Monoclonal antibodies for the treatment of Graves' ophthalmopathy: A systematic review and meta-analysis.

PURPOSE: The traditional standard of care for Graves' ophthalmopathy (GO) is glucocorticoid therapy, which is associated with many long-term side effects. The aim of this systematic review and meta-analysis was to compare the traditional therapy to novel monoclonal antibodies (e.g. rituximab [RTX], teprotumumab, and tocilizumab [TCZ]). METHODS: We searched the Medline, Embase, and Cochrane Central Register of Controlled Trials databases. We included randomized controlled trials (RCTs) that compared different monoclonal antibodies (e.g. RTX, teprotumumab, and TCZ) with glucocorticoids or placebo in patients with GO. We evaluated the clinical activity score (CAS), proptosis, subjective diplopia using the Gorman score, quality of life (QoT), adverse events, change in lid fissure, NOSPECS score, and TSH receptor antibody (TRAb) levels. The odds ratio (OR) was used to represent dichotomous outcomes. The continuous outcomes were represented as standardized mean difference (SMD). Data were pooled using the inverse variance weighting method. Risk of bias was assessed using the revised Cochrane risk-of-bias tool for randomized trials. RESULTS: Six (n = 571) RCTs were deemed eligible. The different monoclonal antibodies were significantly more efficacious than glucocorticoid/placebo in terms of reduction in CAS (SMD = -1.44, 95% confidence interval (CI): -1.91--0.97, P < 0.00001, I2 = 74%), change in proptosis (SMD = -4.96, 95% CI: -8.02--1.89, P = 0.002, I2 = 99%), QoL (SMD = 2.64, 95% CI: 0.50-4.79, P = 0.02, I2 = 97%), and Gorman score for diplopia (OR = 3.42, 95% CI: 1.62-7.22, P = 0.001, I2 = 8%). However, monoclonal antibodies have shown higher rates of adverse events (OR = 2.91, 95% CI: 1.12-7.56, P = 0.03, I2 = 62%). No significant difference was found with respect to lid fissure, NOSPECS, and TRAb levels. CONCLUSION: This meta-analysis demonstrated that monoclonal antibodies were associated with more favorable clinical outcomes than standard steroid therapy or placebo, especially with regard to CAS, change in proptosis, diplopia, and QoL, with teprotumumab being superior. In addition, only minor safety concerns were identified with monoclonal antibodies though less worrisome than using traditional steroids.

Environmental distribution of Cryptococcus species and some other yeast-like fungi in India.

A study of environmental distribution revealed the occurrence of Cryptococcus neoformans and C. gattii in 9% and 3%, respectively, of 611 samples investigated. C. neoformans showed the highest isolation frequency from tree trunk hollows in Delhi (31%), whereas C. gattii occurred in 12% of the samples in Delhi and 5% in Rajasthan. In addition, Cryptococcus laurentii (=Papiliotrema laurentii), C. rajasthanensis (=Papiliotrema rajasthanensis), C. podzolicus (=Saitozyma podzolica) and C. flavescens (=Papiliotrema flavescens) occurred in 0.5% each. The recovery of C. flavescens and C. podzolicus was new findings for India. One more noteworthy finding was isolation of a new yeast, recently classified as Saitozyma cassiae sp. Novo. The previous strain of this yeast came from tree bark debris in South India. Our isolates came from decayed wood inside a trunk hollow of an Acacia tree in, Bharatpur Bird Sanctuary, Rajasthan. The isolations of novel strains of Cutaneotrichosporon moniliiforme from decayed wood of a Pinus tree was another significant finding. Phenotypically, they differed from T. moniliforme by being encapsulated cells, had melanin-like pigment production and were unable to assimilate d-manitol and d-melezitose. AFLP analysis showed a distinctive banding profile vis-a-vis the reference strains of T. moniliiforme and Cryptotrichosporon anacardii.

In vitro Impact of Limited Exposure to Subtherapeutic Concentrations of Chlorhexidine Gluconate on the Adhesion-Associated Attributes of Oral Candida Species.

