their colonization. Materials and methods: Subjective and objective dry mouth scores, stimulated salivary flow rates, pH and buffering capacity were measured in 72 xerostomic post-radiotherapy head and neck cancer patients. Candida counts and species identification were performed using oral rinse samples cultured in Candida Chromagar, followed by polymerase chain reaction and API 20C AUX system. Results: Candida colonization was observed in 87.5% of subjects, with 80.6% and 48.6% of study population colonized by C.albicans and NACS, respectively. NACS was associated with high objective dry mouth scores, denture use, and females (P=0.006, 0.009, and 0.036, respectively). In addition, C.glabrata was detected more in females (P=0.018) and denture wearers (P=0.026), while C.tropicalis was associated with high objective dry mouth scores (p=0.022) and females (P=0.027). Quantity of Candida colonization correlated positively with objective dry mouth scores (r=0.599, P<0.001), and negatively with salivary flow rates (r=-0.258, P=0.041) and pH (r=-0.290, P=0.022). Conclusion: NACS colonization was common in xerostomic head and neck cancer patients. Increased signs of dry mouth, female and dental prostheses may promote NACS colonization. This article is protected by copyright. All rights reserved.

Identification methods of Candida isolated from vaginal infections

Article

Mar 2019

The evaluation of the immune response against three Candida albicans vaccines by using ELISA

Article

Full-text available

Oct 2018

The Candida albicans infection is of minor veterinary significance, however, during the last years a considerable concern has been focused on the illness caused by these yeast.

Yeasts as Distinct Life Forms of Fungi

Chapter

Oct 2017

Detection, identification, and classification of yeasts have undergone major changes since application of gene sequence analyses and genome comparisons. Development of a database of barcodes consisting of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences is leading to a major revision of yeast systematics that will result in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules were recently implemented in the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed.

Isolation of Candida spp. from denture-related stomatitis in Pará, Brazil

Article

Full-text available

Jul 2017

The aim of this study was to isolate and identify Candida species from the oral cavity of denture wearers with denture-related stomatitis who were attended at the University Federal of Pará (Belém City, Pará State, Brazil). A total of 36 denture wearers with denture-related stomatitis were included, and type I (50%), type II (33%) and type III (17%) stomatitis were observed. Candida spp. were isolated from 89% of the cases and included five different Candida species. C. albicans was the most frequently recovered species (78% of the cases), followed by C. famata and C. tropicalis. We observed a significant association between Candida species isolation and unsatisfactory denture condition (p = 0.0017). Our results demonstrated the highly frequency of Candida species isolation in denture wearers with denture-related stomatitis and showed the relationship between these species and poor denture maintenance.

EFICACIA DE UN AGAR CROMOGÉNICO PARA LA IDENTIFICACIÓN DE ESPECIES DE CANDIDA EN AISLAMIENTOS CLÍNICOS

Article

Full-text available

Jul 2013

Adrian Canizalez-Roman

La incidencia creciente de fungemias intrahospitalarias causadas por especies levaduriformes requieren pruebas alternativas que consuman menos tiempo. El medio de cultivo agar cromogénico es de gran utilidad para identi car y diferenciar los principales especies de candida. Objetivo: determinar la e cacia de un agar cromogénico para la identi cación de especies de Candida en aislamientos clínicos. Material y método: mediante un diseño de pruebas diagnósticas estudiamos 204 aislamientos clínicos de cepas de levaduras, que fueron cultivadas primero en medio de Sabouraud dextrosa por 24 horas a 35 °C, después se cultivaron en el medio de cultivo cromogénico Chromoagar Candida (OXOID, UK) por 24, 48 y 72 horas a 37°C. Para estandarizar el medio de cultivo se utilizaron especies del género Candida obtenidos de la American Type Culture colection, C. albicans ATCC 1880., C. glabrata ATCC 66032, C. krusei ATCC 14243, C. ke r ATCC 2512, C. lusitaniae ATCC 34449, C. parapsilosis ATCC 4136, C. tropicalis ATCC 201380, Aspergillus niger ATCC. Resultados: la sensibilidad del cultivo cromogénico para C. albicans y C. tropicalis fue 98.5% con intervalo de con anza a 95% (IC 95%) 95.5% – 100%, y 88.6%, IC 95% de 79.3% – 97.9% respectivamente mientras que la especi cidad para ambas fue de 100%. Para C. glabrata, la sensibilidad fue de 62.5%, IC 95% de 63 – 95.4% y especi cidad de 100%. Conclusiones: los resultados arrojan una e cacia aceptable del agar cromogénico para identi car C. albicans y C. tropicalis, no así para C. parapsilosis y C. glabrata.

MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR- DGGE, A TRADITIONAL MEXICAN BEVERAGE

Article

Mar 2015

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR- DGGE, A TRADITIONAL MEXICAN BEVERAGE

Article

Full-text available

Mar 2015

CANDIDAL INFECTION: EPIDEMIOLOGY, PATHOGENESIS AND RECENT ADVANCES FOR DIAGNOSIS

Article

Full-text available

May 2013

Candidiasis is the commonest fungal disease in human being. The causative agent of the disease is Candida albicans and non- albicans Candida. Candida has 163 acknowledged anamorphic species, present on the different habitat out of which following species are C.albicans, C. tropicalis, C. krusei, C. glabrata, C. guilliermondii, C. parapsilosis, C. lusitaniae, C. kefyr, C. rugosa, C. dubliniensis and C. viswanathii known to causing disease in human beings. The virulence factors of the C. albicans have the great role in the pseudohyphae formation by attached with epithelial cells and endothelial cells. Now a day various techniques like PCR, Candida Detection System, CAND-TEC and Dot Immunobinding Assay are available for detection of candidiasis from various clinical samples.

Literature Cited

Chapter

Jan 2004

The evaluation of the immune response against three Candida albicans vaccines by using ELISA

Conference Paper

Full-text available

Jan 2012

The Candida albicans infection is of minor veterinary significance, however, during the last years a considerable concern has been focused on the illness caused by these yeast. The use of ELISA has been introduced as a new diagnostic tool to evaluate the immune response in case of various diseases including the fungal infections via measuring the level of IgG. Herein, the aim of this study was to identify the C.albicans microscopically and by using PCR techniques. In addition, we have used three types of vaccines (heat killed C. albicans vaccine with adjuvant or with culture filtrate, or mixed vaccines (HK-CA) vaccine and (CFV)) and immune response (level of IgG) of rats was evaluated by ELISA. The data showed that, the immune response of rats was significantly increased after challenge in all three types of vaccines and there was no significant differences between level of immunity emerged by heat killed C. albicans vaccines and culture filtrate of C. albicans. While, immunization by mixed vaccine (HK-CA, CFV) showed significant increase in anti-Candida albicans than heat killed or culture filtrate alone especially after challenge with the virulent C. albicans strain.

Detection, identification and enumeration methods for spoilage yeasts

Article

Mar 2006

Cletus Kurtzman

Methods to identify yeats

Chapter

Dec 2003

Amplification and Direct Sequencing of Fungal Ribosomal Rna Genes for Phylogenetics.

Chapter

Full-text available

Nov 1989

Evaluation of the Baxter-MicroScan 4-hour enzyme-based yeast identification system

Article

Full-text available

May 1991

A new 4-h Yeast Identification Panel (YIP; Baxter-MicroScan, W. Sacramento, Calif.) was compared with the API 20C Yeast Identification System (Analytab Products, Inc., Plainview, N.Y.) in the identification of recent clinical yeast isolates. The YIP had a 94% correlation (288 of 306) in identifying 22 species within the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces, and Torulopsis. Correlation dropped to 65% for those species within the genera of slower growing yeasts, i.e., Blastoschizomyces spp., Crpytococcus spp., Geotrichum spp., Hyphopichia spp., Phaeococcomyces spp., Prototheca spp., and Trichosporon spp. Overall correlation with the API 20C was 92% (365 of 401) for those taxa included in the data base and 85% (373 of 437) for all yeasts encountered in the study. There were 36 (8.2%) discrepant identifications, which were due in part to the limited data base. Expansion of the data base plus the easy inoculation, reading, and rapid results of the YIP should make it an excellent method for yeast identification.

