Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics.

OBJECTIVE: Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. MATERIALS AND METHODS: Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. RESULTS: The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. CONCLUSIONS: Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

Pathogenicity of Candida viswanathii for normal and cortisone-treated mice.

The pathogenicity of Candida viswanathii, PCI 501/1 (CBS 4024), originally isolated from CSF of a fatal case of meningitis in India, is reported. Also, included is a global overview of the occurrence of C. viswanathii in clinical and environmental sources. The investigation was done in normal and cortisone-treated albino mice challenged intravenously with variable doses of 1×10(6), 4×10(6) and 16×10(6) actively growing yeast cells of the fungus. The animals were kept under observation up to 3 weeks when they were sacrificed for a mycological and histopathologic study. As apparent from the data on morbidity and mortality, the species exhibited low virulence for normal mice, whereas it caused significantly higher mortality (P<0.0008) and morbidity (macroscopic lesions) (P<0.0004) in cortisone group. Likewise, there was overall higher recovery of C. viswanathii in culture from the cortisone-treated than in the normal group of mice. These observations are indicative of C. viswanathii being an opportunistic pathogen. It is recognized that a definitive identification of C. viswanathii requires mycological expertise for comprehensive phenotypic characterization or the application of expensive techniques such as Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) and molecular techniques, facilities for which are generally lacking in a vast majority of laboratory diagnostic centers especially in developing countries. Consequently, the prevalence of C. viswanathii in clinical and environmental samples is currently likely to be underestimated.

Species spectrum and antifungal susceptibility profile of vaginal isolates of Candida in Kuwait.

OBJECTIVE: The study was undertaken to determine the prevalence of vulvovaginal candidiasis (VVC) among patients with vaginitis, frequency of different Candida species, and their susceptibility profile. PATIENTS AND METHODS: Over six months period, high vaginal swabs were cultured on Sabouraud's dextrose agar and isolates were identified by culture on CHROMagar Candida and Vitek2 yeast identification system or/and API 20C (BioMerieux, France). Antifungal susceptibility of the Candida isolates was determined by E-test against amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole and caspofungin. RESULTS: One thousand seven hundred and fifty-two women with vaginitis were screened for the prevalence of Candida spp. Vaginal swab cultures of 231 (13.2%) women yielded Candida spp. The isolation rates of different species were as follows: Candida albicans (73.9%), Candida glabrata (19.8%), Candida kefir (1.94%), Candida tropicalis (0.96%), Candida parapsilosis (0.96%), Candida krusei (0.96%), Candida guilliermondii (0.96%), and Saccharomyces cerevisiae (0.52%). All strains of C. albicans and non-C. albicans were susceptible to most of the antifungal agents tested. CONCLUSION: The high frequency with which C. albicans was recovered and its azole susceptibility support the continued use of azole agents for empirical therapy of uncomplicated VVC. However, a larger controlled study is required to determine the role of non-C. albicans in recurrent VVC.

Comparative evaluation of two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems for the identification of clinically significant yeasts.

OBJECTIVES: To prospectively evaluate the performance of two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry systems (MALDI-TOF MS) for the identification of clinically significant yeast isolates compared to the VITEK 2 system. METHODS: One hundred and eighty-eight consecutive yeast isolates were analyzed by Bruker Biotyper and VITEK MS. The results were compared with the conventional VITEK 2 yeast identification system. Discrepant results were resolved by direct sequencing of rDNA. RESULTS: Accurate identification by VITEK 2, VITEK MS, and Bruker Biotyper MS was 94.1% (177/188), 93.0% (175/188), and 92.6% (174/188), respectively. Three isolates were not identified by VITEK MS, while nine Candida orthopsilosis were misidentified as Candida parapsilosis, as this species is not present in its database. Eleven isolates were not identified or were wrongly identified by Bruker Biotyper and although another 14 were correctly identified, the score was unreliable at <1.7. CONCLUSION: The overall accuracy of rapid MALDI-TOF MS systems was essentially comparable to that of the conventional VITEK 2 yeast identification system. However, future expansion of the databases may further improve the outcome and accuracy of identification of yeast species.

The postantifungal effect and phospholipase production of oral Candida albicans from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to subtherapeutic concentrations of chlorhexidine gluconate.