OBJECTIVE: Candida albicans and its non-albicans counterparts, such as C. tropicalis, C. krusei, C. glabrata and C. dubliniensis, are the major etiological agents of oral candidosis. Their adherence to buccal epithelial cells (BEC), denture acrylic surfaces (DAS) and cell surface hydrophobicity (CSH) are attributes associated with yeast colonization and infection. Chlorhexidine gluconate (CG) is a widely used antiseptic in dentistry. When administered, the diluent effect of saliva and the cleansing effect of the oral musculature reduce its bioavailability, compromising its efficacy. Hence, intraorally, Candida undergoes a transient exposure to high CG concentrations, and thereafter it is likely to be subtherapeutic. Therefore, the impact of CG on adhesion to BEC, DAS and CSH of different oral Candida species was investigated following brief exposure to three subtherapeutic concentrations of CG. MATERIALS AND METHODS: Ten oral isolates of each of the above five Candida species obtained in Kuwait from oral rinse samples were exposed to 0.00125, 0.0025 and 0.005% CG for 30 min. Subsequently, the yeast adhesion to BEC, DAS and CSH was determined. The data were analyzed using ANOVA Dunnett's t tests. RESULTS: Exposure to the lowest dilution (0.00125%) of CG did not elicit a noteworthy collective suppression on all three adhesion traits evaluated. Exposure to 0.0025% CG curtailed the adhesion to BEC, DAS and CSH of Candida species by 50.89, 40.79 and 24.58%, respectively (p < 0.001). Exposure to the highest concentration (0.005%) of CG reduced the adhesion to BEC, DAS and CSH of Candida species by 64.68, 54.59 and 50%, respectively (p < 0.001). CONCLUSIONS: Brief exposure to subtherapeutic concentrations of CG suppressed the adhesion to BEC, DAS and CSH of oral Candida species, indicating probable pharmacodynamics that may potentiate its antiseptic properties.

Impact of brief and sequential exposure to nystatin, amphotericin B, ketoconazole, and fluconazole in modulating adhesion traits of oral Candida dubliniensis isolates.

AIM: Candida adherence is implicated in the pathogenesis of oral candidosis. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation, and relative cell surface hydrophobicity (CSH) are colonization attributes of candidal pathogenicity. Candida dubliniensis (C. dubliniensis) is allied with recurrent oral candidosis, which can be treated with nystatin, amphotericin B, ketoconazole, and fluconazole. Due to the diluent effect of saliva and the cleansing effect of the oral musculature in the oral cavity C. dubliniensis isolates undergo brief and sequential exposure to antifungal agents during therapy. Thus, in the present study, we evaluated the adhesion to BEC, GT formation, and the CSH of oral isolates of C. dubliniensis following brief and sequential exposure to nystatin, amphotericin B, ketoconazole, and fluconazole. METHODS: After determining the minimum inhibitory concentration (MIC) of the aforementioned drugs, 20 oral isolates of C. dubliniensis were briefly (1 h), and sequentially (10 days) exposed to subcidal concentrations of these drugs. Following drug removal, adhesion to BEC, GT formation, and CSH of these isolates were determined. RESULTS: The percentage reduction of adhesion to BEC, GT formation, and CSH of the isolates following exposure to antifungal agents were as follows: nystatin: 53.55%, 33.98%, and 29.83% (P < 0.001); amphotericin B: 53.84%, 36.23%, and 28.97% (P < 0.001); ketoconazole: 37.43%, 20.51%, and 16.49% (P < 0.001); and fluconazole: 8.93% (P < 0.001), 1.6%, and 0.63% (P > 0.05). CONCLUSIONS: Brief and sequential exposure of C. dubliniensis to antifungal agents would continue to wield an antifungal effect by altering its adhesion attributes, and elucidate possible pharmacodynamics by which antifungal agents might operate in modulating candidal adherence.

Impact of short-term exposure of antifungal agents on hemolysin activity of oral Candida dubliniensis isolates from Kuwait and Sri Lanka.

