Helicobacter pylori, Protected from Antibiotics and Stresses Inside Candida albicans Vacuoles, Cause Gastritis in Mice.

Due to (i) the simultaneous presence of Helicobacter pylori (ulcer-induced bacteria) and Candida albicans in the stomach and (ii) the possibility of prokaryotic-eukaryotic endosymbiosis (intravacuolar H. pylori in the yeast cells) under stresses, we tested this symbiosis in vitro and in vivo. To that end, intravacuolar H. pylori were induced by the co-incubation of C. albicans with H. pylori under several stresses (acidic pH, non-H. pylori-enrichment media, and aerobic environments); the results were detectable by direct microscopy (wet mount) and real-time polymerase chain reaction (PCR). Indeed, intravacuolar H. pylori were predominant under all stresses, especially the lower pH level (pH 2-3). Interestingly, the H. pylori (an amoxicillin-sensitive strain) inside C. albicans were protected from the antibiotic (amoxicillin), while extracellular H. pylori were neutralizable, as indicated by the culture. In parallel, the oral administration of intravacuolar H. pylori in mice caused H. pylori colonization in the stomach resulting in gastritis, as indicated by gastric histopathology and tissue cytokines, similar to the administration of free H. pylori (extra-Candida bacteria). In conclusion, Candida protected H. pylori from stresses and antibiotics, and the intravacuolar H. pylori were able to be released from the yeast cells, causing gastric inflammation with neutrophil accumulations.

Beta-Glucan from S. cerevisiae Protected AOM-Induced Colon Cancer in cGAS-Deficient Mice Partly through Dectin-1-Manipulated Macrophage Cell Energy.

Although the impacts of Saccharomyces cerevisiae on cancers are mentioned, data on its use in mice with cyclic GMP-AMP synthase deficiency (cGAS-/-) are even rarer. Here, 12 weeks of oral administration of S. cerevisiae protected cGAS-/- mice from azoxymethane (AOM)-induced colon cancers, partly through dysbiosis attenuation (fecal microbiome analysis). In parallel, a daily intralesional injection of a whole glucan particle (WGP; the beta-glucan extracted from S. cerevisiae) attenuated the growth of subcutaneous tumor using MC38 (murine colon cancer cell line) in cGAS-/- mice. Interestingly, the incubation of fluorescent-stained MC38 with several subtypes of macrophages, including M1 (using Lipopolysaccharide; LPS), M2 (IL-4), and tumor-associated macrophages (TAM; using MC38 supernatant activation), could not further reduce the tumor burdens (fluorescent intensity) compared with M0 (control culture media). However, WGP enhanced tumoricidal activities (fluorescent intensity), the genes of M1 pro-inflammatory macrophage polarization (IL-1ß and iNOS), and Dectin-1 expression and increased cell energy status (extracellular flux analysis) in M0, M2, and TAM. In M1, WGP could not increase tumoricidal activities, Dectin-1, and glycolysis activity, despite the upregulated IL-1ß. In conclusion, S. cerevisiae inhibited the growth of colon cancers through dysbiosis attenuation and macrophage energy activation, partly through Dectin-1 stimulation. Our data support the use of S. cerevisiae for colon cancer protection.

Drug-drug interaction between itraconazole capsule and efavirenz in adults with HIV for talaromycosis treatment.

OBJECTIVES: To assess the pharmacokinetic of itraconazole capsule formulation and its active metabolite, hydroxyitraconazole, in adults with HIV diagnosed with talaromycosis in an endemic area, and to evaluate the drug-drug interaction between itraconazole/hydroxyitraconazole (ITC/OH-ITC) and efavirenz. METHODS: Open-label, single arm, sequential pharmacokinetic study. Eligible subjects were adults with HIV, ≥18 years old, with confirmed talaromycosis, initiating itraconazole capsule as part of standard talaromycosis treatment, in whom efavirenz-based ART was anticipated. Steady-state pharmacokinetic assessments (pre-dose and at 1, 3, 4, 5, 6, 8 and 12 h post dose) were performed for itraconazole/hydroxyitraconazole without and with efavirenz use. Mid-dose efavirenz concentrations were also assessed. Pharmacokinetics parameters were calculated using non-compartmental analysis. RESULTS: Ten subjects (70% male) were enrolled. At entry, median (range) age was 29.5 years (22-64), and CD4 cell count was 18.0 (1-39) cells/mm3. Geometric mean (95% CI) of itraconazole and hydroxyitraconazole AUC0-12 without efavirenz were 9097 (6761-12 239) and 11 705 (8586-15 959) ng·h/mL, respectively, with a median metabolic ratio of OH-ITC : ITC of 1.3 (95% CI 0.9-1.9). Intra-subject comparison revealed that both itraconazole and hydroxyitraconazole exposures were significantly reduced with concomitant efavirenz use, with the mean AUC0-12 of itraconazole and hydroxyitraconazole being 86% (71%-94%) and 84% (64%-97%) lower, respectively. With efavirenz, itraconazole trough concentrations were also below the recommended therapeutic level (0.5 µg/mL). All subjects had mid-dose efavirenz concentrations >1000 ng/mL. CONCLUSIONS: Concomitant administration of itraconazole capsule with efavirenz significantly reduced itraconazole and hydroxyitraconazole exposures. The clinical impact of this drug-drug interaction on talaromycosis treatment or prophylaxis in the era of potent ART needs further evaluation.