Candida albicans is the major aetiological agent of oral candidosis and one of its important virulent factors is the production of extracellular phospholipases, which can be modulated by subtherapeutic concentrations of antifungal agents thus decreasing their pathogenicity. Hence, considering that chlorhexidine gluconate (CG) is a common antimicrobial mouthwash used in dentistry and that its concentration in the mouth reaches subtherapeutic levels during dosage intervals due to the diluent effect of saliva and cleansing effect of the oral musculature, the postantifungal effect (PAFE) and the phospholipase production of oral C. albicans following brief exposure to subtherapeutic concentrations of CG was studied. Fifty C. albicans planktonic oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to three subtherapeutic concentrations of CG (0.005%, 0.0025% and 0.00125%) for 1 h. Isolates unexposed to CG was the control group. Thereafter the antiseptic was removed and the PAFE and phospholipase production was determined by a turbidometric method and a plate assay using an egg yolk agar medium respectively. Mean PAFE (hours) of 50 oral isolates of C. albicans following 1-h exposure to 0.005%, 0.0025% and 0.00125% CG was 6.97, 1.85 and 0.62 respectively. The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 21.68, 18.20 and 14.04% following exposure to 0.005%, 0.0025% and 0.00125% CG respectively. Brief exposure of C. albicans isolates to subtherapeutic concentrations of CG would wield an antifungal effect by suppressing growth and phospholipase production, thereby quelling its pathogenicity.

The postantifungal effect of nystatin and its impact on adhesion attributes of oral Candida dubliniensis isolates.

The postantifungal effect (PAFE) has an impact on candidal pathogenicity. However, there is no information on either the PAFE or its impact on adhesion traits of oral Candida dubliniensis isolates. Oral candidosis can be treated topically with nystatin. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation and relative cell surface hydrophobicity (CSH) are all colonisation attributes of candidal pathogenicity. Hence, the main objective of this study was to investigate the in vitro PAFE on 20 C. dubliniensis isolates following exposure to nystatin. In addition, the impact of nystatin-induced PAFE on adhesion to BEC, GT formation and relative CSH of C. dubliniensis isolates were also evaluated. After determining the minimum inhibitory concentration (MIC) of nystatin, C. dubliniensis isolates were exposed to sublethal concentrations of nystatin for 1 h. Following this exposure, the drug was removed and PAFE, adhesion to BEC, GT formation and relative CSH were determined by a previously described turbidometric method, adhesion assay, germ tube induction assay and biphasic aqueous-hydrocarbon assay respectively. MIC (µg/ml) of C. dubliniensis isolates to nystatin ranged from 0.09 to 0.78. The nystatin-induced mean PAFE (hours) on C. dubliniensis isolates was 2.17. Compared with the controls, exposure to nystatin suppressed the ability of C. dubliniensis isolates to adhere BEC, GT formation and relative CSH by a mean percentage reduction of 74.45% (P < 0.0001), 95.92% (P < 0.0001) and 34.81 (P < 0.05) respectively. Hence, brief exposure of C. dubliniensis isolates to nystatin would continue to wield an antifungal effect by suppressing growth as well as its adhesion attributes.

Changes in germ tube formation and cell-surface hydrophobicity of oral Candida dubliniensis isolates following brief exposure to sub-cidal concentrations of polyene and azole antifungal agents.

Adherence of Candida has been implicated as the initial process in the pathogenesis of oral candidosis. Candidal germ tubes and its relative cell-surface hydrophobicity (CSH) are contributory attributes. Candida dubliniensis is currently documented as an opportunistic pathogen allied with recurrent oral candidosis. Oral candidosis can be treated with polyene and azole antifungals such as amphotericin B, ketoconazole and fluconazole. However, the intraoral concentration of these drugs fluctuates and becomes sub-therapeutic because of the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intraorally, the pathogenic yeast may undergo a brief exposure to antifungal drugs. The objective of this study was to investigate the effect of brief exposure to sub-lethal concentrations of these antifungals on the germ tube formation and CSH of C. dubliniensis. After determining the minimum inhibitory concentration of the drugs, 20 oral isolates of C. dubliniensis were exposed to sub-lethal concentrations of these antifungals for 1 h. Following this brief exposure, the drugs were removed, and following subsequent incubation in a germ tube inducing medium and exposure to bi-phasic hydrocarbon assay, the germ tube formation and CSH of these isolates was quantified respectively. Compared with controls, exposure to amphotericin B almost completely suppressed the ability to form germ tubes with a mean percentage reduction of 95.91% (P < 0.0001), whereas ketoconazole and fluconazole also significantly inhibited germ tube formation but to a lesser degree with a mean percentage reduction of 18.73% and 12.01% respectively (P < 0.05). Compared with controls, exposure to amphotericin B and ketoconazole elicited a significant suppression on CSH with a mean percentage reduction of 33.09% and 21.42%, respectively (P < 0.001), whereas exposure to fluconazole did not elicit a significant suppression on CSH (9.21%; P > 0.05). In clinical terms it appears that, even a short exposure to sub-lethal concentrations of these drugs, a situation all too familiar in the oral environment, would continue to exert an antifungal effect by suppressing the pathogenic potency of C. dubliniensis.