OBJECTIVE: Ability to produce hemolysin by Candida species is an important determinant of its pathogenicity. Candida dubliniensis is implicated in the causation of oral candidosis, which can be treated with polyene, echinocandin, and azole groups of antifungal agents as well as chlorhexidine. After oral application, however, the concentrations of these agents tend to decrease quickly to subtherapeutic levels due to the peculiarity of the oral environment. In this study, we have evaluated the effect of short-term exposure of sublethal concentrations of these drugs on hemolysin production by oral C. dubliniensis isolates obtained from two different geographical locale. MATERIALS AND METHODS: Twenty C. dubliniensis oral isolates obtained from Kuwait and Sri Lanka were exposed to sublethal concentrations of nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine for 1 h. Thereafter, the drugs were removed by dilution and the hemolysin production determined by a previously described plate assay. RESULTS: Hemolysin production of these isolates was significantly suppressed with a percentage reduction of 17.09, 16.45, 17.09, 11.39, 8.23 and 12.03 following exposure to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine, respectively. CONCLUSION: Brief exposure to sublethal concentrations of drugs with antifungal properties appears to reduce the pathogenic potential of C. dubliniensis isolates by suppressing hemolysin production.

In vitro postantifungal effect, adhesion traits and haemolysin production of Candida dubliniensis isolates following exposure to 5-fluorocytosine.

The phenomenon of postantifungal effect (PAFE), which is the suppression of candidal growth following brief exposure to antifungal agents, is linked with candidal pathogenicity. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation and relative cell surface hydrophobicity (CSH) are all adhesion traits of candidal pathogenicity. Ability to produce haemolysin by Candida species is also a determinant of its pathogenicity. There is no information on either the PAFE or its impact on adhesion traits and haemolysin production of oral Candida dubliniensis isolates following exposure to 5-fluorocytosine (5-FC). Hence, the focus of this investigation was to research the in vitro PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production on 20 C. dubliniensis isolates following exposure to 5-FC. Following obtaining the minimum inhibitory concentration (MIC) of 5-FC, isolates of C. dubliniensis were exposed to sub-lethal concentrations (×3 MIC) of 5-FC for 1 h. After this brief exposure, the antimycotic was removed and PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production was determined by formerly described in vitro methods. MIC (µg/ml) of C. dubliniensis isolates to 5-FC ranged from 0.002 to 0.125. The mean PAFE (hours) elicited by 5-FC on C. dubliniensis isolates was approximately 1 h. Exposure to 5-FC suppressed the ability of C. dubliniensis isolates to adhere BEC, GT formation, relative CSH and haemolysin activity by a mean percentage reduction in 50.98%, 29.51%, 36.79% and 12.75% (P < 0.001 for all) respectively. Therefore, brief exposure of C. dubliniensis isolates to 5-FC appears to exert an antifungal effect by subduing its growth, adhesion traits as well as haemolysin production.

Post-antifungal effect and adhesion to buccal epithelial cells of oral Candida dubliniensis isolates subsequent to limited exposure to amphotericin B, ketoconazole and fluconazole.

AIM: The post-antifungal effect (PAFE) of Candida and its adherence to oral mucosal surfaces are important determinants of candidal pathogenicity. Candida dubliniensis is allied with recurrent oral candidosis. Oral candidosis can be treated with amphotericin B, ketoconazole and fluconazole. There is no information on the PAFE and its impact on adhesion to oral buccal epithelial cells (BEC) of oral C. dubliniensis isolates. Therefore, the main objective was to reconnoiter the PAFE and adhesion to BEC of 20 C. dubliniensis isolates following brief exposure to aforementioned antimycotics. METHODS: After determining the minimum inhibitory concentration (MIC), C. dubliniensis isolates were exposed to sub-lethal concentrations of these drugs for 1 h. Following subsequent drug removal, the PAFE and adhesion to BEC, was determined by a turbidometric method, and an adhesion assay, respectively. RESULTS: Minimum inhibitory concentration (µg/mL) to amphotericin B, ketoconazole and fluconazole, ranged from 0.002 to 0.125, 0.002 to 0.012 and 0.016 to 0.38, respectively. Amphotericin B and ketoconazole induced mean PAFE (hours) were 2.21 and 0.6, respectively. Fluconazole failed to produce a detectable PAFE. Compared to controls, amphotericin B, ketoconazole and fluconazole suppressed the ability to adhere to BEC with a mean percentage reduction of 74.31%, 49.80% (P < 0.0001) and 29.36% (P < 0.05), respectively. CONCLUSIONS: Brief exposure to sub-lethal concentrations of aforementioned drugs would exert an antifungal effect by modifying the growth and adhesion of C. dubliniensis isolates.

