Oropharyngeal yeast colonization in HIV-infected outpatients in southern Taiwan: CD4 count, efavirenz therapy and intravenous drug use matter.

To understand the status of oropharyngeal yeast colonization in human immunodeficiency virus (HIV) -infected outpatients in the era of highly active antiretroviral therapy (HAART), we conducted a prospective, cross-sectional study from October 2009 to January 2010 at a medical centre in southern Taiwan. Fungal cultures of the oropharyngeal swabs were performed on 327 enrolled patients. At enrolment, 258 (79%) patients had been receiving HAART, and 42 (12.8%), 73 (22.3%) and 212 (64.8%) patients had CD4 cell counts ≤200, 201-350, and >350 cells/mm(3) , respectively. Oral yeast colonization was detected in 193 (59%) patients, among whom 157 (81.3%), 25 (13.0%), and 11 (5.7%) were colonized by a single, two and more than two species, respectively. Multivariate analysis showed that receipt of efavirenz-containing regiments and CD4 cell counts >200 cells/mm(3) were associated with lower risks of oral yeast colonization, while intravenous drug users were at a higher risk. Among the 241 isolates recovered, Candida albicans accounted for 69.7%, followed by C. dubliniensis (9.5%), C. glabrata (8.3%), C. tropicalis (3.3%), C. intermedia (2.1%), C. parapsilosis (1.7%), and 11 other species (5.4%). Overall, 230 (95.4%), 236 (97.9%) and 240 (99.6%) isolates were susceptible to fluconazole, voriconazole and amphotericin B, respectively. In conclusion, colonization by C. dubliniensis has emerged in recent years. In addition to a CD4 cell count ≤200 cells/mm(3) , which is a known risk factor for oropharyngeal yeast colonization in HIV-infected patients that was identified in our previous studies, two risk factors, non-receipt of efavirenz-based combinations and intravenous drug use, were first identified in the present study. Fluconazole remained effective in vitro against the yeasts colonizing the oropharynx in this population.

Characterization of yeasts colonizing in healthy individuals.

Opportunistic yeast pathogens may switch from harmless commensal to pathogenic relationships with the host under different conditions. They usually cause superficial infections, but may be the agents of more significant infections in immunocompromised patients. To investigate yeast colonization in the oral cavities of clinically healthy individuals, we collected oral swabs from 323 students and staff at the National Health Research Institutes, Taiwan. A total of 49 (15.2%) volunteers were colonized by low levels of yeasts and of these, only 3 (6.1%) were co-colonized by more than one species. Among the 52 isolates, comprising seven fungal genera and 13 species, Candida albicans (57.7%) was the dominant species, followed by Candida parapsilosis (15.4%). There was only one isolate of C. parapsilosis that showed, in vitro, a high (2 µg/ml) minimum inhibitory concentration (MIC) to amphotericin B. There were six (11.5%) isolates with fluconazole MICs ≥ 64 µg/ml and all of them were non-Candida species. With the exception of Cryptococcus albidus, the remaining five isolates had voriconazole MICs ≥ 4 µg/ml. In addition, there was one C. albicans isolate with relatively high fluconazole (32 µg/ml) and voriconazole (4 µg/ml) MICs.

The distribution of species and susceptibility of amphotericin B and fluconazole of yeast pathogens isolated from sterile sites in Taiwan.

To study the demographic changes of yeasts causing invasive infections in Taiwan, especially with respect to species distribution and antifungal susceptibility, we analyzed isolates obtained from four sterile sites of patients in 19 hospitals in 2002 (155 strains) and again from the same hospitals in 2006 (208 strains). Blood was the most common source of the yeasts, accounting for 73.8% of the total isolates, followed by ascites (21.5%), cerebrospinal fluid (3%), and synovia (1.7%). Candida albicans was the most frequently recovered species (50.1% of the total), followed by Candida tropicalis (20.7%), Candida glabrata (11.6%), Candida parapsilosis (8.5%), Cryptococcus neoformans (3.9%), Candida krusei (0.8%), and nine other species (4.3%). There were one (0.3%) and seven (1.9%) isolates with minimum inhibitory concentrations (MICs) of amphotericin B > or =2 mg/l after 24 h and 48 h incubation, respectively. In addition, there were 15 (4.3%) and 31 (8.6%) isolates with MICs of fluconazole > or =64 mg/l under the same conditions. The MIC(90) value of amphotericin B was 1 mg/l. The MIC(90) values of fluconazole were 4 mg/l after 24 h incubation and 32 mg/l after 48 h incubation. Interestingly, MICs for fluconazole > or =64 mg/l after 24 h were significantly higher for isolates obtained in 2006 than those in 2002 after 24 h (7.1% vs. 0.7%, p =0.009) and 48 h (13.5% vs. 2%, p =0.0003) incubations. The demographic difference between these two surveys is mainly due to one species, C. tropicalis.