Cell surface hydrophobicity of oral Candida dubliniensis isolates following limited exposure to sub-therapeutic concentrations of chlorhexidine gluconate.

Candidal adhesion has been implicated as the initial step in the pathogenesis of oral candidiasis and cell surface hydrophobicity (CSH) has been implicated in adhesion to mucosal surfaces. Candida dubliniensis is an opportunistic pathogen associated with recurrent oral candidiasis. Chlorhexidine gluconate is by far the commonest antiseptic mouth wash prescribed in dentistry. At dosage intervals the intraoral concentration of this antiseptic fluctuates considerably and reaches sub-therapeutic levels due to the dynamics of the oral cavity. Hence, the organisms undergo only a limited exposure to the antiseptic during treatment. The impact of this antiseptic following such exposure on CSH of C. dubliniensis isolates has not been investigated. Hence, the main objective of this study was to investigate the effect of brief exposure to sub-therapeutic concentrations of chlorhexidine gluconate on the CSH of C. dubliniensis isolates. Twelve oral isolates of C. dubliniensis were briefly exposed to three sub-therapeutic concentrations of 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate for 30 min. Following subsequent removal of the drug, the CSH of the isolates was determined by a biphasic aqueous-hydrocarbon assay. Compared with the controls, exposure to 0.005% and 0.0025% chlorhexidine gluconate suppressed the relative CSH of the total sample tested by 44.49% (P < 0.001) and 21.82% (P < 0.018), respectively, with all isolates being significantly affected. Although exposure to 0.00125% of chlorhexidine gluconate did not elicit a significant suppression on the total sample tested (7.01%; P > 0.05), four isolates of the group were significantly affected. These findings imply that exposure to sub-therapeutic concentrations of chlorhexidine gluconate may suppress CSH of C. dublinienis isolates, thereby reducing its pathogenicity and highlights further the pharmacodynamics of chlorhexidine gluconate.

Changes in the cell surface hydrophobicity of oral Candida albicans from smokers, diabetics, asthmatics, and healthy individuals following limited exposure to chlorhexidine gluconate.

OBJECTIVE: The objective of this study was to determine the cell surface hydrophobicity of 40 oral Candida albicans isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, and healthy individuals, following brief exposure to subtherapeutic concentrations of chlorhexidine gluconate. MATERIALS AND METHODS: Forty C. albicans oral isolates (10 isolates each from smokers, diabetics, asthmatics using steroid inhalers, and healthy individuals) were exposed to 3 subtherapeutic concentrations of chlorhexidine gluconate (0.00125, 0.0025, and 0.005%) for 30 min. Thereafter, the antiseptic was removed and the cell surface hydrophobicity was measured by a biphasic aqueous-hydrocarbon assay. RESULTS: Compared to the unexposed controls, the cell surface hydrophobicity of C. albicans isolates was suppressed by 5.40% (p > 0.05), 21.17% (p < 0.05), and 44.67% (p < 0.05) following exposure to 0.00125, 0.0025, and 0.005% chlorhexidine gluconate, respectively. CONCLUSIONS: A brief period of transient exposure to subtherapeutic concentrations of chlorhexidine gluconate may modulate the cell surface hydrophobicity of C. albicans isolates and thereby may reduce candidal pathogenicity.