In vitro adhesion of oral Candida dubliniensis isolates to acrylic denture surfaces following brief exposure to sub-cidal concentrations of polyenes, azoles and chlorhexidine.

OBJECTIVES: We aimed to investigate the effect of brief exposure to sub-cidal concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and chlorhexidine gluconate on the adhesion of oral Candida dubliniensis isolates to the surface of acrylic dentures. METHODS: After determining the minimum inhibitory concentration of each drug, 20 oral isolates of C. dubliniensis were exposed to sub-cidal concentrations of the drugs for 1 h. The drugs were then removed by dilution, and the adhesion of the isolates to denture acrylic strips was assessed by an in vitro adhesion assay. RESULTS: Compared to the controls, exposure to nystatin, amphotericin B, ketoconazole, fluconazole and chlorhexidine gluconate suppressed the ability of C. dubliniensis isolates to adhere to acrylic denture surfaces with a reduction of 74.68, 74.27, 57.31, 44.57 and 56.53% (p < 0.001 for all drugs), respectively. CONCLUSIONS: Brief exposure to sub-cidal concentrations of anti-mycotics suppressed the adhesion of C. dubliniensis oral isolates to acrylic denture surfaces.

Isolation of Lodderomyces elongisporus from the Catheter Tip of a Fungemia Patient in the Middle East.

Lodderomyces elongisporus is phenotypically closely related to Candida parapsilosis and has recently been identified as an infrequent cause of bloodstream infections in patients from Asia and Mexico. We report here the isolation of Lodderomyces elongisporus from the catheter of a suspected case of fungemia. The identity of the isolate was confirmed by phenotypic characteristics and ribosomal DNA sequencing.

Genotypic heterogeneity and molecular basis of 5-flucytosine resistance among Candida dubliniensis isolates recovered from clinical specimens in Kuwait.

There is a paucity of information about genotypic heterogeneity among Candida dubliniensis isolates recovered from different geographic regions. This study explored genotypic heterogeneity among 103 C. dubliniensis strains obtained over a six-year period from clinical specimens in Kuwait. Genotype assignment was based on amplification with genotype-specific primers and sequencing of rDNA. Susceptibility to 5-flucytosine was determined by means of the Etest. DNA sequencing of cytosine deaminase was performed to determine the molecular basis of resistance to 5-flucytosine. DNA sequencing of rDNA identified seven different genotypes, i.e., 68 (66%) isolates were found to belong to genotype 1, 25 to genotype 4, six to genotype 5 and one each to genotypes 6-9. Strains of genotype 2 or genotype 3 were not detected. All isolates of genotype 4 but none of other genotypes were resistant to 5-flucytosine and the resistant strains all contained S29L mutation. Isolates of all other genotypes contained wild-type codon 29 in cytosine deaminase. A simple, PCR-RFLP-based method has been developed to facilitate rapid detection of S29L mutation in cytosine deaminase. A noteworthy observation of our study is the identification of five new genotypes of C. dubliniensis isolates, recovered from oral/respiratory specimens from patients of Middle Eastern origin. Furthermore, all 5-flucytosine resistant C. dubliniensis isolates in Kuwait belonged to genotype 4 only.