A computer program for choosing optimal oligonudeotides for filter hybridization, sequencing and in vitro amplification of DNA

Article

Full-text available

Dec 1989

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A.(1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4°C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.

Comparison of the Quantum II, API Yeast Ident, and AutoMicrobic systems for identification of clinical yeast isolates

Article

Full-text available

Nov 1988

The Quantum II Yeast Identification System (Abbott Laboratories) is a microprocessor-based spectrophotometric system for identification of clinical yeast isolates within 24 h. We compared the Quantum II system with the API Yeast Ident (Analytab Products) and the AutoMicrobic System Yeast Biochemical Card (AMS-YBC; Vitek Systems, Inc.) for the identification of 221 clinical yeast isolates, including 120 common clinical isolates (Candida albicans, C. tropicalis, C. parapsilosis, Torulopsis glabrata, and Cryptococcus neoformans) and 101 relatively uncommon clinical isolates. The API 20C (Analytab) was used as the reference system. The Quantum II and AMS-YBC systems correctly identified 181 (82%) and 184 (83%) isolates, respectively, whereas the Yeast Ident system correctly identified 132 (60%) isolates. Of the 120 common clinical isolates, 113 (94%) were correctly identified by Quantum II, 103 (86%) were correctly identified by AMS-YBC, and 83 (69%) were correctly identified by Yeast Ident. Of the 101 uncommon clinical isolates tested, 68 (67%) were correctly identified by Quantum II, 81 (80%) were correctly identified by AMS-YBC, and 49 (49%) were correctly identified by Yeast Ident. The overall accuracy of the Quantum II, AMS-YBC, and API Yeast Ident was not sufficient to recommend any of these systems for routine use in the clinical microbiology laboratory without substantial expansion of the respective data bases.

Comparison of relative susceptibities of Candida species to three antifungal agents as determined by unstandardized methods

Article

Full-text available

Oct 1987

Three groups of isolates, each comprising four isolates of Candida species, were selected for their diversity of susceptibilities to amphotericin B, flucytosine, or ketoconazole. The isolates were distributed in duplicate and in blinded fashion to three laboratories where a total of eight procedures were performed for each drug. From the decoded results, intralaboratory variability among replicate determinations was found usually to fall within a fourfold range. Interlaboratory variation, however, was 16-fold or greater for all isolates, ranging to 50,000-fold differences for some isolates. Relative susceptibilities of isolates within each method could be determined in 11 of the 24 drug-method combinations and agreed with the reference rank order in all but one instance. Our findings underscore the lack of agreement among laboratories for the susceptibility testing of yeasts but indicate that such differences could likely be resolved by standardization without loss of clinical value.

Phylogenetic Relationships of the Soybean Sudden Death Syndrome Pathogen Fusarium solani f. sp. phaseoli Inferred from rDNA Sequence Data and PCR Primers for Its Identification

Article

Full-text available

Sep 1995
MOL PLANT MICROBE IN

Phylogenetic relationships of several species within the Fusarium solani-complex were investigated using characters from the nuclear ribosomal DNA. Genetic variation within 24 isolates, including 5 soybean sudden death syndrome (SDS) strains, was assessed using rDNA sequence data and restriction fragment length polymorphic markers. By these techniques, the causal agent of soybean SDS was identified as F. solani f. sp. phaseoli. In separate cladistic analyses, Plectosphaerella cucumerina and Nectria cinnabarina or F. ventricosum were used for rooting purposes. Monophyly of the F. solani-complex was strongly supported by bootstrap and decay analyses. Parsimony analysis indicates that this complex is composed of a number of phylogenetically distinct species, including Neocosmospora vasinfecta, F. solani f. sp. phaseoli, and biological species designated as MPI, MPV, and MPVI of N. haematococca. The results demonstrate complete congruence between biological and phylogenetic species within the N. haematococca-complex. In addition, DNA sequence data were used to design a PCR primer pair which could specifically amplify DNA from isolates of the SDS pathogen from infected plants.