Rapid differentiation of Candida dubliniensis from Candida albicans by early D-xylose assimilation.

OBJECTIVE: To determine if D-xylose (XYL) and/or α-methyl-D-glucoside (MDG) assimilation can be used reliably as a rapid test to differentiate Candida dubliniensis from Candida albicans at an earlier time point such as 2 h after inoculation. MATERIALS AND METHODS: Thirty isolates of C. albicans and C. dubliniensis recovered from anatomical sites and clinical specimens were used. Isolates were inoculated into the API 20C AUX yeast identification system, and incubated at 30°C. XYL and MDG assimilations were read at 2-hour intervals beginning 2 h after the initial inoculation and up to 24 h of incubation; thereafter, results were read after 48 and 72 h. RESULTS: Twenty-nine (97%) C. albicans isolates had assimilated XYL at 16 h and, by 24 h, all isolates were positive for XYL assimilation. None of the C. dubliniensis isolates assimilated XYL. The MDG assimilation revealed that 24, 40, 92 and 100% of C. albicans isolates became positive after 16, 24, 48 and 72 h of incubation, respectively, whereas only 3% of C. dubliniensis isolates assimilated MDG after 72 h. CONCLUSIONS: The findings showed that it is possible to rapidly differentiate C. albicans from C. dubliniensis isolates using the API 20C AUX carbohydrate assimilation kits after 16 h of incubation at 30°C based on the XYL assimilation.

Effects of subtherapeutic concentrations of chlorhexidine gluconate on germ tube formation of oral Candida.

OBJECTIVE: The main objective of this study was to investigate the effect of brief exposure to subtherapeutic concentrations of chlorhexidine gluconate on germ tube formation of Candida albicans isolates obtained from smokers, diabetics, asthmatics using steroid inhalers and healthy individuals. MATERIALS AND METHODS: Forty isolates of C. albicans were used in this study. All these isolates were quantified for germ tube formation without exposure to the drug and were used as the control group for data analysis. Isolates were also exposed to three subtherapeutic concentrations of chlorhexidine gluconate (0.00125, 0.0025 and 0.005%) for 30 min (limited exposure); the antiseptic was then removed and germ tube formation of these isolates was quantified microscopically following incubation in a germ tube-inducing medium. RESULTS: Compared with the unexposed controls, brief exposure to all concentrations of chlorhexidine gluconate suppressed the ability of the C. albicans isolates to form germ tubes in increasing order by 13.72% (p < 0.001 to p = 0.02), 46.16% (p < 0.001) and 72.46% (p <0.001). CONCLUSIONS: These findings show that brief exposure to subtherapeutic concentrations of chlorhexidine gluconate may modulate germ tube formation of C. albicans isolates, thereby suppressing their pathogenicity, and further elucidate the pharmacodynamic mechanisms by which chlorhexidine gluconate may operate in vivo.

Value of (1-3)-ß-d-glucan, Candida mannan and Candida DNA detection in the diagnosis of candidaemia.

This study determined the value of (1,3)-ß-d-glucan (BDG), Candida mannan (MN) and Candida species-specific DNA as surrogates for diagnosis of candidaemia. Thirty-nine patients yielding Candida species in blood cultures were investigated for presence of BDG, MN and Candida species-specific DNA in serum samples. The Candida spp. bloodstream isolates included C. albicans (n = 16), C. tropicalis (n = 10), C. parapsilosis (n = 7), C. glabrata (n = 3) and C. dubliniensis (n = 3). Positivity of the three markers was as follows: Candida DNA for corresponding Candida species, 100%; BDG, 87%; MN, 59%. Despite varying sensitivities of these biomarkers, they provided a useful adjunct to the diagnosis of candidaemia.

Bipolaris hawaiiensis as etiologic agent of allergic bronchopulmonary mycosis: first case in a paediatric patient.