Prevalence of Candida dubliniensis among oral Candida isolates in patients attending the Kuwait University Dental Clinic.

OBJECTIVES: The aim of this study was to determine the oral candidal carriage of patients seeking dental treatment at the Kuwait University Dental Clinic and to ascertain the Candida species composition among them. METHODS: 370 oral rinse samples were collected from patients. The germ tube test, CHROMagar Candida medium and VITEK 2 yeast identification system were used for species identification. C. dubliniensis isolates were confirmed by the production of rough colonies with hyphal fringes and chlamydospores on simplified sunflower seed agar. RESULTS: Of the 370 samples investigated, 160 (43.24%) showed Candida in culture. The isolation of Candida was significantly higher in individuals who were smokers or were under medication for either diabetics or asthma [99 (62%)] compared to healthy individuals [61 (38%)]. Of the 210 samples which did not yield Candida, 131 (62.38%) were healthy and 79 (37.62%) were associated with smoking or with usage of drugs for aforementioned conditions. Species isolated were C. albicans [102 (63.7%)], C. dubliniensis [23(14.3%)], C. krusei [13 (8.1%)], C. tropicalis [12 (7.5%)] and C. glabrata [10 (6.2%)]. CONCLUSIONS: Candida species were more prevalent in patients having predisposing factors implicated in oral candidosis, such as in smokers, diabetic patients and asthmatic patients using inhalation steroids. C. albicans was the most prevalent species isolated, followed by C. dubliniensis.

Kodamaea ohmeri as an emerging pathogen: a case report and review of the literature.

Kodamaea (Pichia) ohmeri is a yeast-like fungus that has recently emerged as an important etiologic agent of fungemia in immunocompromised patients. We report such a case in a premature neonate born at 29 weeks of gestation. Prior to developing fungemia, she had two episodes of bacterial sepsis on day 13 and day 32 due to Enterobacter cloacae and Staphylococcus epidermidis, respectively. Kodamaea ohmeri was repeatedly isolated from blood cultures and its identity was determined by phenotypic characteristics and sequencing of the ITS and D1/D2 regions of rDNA. The neonate was successfully treated with amphotericin B. The published cases of K. ohmeri fungemia reported in pediatric patients are reviewed highlighting its increasing importance as a bloodstream pathogen.

A comparison of the antifungal activity of herbal toothpastes against other brands of toothpastes on clinical isolates of Candida albicans and Candida dubliniensis.

OBJECTIVES: To evaluate the anti-Candida activity on Candida albicans and Candida dubliniensis species of 2 herbal and 7 other brands of toothpastes commonly used in Kuwait. MATERIALS AND METHODS: Antifungal activity was determined by agar diffusion test on 65 isolates of C. albicans and 21 isolates of C. dubliniensis for each toothpaste. A uniform quantity of toothpaste was filled into wells punched into Sabouraud dextrose agar medium plates inoculated with the test isolates, incubated at 37°C; inhibition zone diameters were read after 24 h. RESULTS: The mean inhibition zone diameters ranged between 12 and 23 mm for C. albicans and between 12 and 27 mm for C. dubliniensis. A herbal toothpaste brand manufactured in the Middle Eastern region (United Arab Emirates) consisting of many herbal ingredients compared to other brands was found to be the most active (p < 0.001) against both Candida species tested, which also demonstrated higher inhibitory activity against C. dubliniensis isolates compared to C. albicans. CONCLUSIONS: The herbal toothpaste brand presented significant anticandidal activity over conventional toothpastes and may be useful in reducing the pathogenic potential of Candida species.

Prevalence of Candida dubliniensis among cancer patients in Kuwait: a 5-year retrospective study.