Resistance of Candida species to fluconazole

Article

Full-text available

Feb 1995

Detection of Candida albicans and other yeast in blood by PCR

Article

Full-text available

Feb 1994

Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.

Rapid identification of yeast species using three primers in polymerase chain reaction

Article

Full-text available

Jul 1993
Mol Mar Biol Biotechnol

Jack W Fell

Classic methods for identification of yeasts rely on a variety of morphological and physiological tests that often take days to weeks to complete. We have been able to reduce the time to less than one day through the use of multiple segment-specific oligonucleotide priming of a region of the large subunit rDNA in a polymerase chain reaction. The "hot start" reaction was used with two universal external delimiting primers and one internal species-specific primer. Five specific primers were tested: a primer for a biologically similar group of Rhodotorula species, a generic (Cystofilobasidium) primer, and 3 species-specific primers (Leucosporidium scottii, Cryptococcus muscorum, and Rhodotorula mucilaginosa). In the absence of specific target DNA, the universal rDNA segment is amplified; in the presence of target DNA, the specific primer region is amplified. The technique is accurate within two base position differences when a 24 nucleotide-specific primer is used. The technique should be applicable to other marine eukaryotes.

Evaluation of commercially available acridinium ester-labeled chemiluminescent DNA probes for culture identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum

Article

Full-text available

Apr 1993

Four commercially available acridinium ester-labeled DNA probes directed against rRNA were evaluated for their ability to identify Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans in culture. rRNA was extracted by sonication of 1- to 2-mm2 portions of cultures of fungi in two chaotropic reagents with glass beads. Following a heat inactivation step, the extracts were hybridized in solution with probes specific for each pathogen. The acridinium ester reporter moiety of nonhybridized probe was selectively hydrolyzed, and chemiluminescence of specific DNA:RNA hybrids was quantitated in relative light units with a luminometer. A positive identification required a relative light unit value of > or = 50,000. Sensitivity and specificity of the probes were determined by probing cultures of the respective pathogenic fungi (target) and nontarget fungi. Both mycelial and yeast forms of the dimorphic fungi (B. dermatitidis and H. capsulatum) were tested. For B. dermatitidis, sensitivity and specificity were 87.8 and 100%, respectively (74 target and 219 nontarget fungi tested). For C. immitis, sensitivity and specificity were 99.2 and 100%, respectively (122 target and 164 nontarget fungi tested). For H. capsulatum, sensitivity and specificity were 100 and 100%, respectively (86 target and 154 nontarget fungi tested). For C. neoformans, sensitivity and specificity were 97 and 100%, respectively (100 target and 230 nontarget fungi tested). For B. dermatitidis, C. immitis, and C. neoformans, repeat testing increased the respective sensitivities to 97.3, 100, and 100%. The high sensitivities and specificities of the probes, the relatively short time (less than 1 h) required to perform the assay, and the availability of standardized reagent kits make the acridinium ester-labeled DNA probes well suited to laboratories in need of a rapid method to identify these fungal pathogens. Further, use of the probes to identify pathogenic fungi as soon as colonies appear on primary recovery media significantly shortens the time to reporting.

Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro

Article

Full-text available

Apr 1997

Candida dubliniensis is a recently described species of Candida associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. Nineteen oral isolates of C. dubliniensis recovered from 10 HIV-positive and 4 HIV-negative individuals and one vaginal isolate from an additional HIV-negative subject were assessed for fluconazole susceptibility by broth microdilution (BMD), hyphal elongation assessment, and Etest. The susceptibilities of these 20 isolates to itraconazole and amphotericin B and of 10 isolates to ketoconazole were also determined by BMD only. Sixteen of the C. dubliniensis isolates were susceptible to fluconazole (MIC range, 0.125 to 1.0 microgram ml-1), and four (recovered from two AIDS patients) were fluconazole resistant (MIC range, 8 to 32 micrograms ml-1). Fluconazole susceptibility data obtained by hyphal elongation assessment correlated well with results obtained by BMD, but the corresponding Etest MIC results were one to four times higher. All of the isolates tested were found to be sensitive to itraconazole, ketoconazole, and amphotericin B. Sequential exposure of two fluconazole-sensitive (MIC, 0.5 microgram ml-1) C. dubliniensis isolates to increasing concentrations of fluconazole in agar medium resulted in the recovery of derivatives which expressed a stable fluconazole-resistant phenotype (BMD-determined MIC range, 16 to 64 micrograms ml-1), even after a minimum of 10 consecutive subcultures on drug-free medium and following prolonged storage at -70 degrees C. The clonal relationship between the parental isolates and their respective fluconazole-resistant derivatives was confirmed by genomic DNA fingerprinting and karyotype analysis. The results of this study demonstrate that C. dubliniensis is inherently susceptible to commonly used antifungal drugs, that fluconazole resistance does occur in clinical isolates, and that stable fluconazole resistance can be readily induced in vitro following exposure to the drug.

Transcriptional Analyses of Antifungal Drug Resistance in Candida albicans

Article

Full-text available

Sep 2000
ANTIMICROB AGENTS CH

Oral infections with the pathogenic yeast Candida albicans are one of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug-resistant isolates. Several molecular mechanisms that contribute to drug resistance have been identified, including increased mRNA levels for two types of efflux pump genes: the ATP binding cassette transporter CDRs (CDR1 and CDR2) and the major facilitator MDR1. Using Northern blot analyses, the expression patterns of these genes have been determined during logarithmic and stationary phases of cell growth and during growth in different carbon sources in a set of matched susceptible and fluconazole-resistant isolates that have been characterized previously.MDR1, CDR1, and CDR2 are expressed early during logarithmic growth, CDR4 is expressed late during logarithmic growth, and CDR1 is preferentially expressed in stationary-phase cells. There is a small decrease in expression of these genes when the cells are grown in carbon sources other than glucose. While increased mRNA levels of efflux pump genes are commonly associated with azole resistance, the causes of increased mRNA levels have not yet been resolved. Southern blot analysis demonstrates that the increased mRNA levels in these isolates are not the result of gene amplification. Nuclear run-on assays show thatMDR1 and CDR mRNAs are transcriptionally overexpressed in the resistant isolate, suggesting that the antifungal drug resistance in this series is associated with the promoter andtrans-acting factors of the CDR1,CDR2, and MDR1 genes.

Paup: phylogenetic analysis using parsimony version 3.1. computer program distributed by the illinois natural history Survey

Article

Jan 1993

D.L. Swofford

Antifungal drug resistance in Candida albicans

Article

Jan 1997
Am Soc Microbiol News

Theodore Curtis White

Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells

Article

Feb 1988

Examination of collections of 16S rRNA sequences revealed sequence domains that were unique to (and invariant within) the three primary lines of cellular descent: the archaebacteria, the eubacteria, and the eucaryotes. Oligodeoxynucleotides complementary to these conserved sequence domains were synthesized and used as hybridization probes. Each of the radiolabeled probes specifically hybridized to nylon membrane-bound 16S rRNA from the targeted kingdom. A probe complementary to a universally conserved sequence in 16S rRNAs was used as a positive control, while its complement provided a negative control for nonspecific binding. The abilities of the probes to bind specifically to whole, fixed cells representing a broad array of phylogenetic diversity were tested in whole-cell dot blot assays. Again, all of the probes specifically bound the targeted groups. By microautoradiography, the method was extended to permit phylogenetic identification of single cells microscopically.