Allergic bronchopulmonary mycosis (ABPM) is a worldwide hypersensitivity lung disease of multiple etiologies with Aspergillus fumigatus as the most common etiologic agent. We report the first instance of Bipolaris hawaiiensis causing ABPM in a paediatric patient. A six-year-old girl presented in June 2009 with productive cough, exertional dyspnoea, occasional wheezing, restricted air entry in left infra-scapular and infra-axillary areas, 7% eosinophils (absolute count 540/mm(3)) and total IgE 1051.3 IU/m in the sera. Bronchoscopy revealed narrowing of left main bronchus and mucoid impaction of the left lower lobe segmental bronchi. Cytological examination of BAL revealed few eosinophils, Charcot-Leyden crystals and mucus embedded hyphae. Examination of KOH wet mounts of repeated sputum and BAL specimens revealed septate, brownish hyphae and culture of the specimens resulted in the isolation of multiple colonies of a fungus later identified as B. hawaiiensis based on phenotypic characters and sequencing of internal transcribed spacer and D1/D2 regions of rDNA. In addition, (1-3)-ß-D-glucan was demonstrated in serum (316 pg/ml) by Fungitell kit, supportive of fungal infection/colonization. Histopathologic studies of a bronchial biopsy revealed necrotic debris, macrophage aggregates, lymphocytes, polymorphs and PAS positive hypae. The patient was administered oral itraconazole for 12 weeks, intravenous liposomal amphotericin B for one month, weekly bronchoscopic suctioning and voriconazole instillation, resulting in reduced mucopurulent secretions and considerable clinical improvement. A serum sample collected on 5 November demonstrated precipitins against antigens of the B. hawaiiensis isolate. In March 2010, intradermal skin testing revealed a strong, type I hypersensitivity (induration diam-12 mm) against B. hawaiiensis. The patient relapsed with wheezing and difficulty in respiration in April 2010. Considering the positive type I cutaneous hypersensitivity, the aforementioned laboratory and clinical observations, the patient was finally diagnosed as having ABPM and was successfully treated with oral prednisone. A high index of clinical suspicion with requisite investigations is crucial for early diagnosis and appropriate therapy of ABPM in order to prevent the late sequelae of irreversible broncho-pulmonary damage.

Seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of diverse tree species in north-western India: a retrospective study.

This study presents a 7-year retrospective analysis of seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of 518 trees belonging to 20 species in north-western India during 2000-2007. Of the 1,439 wood samples investigated, 406 (28.2%) were found to be positive for the Cryptococcus neoformans species complex which included 247 samples from which C. neoformans var. grubii was recovered and 171 which yielded C. gattii. While both of the pathogens were isolated through all the seasons, the overall prevalence of C. neoformans var. grubii was significantly higher (17.2%) than that of C. gattii serotype B (11.9%, P < 0.0001), indicating that decayed wood was as good, if not better, a natural habitat of C. neoformans var. grubii as that of C. gattii. The highest recovery of both yeasts was in the autumn, followed by that in the summer. For C. gattii, the lowest prevalence occurred during the winter and for C. neoformans var. grubii during the rainy season. The low prevalence of C. gattii during winter is similar to that reported from Bogota, Colombia, where C. gattii had a low population density in bark samples but it was not found in decayed wood of trunk hollows investigated during the period of January and February. The prevalence of C. neoformans var. grubii was significantly lower in the rainy season than in the other portions of the year. This finding is similar to the reported low isolation frequency (4%) of C. neoformans var. grubii from chicken feces in the rainy season in northern Thailand. Further investigations are warranted to determine the clinical significance of seasonal variations in the prevalence of C. neoformans var. grubii and C. gattii in decayed trunk wood of various trees in climatically divergent regions of India.

Invasive pulmonary aspergillosis due to Aspergillus terreus: value of DNA, galactomannan and (1->3)-beta-D-glucan detection in serum samples as an adjunct to diagnosis.

A case of invasive pulmonary aspergillosis caused by Aspergillus terreus is described. The diagnosis was based on demonstration of branched septate hyphae in a sputum specimen and isolation of the fungus in culture. The diagnosis was further supported by detection of A. terreus-specific DNA, galactomannan (GM) and (1→3)-ß-D-glucan (BDG) in consecutive serum specimens. The patient was treated for about 10 weeks with voriconazole. The decreasing levels of GM and BDG in serum samples were accompanied by symptomatic and radiological improvement. The report highlights the value of surrogate markers in the diagnosis and for monitoring the course of invasive aspergillosis during therapy.

Efficiency of gene transfection reagents in NG108-15, SH-SY5Y and CHO-K1 cell lines.

Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells.

Distribution of D1 and D5 dopamine receptors in the primate nucleus accumbens.