Non-lethal Candida albicans cph1/cph1 efg1/efg1 transcription factor mutant establishing restricted zone of infection in a mouse model of systemic infection.

The cph1/cph1 efg1/efg1 Candida albicans mutant cells were non-lethal in a mouse model of systemic infection. We investigated in vivo proliferation and invasion of C. albicans cells in infected mice to elucidate the interaction between the host and the pathogen. Homogenates of kidneys from the mice infected with the wild-type and the mutant C. albicans cells yielded a mean of 2.1 x 10 7 CFU/g and 2.2 x 10 6 CFU/g, respectively. The kidneys from the mice infected with the wild-type cells showed extensive renal cortical necrosis associated with neutrophilic infiltration. There were also wild-type hyphal cells present in abundance. Hence, tubular necrosis leading to renal failure in the mice may be the cause of death. Although the cph1/cph1 efg1/efg1 mutant cells were not lethal, they were capable of establishing restricted zones of infection and colonization near the renal pelvis instead of simply being cleared by the immune system in mice.

Molecular epidemiology of long-term colonization of Candida albicans strains from HIV-infected patients.

Twenty-one Candida albicans isolates from three HIV-infected patients were collected over a period of 3 years and characterized for fluconazole susceptibility, infectivity and genetic relatedness. Fluconazole resistance was found in five isolates, four exhibited dose-dependent susceptibility and the remainder were fully susceptible to this agent. Pulsed-field gel electrophoresis of SfiI restriction digests of the genomic DNA from the isolates revealed that isolates from the same swab specimen were identical despite differences in susceptibility to fluconazole and isolates recovered over time from the three patients retained clonally related DNA fingerprints within each patient. This small-scale study confirms the persistence of oral colonization of C. albicans strains in HIV-infected patients. Clinical data also suggests that the primary infecting strain may become a persistent colonist in the oral cavity once the immune function of the patient has been restored.

Distribution and susceptibility to amphotericin B and fluconazole of Candida spp. isolated from Taiwan.

Susceptibilities to amphotericin B and fluconazole of 628 clinical yeast strains collected from 22 hospitals in Taiwan were determined. A total of 53 isolates (8.4%) were resistant to fluconazole. Each hospital had different resistance rate to fluconazole ranging from 0% to 24%. None of the 186 isolates from eight of the 22 hospitals was resistant to fluconazole. In contrast, isolates from nine of the remaining 14 hospitals had greater than 10% resistance rate to fluconazole. Consistently, 88.9% (8/9) fluconazole-resistant C. albicans isolates were from hospitals having a high resistance rate to fluconazole. The prevalence of various Candida spp. in each hospital was different. A positive association was found between the prevalence of C. tropicalis and the resistance rate to fluconazole for individual hospitals. Although only three isolates (0.5%) were resistant to amphotericin B, a co-resistance to both amphotericin B and fluconazole was observed, which highlights the emerging problem of drug resistance.

Factors accounting for misidentification of Candida species.

From April 15 through June 15, 1999, a total of 660 yeast isolates were collected from 22 hospitals in Taiwan to investigate factors determining the accuracy of yeast identification. The germ tube test was the method most frequently used by hospitals for yeast identification, followed by the API-32C, cornmeal agar window test, and assimilation method. All of the submitted isolates were re-speciated in the National Health Research Institutes laboratory. The frequencies of inconsistent identification of isolates between hospitals and the National Health Research Institutes laboratory varied with the location and the type of hospital. The sensitivity and specificity of the germ tube test were 95% and 98.6%, respectively. This study showed that hospitals using the germ tube test as the first step in yeast identification had fewer inconsistent identifications of isolates than those using other methods. The VITEK Yeast Biochemical Card and API-32C had a sensitivity of 92.6% and 98.3%, respectively. No single method consistently identified all yeast isolates. Thus, every laboratory should have at least 2 methods available for yeast identification.

Emergence of reduced susceptibility and resistance to fluoroquinolones in Escherichia coli in Taiwan and contributions of distinct selective pressures.