Yeasts pathogenic for humans

Chapter

Dec 1998

D.G. Ahearn

This chapter focuses on various pathogenic yeasts in humans. The principal yeasts pathogenic for humans are Candida albicans and Cryptococcus neoformans. Candida albicans is an asexual, diploid (possibly aneuploid), plemorphicfungus with an ascomycetous-type cell wall. This species is endogenous in the oral-, gastrointestinal-, or urinogenital tracts of humans and other warm blooded animals. The syndromes produced by Candida albicans are highly diversified including vulvovaginitis, dermatitis, cystitis, fever, myositis, hepatic disfunction, and mental confusion. These may occur singly or in combinations dependent upon the superficial, locally invasive, or deep nature of the infection. Cryptococcus neoformans, which is basidiomycetous yeast, includes two varieties that are often haploid when isolated from nature. Cryptococcus neoformans var. neoformans includes serotypes A, D, and AD. C.neoformans var. gatti includes serotypes B and C. Serotypes A and D are usually associated with soil enriched with pigeon droppings. Serotype A is most common in the U.S.A. and serotype D is reported mostly from Europe. Serotypes B and C seem confined to warm regions, particularly serotype B has been associated with the flowering of Eucalyptus camaldulensis. Cryptococcus neoformans most often primarily infects the lungs with mild symptoms. In stressed patients, pneumococcal-type pneumonia is not uncommon. Candida tropicalis is probably the third most important yeast pathogen of humans. Normally, this species does not produce germ tubes or chlamydoconidia, but elongated pseudohyphal cells may develop commonly in some strains in serum at 37°C. Candida tropicalis, like C. albicans, is diploid ascomycetous-type yeast, but it is commonly isolated from host-free habitats.

Molecular cloning: A laboratory manual, second ed

Article

Jan 1989

Molecular taxonomy of the yeasts

Article

Dec 1994
YEAST

Cletus Kurtzman

The term ‘yeast’ is often taken as a synonym for Saccharomyces cerevisiae, but the phylogenetic diversity of yeasts is illustrated by their assignment to two taxonomic classes of fungi, the ascomycetes and the basidiomycetes. Subdivision of taxa within their respective classes is usually made from comparisons of morphological and physiological features whose genetic basis is often unknown. Application of molecular comparisons to questions in yeast classification offers an unprecedented opportunity to re-evaluate current taxonomic schemes from the perspective of quantitative genetic differences. This review examines the impact of molecular comparisons, notably rRNA/rDNA sequence divergence, on the current phenotypically defined classification of yeasts. Principal findings include: 1) budding ascomycetous yeasts are monophyletic and represent a sister group to the filamentous ascomycetes, 2) fission yeasts are ancestral to budding and filamentous ascomycetes, 3) the molecular phylogeny of basidiomycetous yeasts is generally congruent with type of hyphal septum, presence or absence of teliospores in the sexual state, and occurrence of cellular xylose.

Molecular Cloning: A Laboratory Manual

Chapter

Jan 1983

Partial rRNA sequences in marine yeasts: a model for identification of marine eukaryotes

Article

Jul 1992
Mol Mar Biol Biotechnol

The V3 variable region of the large subunit rRNA was examined for nucleotide sequence signatures as potential taxonomic tools. Data are presented on 117 species, representing 23 genera of basidiomycetous yeasts. The results of nucleotide sequence alignments indicate that strains within species have identical base sequences and that species may differ from one another by one to more than 100 base positions. Phylogenetic analyses of the alignments indicates relationships among species, including the prediction of synonymous species and the clustering of species belonging to the Ustilaginales and Tremellales. These results suggest that species-specific nucleotide sequences can be used for the development of techniques for population analyses of a variety of marine and other microeukaryotes.