The D1 family of dopamine receptors (D1R) play a critical role in modulating reward in the nucleus accumbens (NAc). A better understanding of how D1Rs modulate NAc function must take into account the contributions of the two D1R subtypes, D(1) and D(5). In order to determine how these two subtypes contribute to dopamine's actions in the NAc, we utilized subtype specific antibodies and immunoelectron microscopy to quantitatively determine the localization of D(1) and D(5) in the neuropil of the primate NAc. We found that D(1) was more commonly found in dendritic shafts and spines, while D(5) was more commonly found in axon terminals, preterminal axons and glial processes. However, D(5) is well positioned to play an important role in postsynaptic modulation of inputs onto NAc medium spiny neurons. Approximately one third of spines contained D(1) and one quarter contained D(5), and as we have previously observed in the prefrontal cortex (PFC) and amygdala, these receptors overlapped extensively in dendritic spines. Similarly, we found overlap of the two D1R in axon terminals in the NAc; however, here D(5) labeled the larger population of terminals and D(1) was found in a subpopulation of D(5) containing terminals. Given the higher affinity of D(5) for dopamine, this suggest that presynaptic modulation of inputs by dopamine may be more easily evoked than in PFC where D(1) is the dominate presynaptic receptor. Finally, we investigated differences between the NAc and the dorsal striatum. We found that in the caudate half of dendritic spines contain D(1), significantly more than in the NAc. This suggests differences in how receptor is translated and distributed in D(1) mRNA expressing medium spiny neurons in the NAc and caudate.

Rhinoentomophthoromycosis due to Conidiobolus coronatus. A case report and an overview of the disease in India.

Rhinoentomophthoromycosis due to Conidiobolus coronatus is a rare, chronic, granulomatous disease, occurring mainly in tropical Africa, South and Central America and south-east Asia, including India. We report a case of rhinoentomophthoromycosis in a 30-year-old male farmer, a resident of Gorakhpur city in Uttar Pradesh, which was diagnosed by histopathology and isolation C. coronatus in culture. The patient presented with a swollen nose with obstruction that had progressed slowly over one year. His nasal swelling was bilateral, diffuse, mildly tender, erythematous, non-pitting, with mucosal crusting and hypertrophy of inferior turbinates but no regional lympha-denopathy. A contrast-enhanced computed tomography (CECT) scan revealed bilateral pan-sinusitis with nasoethmoid polyposis. Culture of tissue from the nasal biopsy on Sabouraud glucose agar yielded multiple colonies of a mold with satellite smaller colonies at periphery. The isolate demonstrated the macroscopic and microscopic morphologic characteristics of C. coronatus. Its identity was further confirmed by direct DNA sequencing of internal transcribed spacer (ITS) and D1/D2 regions of rDNA. Haemotoxylin and eosin stained tissue sections of the skin biopsy revealed irregular epidermal acanthosis, marked inflammatory and granulomatous reaction with sparse, non-septate hyphae. The patient was treated successfully with a combination therapy of oral saturated potassium iodide solution, itraconazole, and intravenous infusion of amphotericin B. An overview of rhinoentomophthoromycosis cases reported to-date in India is presented.

First isolations in India of Candida nivariensis, a globally emerging opportunistic pathogen.

We randomly screened 363 yeast isolates during 2008 for their ability to form white colonies on CHROM agar Candida medium. Two of these isolates (0.5%) were identified as Candida nivariensis based on detailed phenotypic characterization and DNA sequencing. One was recovered from the sputum of an HIV-positive patient with a pneumonic lesion and the second from the blood of a diabetic with oropharyngeal lesions. Direct DNA sequencing of the D1/D2 region of 28S rRNA gene and/or the internal transcribed spacer (ITS) regions of rDNA confirmed that both of the isolates were C. nivariensis. The carbohydrate assimilation profiles with the ID 32 C and VITEK 2 yeast identification systems revealed only glucose assimilation. In vitro antifungal susceptibility profiles by broth microdilution and Etest methods revealed susceptibility of both isolates to fluconazole, itraconazole, voriconazole, amphotericin B and 5-flucytosine, with low MICs for posaconazole and caspofungin. These results document the occurrence of Candida nivariensis for the first time in India and focus on its potential as an opportunistic human pathogen.