A survey of 1,203 Escherichia coli isolates from 44 hospitals in Taiwan revealed that 136 (11.3%) isolates were resistant to fluoroquinolones and that another 261 (21.7%) isolates had reduced susceptibility. Resistance was more common in isolates responsible for hospital-acquired (mostly in intensive care units) infections (17.5%) than in other adult inpatient (11.4%; P = 0.08) and outpatient isolates (11.9%; P > 0.1). Similarly, reduced susceptibility was more common in isolates responsible for hospital-acquired infections (30.9%) than in other adult inpatient (21.0%; P = 0.04) and outpatient (21.4%; P = 0.06) isolates. Isolates from pediatric patients were less likely to be resistant (1.3 versus 12.0%; P < 0.01) but were nearly as likely to have reduced susceptibility (17.7 versus 21.9%; P > 0.1) as nonpediatric isolates. There was an inverse relationship in the proportion of isolates that were resistant versus the proportion that had reduced susceptibility among isolates from individual hospitals (R = 0.031; P < 0.05). In an analysis of isolates from two hospitals, all 9 resistant strains possessed double point mutations in gyrA and all 19 strains with reduced susceptibility strains had single point mutations; no mutations were found among fully susceptible strains. Risk factors for resistance included underlying cancer (odds ratio [OR], 83; 95% confidence interval [CI(95)], 7.3 to 2,241; P < 0.001), exposure to a quinolone (OR, undefined; P = 0.02), and exposure to a nonquinolone antibiotic (OR, 20; CI(95), 2.2 to 482; P < 0.001); underlying cancer was the only independent risk factor (OR, 83; CI(95), 8.6 to 807; P < 0.001). There were no significant associations between any of these factors and reduced susceptibility. Whereas acute and chronic quinolone use in cancer patients is a major selective pressure for resistance, other undetermined but distinct selective pressures appear to be more responsible for reduced susceptibility to fluoroquinolones in E. coli.

Mechanisms of antifungal agent resistance.

During the past decade, yeast infections have had an important role in nosocomial infections due to alterations in the immune status of patients. Coincidentally with the increased usage of antifungal agents, the number of reports of drug resistance has increased, which highlights the need for understanding the molecular mechanisms of antifungal agent resistance. This review describes the mechanisms of action of antifungal agents, cellular factors contributing to drug resistance, the known molecular mechanisms of drug resistance, and proposed but unproved molecular mechanisms of drug resistance. This review also proposes possible strategies for preventing drug resistance.

Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake.

The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis.

MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene.

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP-regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP-mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/ Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.

Spontaneous oscillations of cerebral blood flow velocity in the middle cerebral arteries of normal subjects and schizophrenic patients.

Although many regional cerebral blood flow (rCBF) studies of schizophrenic patients have been carried out, only a few studies have investigated real-time hemodynamic changes in schizophrenic patients. In the present study, we used long-term monitoring of the middle cerebral artery (MCA) by non-invasive transcranial Doppler ultrasonography to obtain real-time CBF data in 55 schizophrenic patients and 20 normal comparison subjects. The mean blood flow velocity and pulsatility index (PI) of the MCA were not constant during long-term monitoring. They showed sinusoidal oscillations similar to those described in previous reports. The amplitude variations of these oscillations in both drug-naive and medicated schizophrenic patients were significantly decreased compared with findings in normal control subjects. The averaged PI values were found to be decreased in patients with illness durations of more than 10 years. After withdrawal of antipsychotic medication, both the amplitude variations of oscillations and the PI values in the drug-withdrawn patients were significantly decreased relative to findings in normal control subjects. Our results show a decreased adjustment ability of cerebral vessel resistance not only in neuroleptic-naive schizophrenic patients but also in patients with longer illness duration. Neuroleptics could affect the adjustment ability of vessel resistance.

RNA polymerase I-promoted HIS4 expression yields uncapped, polyadenylated mRNA that is unstable and inefficiently translated in Saccharomyces cerevisiae.

The HIS4 gene in Saccharomyces cerevisiae was put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. The rhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-type HIS4 mRNA. Its 3' end maps to the HIS4 3' noncoding region, and it is polyadenylated. The second transcript, rhis4l, is bicistronic. It encodes the HIS4 coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3' end of rhis4l maps to the predicted 3' end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4 mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5' end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5'-3' exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-type HIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1delta genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5' cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

Nonfilamentous C. albicans mutants are avirulent.

Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.

Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells.

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.