Azole Antifungal Agents

Article

Apr 1992

Gerald P. Bodey

The discovery of the antifungal activity of azole compounds represented an important therapeutic advance. Miconazole, ketoconazole, and fluconazole are currently commercially available, and itraconazole has undergone extensive clinical evaluation. Because of its limited activity and toxicity, miconazole has been replaced by newer agents. Ketoconazole has proven useful in therapy for superficial infections and invasive infections caused by the pathogenic fungi. Among its disadvantages are limited absorption in the absence of gastric acid and its potential for drug-drug interactions. Fluconazole is the only azole available as oral and intravenous preparations. Unlike other azoles, it is only minimally metabolized in the liver and largely excreted in the urine as active drug. It is more effective than ketoconazole against superficial candidal infections and is the drug of choice for maintenance therapy for cryptococcal meningitis in patients infected with human immunodeficiency virus. An advantage of itraconazole is its activity against aspergillosis. It is also active against many infections caused by pathogenic fungi. Other azole compounds are at varying stages of preclinical and clinical investigation.

Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms

Article

Mar 1992

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.

Huang, M.-M. , Arnheim, N. & Goodman, M. F. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res. 20, 4567-4573

Article

Oct 1992

Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3′-termini. The transition mispairs, A(primer)·C, C·A, G·T, and T·G were extended 10−3 to 10−4-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10−4 to 10−5 for T·C and T·T, about 10−6 for A·A, and less than 10−6 for G·A, A·G, G·G and C·C. The transversion mispair C(primer)·T was extended with high efficiency, about 10−2 compared to a correct A·T basepair. The unexpected ease of extending the C·T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3′-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an Intrinsic property of the enzyme and not due to an inability to bind to 3′-terminal mispairs. For almost all of the mispairs, C·T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myelobiastosis reverse transcriptase and HIV-1 reverse transcrlptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45°C, 55°C and 70°C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.

Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles

Article

Mar 1992

The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.

Evaluation of the MicroScan Rapid Yeast Identification panel

Article

Nov 1991

The MicroScan Rapid Yeast Identification (RYI) panel is a 4-h microdilution system for identification of clinical yeastlike isolates. Its accuracy was evaluated by using 357 isolates encompassing 11 genera and 30 species. The RYI panel identifications were compared with those obtained by the API 20C system assisted with morphological characterization on cornmeal-Tween 80 agar. The panels were read both visually and with the AutoScan-4, a computer-controlled microplate reader. Both the RYI panel and the API 20C system correctly identified 78% of the strains within 4 and 72 h, respectively, with no additional tests. Supplementary tests recommended by the manufacturers made it possible to identify up to 96.6% (AutoScan-4) and 98.9% (API 20C) of the strains. The accuracy of the RYI panel was 99.5% with common strains and 92.1% with less common strains. The RYI panel misidentified 10 or 12 strains and failed to identify 2 or 3 strains, depending on whether it was read with the AutoScan-4 or visually. Errors occurred with one strain of Torulopsis glabrata and the less common yeasts T. candida, Candida lusitaniae, C. lambica, C. rugosa, C. stellatoidea, Cryptococcus albidus, C. laurentii, and C. uniguttulatus. Overall, the RYI panel appears to be a reliable system for identification of the more common clinical yeast isolates.

Giovannoni SJ, Delong EF, Olsen GJ, Pace NR.. Phylogenetic group-specific oligonucleotide probes for identification of single microbial cells. J Bacteriol 170: 720-726

Article

Mar 1988

Treatment of systemic fungal infections: Recent progress and current problems

Article

Sep 1988

Systemic mycoses continue to emerge as life-threatening infections. Considerable progress in treating these infections is being achieved through better application of established available antifungal agents (amphotericin B, flucytosine, miconazole and ketoconazole), and through development of promising investigational agents (fluconazole, itraconazole). Systemic fungal infections, however, continue to present major problems, including clinical resistance, microbiological resistance, emergence of new pathogens, and involvement of more immunocompromised patients. The purpose of this paper, therefore, is to review the recent progress and current problems in treatment of systemic fungal infections.