Despite close genetic and phenotypic relationship of Candida dubliniensis with Candida albicans, its role in human disease is mostly restricted to oral colonisation, particularly among HIV-infected patients. The prevalence of C. dubliniensis in association with other disease conditions has been infrequently reported. In this study, we present data on the prevalence of C. dubliniensis among yeast species isolated from cancer patients over a 5-year period. A total of 1445 yeast isolates recovered from respiratory specimens, blood, urine and oral swabs were analysed. Candida dubliniensis isolates were provisionally identified by phenotypic methods and their identity was further confirmed by species-specific amplification and/or sequencing of internally transcribed spacer region of rDNA. Antifungal susceptibility for fluconazole was determined by Etest. The number of isolates identified as C. dubliniensis, C. albicans and other yeast species were 71 (4.9%), 862 (59.6%) and 512 (35%) respectively. All the C. dubliniensis isolates originated from respiratory (5.9%) or oral (3.2%) specimens with an overall prevalence of 4.9%, and were found to be susceptible to fluconazole. The isolation of C. dubliniensis from respiratory or oral specimens and not from blood or urine specimens suggests that this species has preference to colonise these sites of human body.

Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli.

The diagnostic potential of secretory proteins of Aspergillus fumigatus is limited by their availability in pure form. We have constructed a vector (pGES-PH-1) to express genes encoding secretory proteins of A. fumigatus as fusion proteins with glutathione S-transferase (GST) in Escherichia coli. The mitogillin, a secretary protein of A. fumigatus, was expressed and purified to homogeneity by using pGES-PH-1. Mitogillin gene was PCR amplified from A. fumigatus DNA, cloned in pGES-PH-1 and expressed in E. coli as fusion protein with GST at N-terminal and 6xHis tag at C-terminal end. Pure mitogillin was obtained by purification on glutathione-Sepharose, cleavage of column-bound fusion protein by PreScission protease and by further purification on Ni-NTA-agarose. Polyclonal anti-mitogillin antibodies were raised in rabbits and were used to study its secretion during in vitro growth of A. fumigatus. The mitogillin was detectable in culture filtrate after 24 h of A. fumigatus growth and thereafter its amount increased progressively until 96 h in both, Sabouraud dextrose broth and potato dextrose broth. However, the secretion of mitogillin in culture medium was slightly delayed when A. fumigatus was grown in a minimal medium as mitogillin was detected only after 36 h of growth. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags and PreScission protease cleavage site for high-level expression and efficient purification of a recombinant A. fumigatus secretory protein expressed in E. coli, which could be used for further studies.

Levels of (1→3)-ß-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species.

BACKGROUND: Surveillance cultures may be helpful in identifying patients at increased risk of developing invasive candidiasis. However, only scant information exists on the effect of Candida colonization on serum levels of diagnostic biomarkers. This prospective surveillance study determined the extent of Candida colonization among pediatric cancer patients and its possible impact on serum levels of (1-3)-ß-D-glucan (BDG), Candida mannan and Candida DNA. METHODS: A total of 1075 swabs originating from oropharynx (n = 294), nostrils (n = 600), rectum (n = 28), groin (n = 50), ear (n = 54), and axilla (n = 49) of 63 pediatric cancer patients were cultured for the isolation of Candida spp. Patients yielding Candida spp. from any sites were considered as colonized. Serum samples were collected from patients at the time of first surveillance culture for detection of BDG by Fungitell kit and Candida mannan by Platelia Candida Ag. Candida DNA was detected by using panfungal primers and identification was carried out by using species-specific primers and DNA sequencing. RESULTS: Seventy-five (7.6%) swab cultures from 35 (55.5%) patients yielded Candida spp. These isolates included C. albicans (n = 62), C. dubliniensis (n = 8), C. glabrata and C. tropicalis (n = 2 each) and C. krusei (n = 1). Eleven patients were colonized at three or more sites. Eight of 36 serum samples from 6 colonized patients yielded BDG values higher than the currently recommended cut-off value of ≥80 pg/ml. However, none of the serum samples yielded Candida mannan levels ≥0.5 ng/ml and PCR test for Candida DNA was also negative in all the serum samples of colonized patients. During the study period, only two colonized patients subsequently developed candidemia due to C. tropicalis. Besides positive blood cultures, C. tropicalis DNA, BDG and Candida mannan were also detected in serum samples of both the patients. CONCLUSIONS: The present study demonstrates that while mucosal colonization with Candida species in pediatric cancer patients is common, it does not give rise to diagnostically significant levels of Candida mannan or Candida DNA in serum specimens. However, BDG values may be higher than the cut-off value in some pediatric patients without clinical evidence of invasive Candida infection. The study suggests the utility of Candida mannan or Candida DNA in the diagnosis of invasive candidiasis, however, the BDG levels in pediatric cancer subjects should be interpreted with caution.

Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi, India.

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37degrees C), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, L: -rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate L: -sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).

Development of a nested PCR assay for the detection of Fusarium solani DNA and its evaluation in the diagnosis of invasive fusariosis using an experimental mouse model.

Fusarium infections are increasingly being encountered in immunocompromised patients. Fusarium solani accounts for nearly half of these infections. A specific nested PCR (nPCR) assay has been developed by using DNA isolated from several Fusarium species and other common fungi. Furthermore, DNA samples isolated from bronchoalveolar lavage (BAL) and serum samples from mice infected intravenously with F. solani conidia and sacrificed on every third day post infection were used for the evaluation of the established nPCR protocol. The lung homogenate, BAL and blood from infected animals were also cultured. The nPCR assay was specific for F. solani and detected 450 fg of DNA corresponding roughly to 11 F. solani cells. Cultures of lung homogenate of infected animals up to day 16 yielded F. solani with decreasing fungal load and were negative thereafter. The nPCR positivity in BAL was 100% concordant with lung tissue culture results. Although detection of F. solani DNA in serum was less sensitive than in BAL, it remained positive for longer duration. Our data from an experimental mouse model show that detection of DNA in BAL and to a lesser extent in serum by nPCR offers a sensitive and specific diagnostic approach to invasive F. solani infection.

Rapid molecular differentiation and genotypic heterogeneity among Candida parapsilosis and Candida orthopsilosis strains isolated from clinical specimens in Kuwait.

Recent molecular studies have led to the recognition of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. As currently available yeast identification systems fail to differentiate these species, there is a paucity of information on their occurrence in different geographical regions. This study describes a simple PCR-based protocol for rapid discrimination among C. parapsilosis, C. orthopsilosis and C. metapsilosis strains by using primers derived from unique sequences within the internally transcribed spacer 1 (ITS1)-5.8 rRNA-ITS2 region. Retrospective analysis of 114 C. parapsilosis-complex isolates recovered from clinical specimens in Kuwait identified 109 as C. parapsilosis, five as C. orthopsilosis and none as C. metapsilosis. The results were further validated by PCR-RFLP patterns of the secondary alcohol dehydrogenase gene fragment. DNA sequencing of the ITS region and the D1/D2 regions of the 28S rRNA gene confirmed the species-specific identification of all five C. orthopsilosis strains. The amplicon length of the intergenic spacer between the 28S and 5S rRNA genes (IGS1) was also species-specific, and PCR-RFLP analyses of the IGS1 region identified two distinct genotypes among the five C. orthopsilosis strains, which corresponded with the ITS region sequence data. The three bloodstream C. orthopsilosis strains were confined to a single genotype. Among 81 randomly selected C. parapsilosis strains, two genotypes were detected by IGS1 region analyses, indicating limited genotypic heterogeneity among C. parapsilosis sensu stricto strains. As far as is known, this is the first report on the identification of C. orthopsilosis from a bloodstream infection in the Arabian Gulf region.

Mucor circinelloides as a cause of invasive maxillofacial zygomycosis: an emerging dimorphic pathogen with reduced susceptibility to posaconazole.

A case of maxillofacial zygomycosis caused by Mucor circinelloides, identified by phenotypic and molecular methods and treated successfully with liposomal amphotericin B (AmBisome) and surgical debridement, is described. The isolate was resistant to posaconazole. This report underscores the importance of prior susceptibility testing of zygomycetes to guide therapy with the most effective antifungal agent for an improved prognosis.