Application of the polymerase chain reaction to the diagnosis of candidosis by amplification of an HSP 90 gene fragment

Article

Oct 1993

A 317-base pair (bp) fragment of the Candida albicans heat shock protein 90 (HSP 90) gene was amplified by the polymerase chain reaction (PCR) for detection of C. albicans DNA in clinical specimens. One hundred specimens were examined including swabs (39), urines (36), peritoneal fluid (9), pus (8) and blood or serum (8): 23% gave positive results with routine culture, 31% with extended broth culture and 37% with PCR. The amplified product was identified by hybridisation with a radiolabelled internal probe and their restriction enzyme digest patterns (SspI, HaeIII, EcoRI, RsaI and XhoI), which could be predicted from the known sequence of HSP 90. C. albicans DNA gave the characteristic 317-bp band and specifically hybridised with restriction enzyme-digested candidal DNA. DNA from other sources intermittently gave multiple faint bands especially in the presence of high concentrations of DNA, but these could be readily distinguished. The method was sensitive to 50 pg of DNA (5 pg with radiolabelled probing) and 100 cfu of C. albicans.

Evaluation of Gen-Probe's Histoplasma capsulatum and Cryptococcus neoformans AccuProbes

Article

Mar 1993

Gen-Probe's DNA probes were evaluated for use in the identification of clinical isolates of Histoplasma capsulatum var. capsulatum and Cryptococcus neoformans. Ninety-five mould-phase fungi were probed, including 41 isolates of H. capsulatum var. capsulatum. Similarly, 98 yeasts, including 42 C. neoformans isolates, were examined by using the C. neoformans DNA probe. In the study, both probes demonstrated 100% specificity and 100% sensitivity. Their use in the clinical laboratory may significantly reduce the time required for definitive identification of fungi.

Resistance of yeasts to azole: Derivative antifungals

Article

May 1993

Frank C. Odds

There are relatively few antifungal agents available for the treatment of systemic mycoses. The incidence of these infections, particularly among the immunocompromised, has increased significantly in recent years. Amphotericin B, flucytosine and the azole-derivatives--fluconazole, itraconazole and ketoconazole--are the only drugs of value in the treatment of systemic yeast infections currently available. To date resistance among individual yeast species or strains has only been a serious problem with flucytosine. However, resistance among Candida spp. to orally administered azole-derivatives has been observed. The frequency with which resistance has been described in clinical practice among yeasts differs considerably between the three azole antifungal agents. Fluconazole has been implicated in emergent resistance more frequently than ketoconazole, and ketoconazole more often than itraconazole. It must be a matter for concern that, by analogy with the known emergence of antibiotic-resistance among bacteria, that the widespread use of a drug inactive against a particular species may lead to an increased incidence of such infections. An international epidemiological survey is required to establish the extent and degree of resistance to the azole antifungals.

Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' End of the Large-Subunit (26S) Ribosomal DNA Gene

Article

Jun 1997

Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented.

Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Drug Resistance

Article

May 1998

In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

PAUP: phylogenetic analysis using parsimony, version 3.1.1

D L Swofford

Swofford, D. L. 1993. PAUP: phylogenetic analysis using parsimony, version 3.1.1. Illinois Natural History Survey, Champaign, Ill

Morphology taxonomy and classification of the fungi Manual of clinical microbiology 6th

D M Dixon
R A Fromtling

Manual of clinical microbiology 6th ed

P R Murray
E J Baron
M A Pfaller
F C Tenover
R H Yolken

Rapid preparation of DNA from filamentous fungi

Jan 1985
17

U Raeder
P Broda
Raeder U.

Extension of base mispairs by Taq polymerase: implications for single nucleotide discrimination in PCR

Jan 1992
4567

M Huang
N Arnheim
M F Goodman
Huang M.

Rapid Identification of Candida albicansand Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR

Abstract

No full-text available

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