In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents.

Human pythiosis is a life-threatening human disease caused by Pythium insidiosum In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 µg/ml), minocycline MICs (1 to 4 µg/ml), tigecycline MICs (1 to 4 µg/ml), azithromycin MICs (1 to 16 µg/ml), and clarithromycin MICs (0.125 to 8 µg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.

Human Pythiosis: Emergence of Fungal-Like Organism.

Pythiosis is an emerging infectious disease caused by the aquatic oomycete Pythium insidiosum, a fungal-like organism. It is believed that P. insidiosum's zoospores, its infected form, play major role in pathogenesis. Vascular and ocular infections are the most common clinical manifestation in humans. It is difficult to establish the diagnosis given its relatively rarity and difficulty to distinguish P. insidiosum from other molds. Delay in diagnosis and treatment has been associated with poor outcomes. High index of suspicion is the key, particularly in thalassemia patients with arterial insufficiency and patients with fungal keratitis/endophthalmitis without improvement on antifungal therapy. Tissue culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The combination of radical surgery, antifungal agents, and immunotherapy has been recommended. It was previously believed that surgery with negative surgical margins was the essential to survive in vascular pythiosis; however, it was recently found that patients could have residual disease despite documented negative surgical margins as infected clot may be dislodged to proximal arterial sites prior to surgery. Serum ß-D-glucan (BG) has been used to monitor disease response after treatment initiation in vascular pythiosis. A significant decrease in BG levels within 2 weeks after surgery is indicative of the absence of residual infection. Unfortunately, monitoring tools for ocular pythiosis are not yet available. Itraconazole plus terbinafine have generally been used in P. insidiosum-infected patients; however, antibacterial agents, including azithromycin and linezolid, have also been used with favorable outcomes in ocular disease. Recently, azithromycin or clarithromycin plus doxycyclin were used in two relapsed vascular pythiosis patients with good outcomes.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

Database establishment for the secondary fungal DNA barcode translational elongation factor 1α (TEF1α) 1.

With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/ . We encourage the mycology community for active contributions.

Clinical outcomes in ocular pythiosis patients treated with a combination therapy protocol in Thailand: A prospective study.

Ocular pythiosis is the second most common form of human pythiosis, and the rates of evisceration/enucleation in Thailand are 55-79%. This prospective study was conducted to evaluate treatment outcomes of the combination therapy protocol and the potential use of serum (1→3)-ß-glucan (BG) and Pythium insidiosum-specific antibody (Pi-Ab) as an aid to diagnosis and monitoring of ocular pythiosis. Thirty patients were enrolled in the study and 14 (non-globe salvage) required evisceration/enucleation. The globe salvage group was significantly younger, and first ocular surgeries were performed significantly sooner than in the non-globe salvage group. Serum BG and Pi-Ab levels were similar among the 2 groups over time. In vitro susceptibility testing of antifungal agents revealed relatively high minimum inhibitory concentrations and lack of synergistic effect. Serum BG and Pi-Ab would not be useful in diagnosis and monitoring of ocular pythiosis. Until effective antimicrobial agents are discovered, ocular surgeries are still the mainstay therapy in Thailand.

The epidemiology of non-Candida yeast isolated from blood: The Asia Surveillance Study.

BACKGROUND: Current guidelines recommend echinocandins as first-line therapy for candidemia. However, several non-Candida yeast are non-susceptible to echinocandins (echinocandin non-susceptible yeast, ENSY), including Cryptococcus, Geotrichum, Malassezia, Pseudozyma, Rhodotorula, Saprochaete, Sporobolomyces and Trichosporon. In laboratories that are not equipped with rapid diagnostic tools, it often takes several days to identify yeast, and this may lead to inappropriate presumptive use of echinocandins in patients with ENSY fungemia. The aim of this study was to determine the distribution of ENSY species during a 1-year, laboratory surveillance programme in Asia. METHODS: Non-duplicate yeast isolated from blood or bone marrow cultures at 25 hospitals in China, Hong Kong, India, Singapore, Taiwan and Thailand were analysed. Isolates were considered to be duplicative if they were obtained within 7 days from the same patient. RESULTS: Of 2155 yeast isolates evaluated, 175 (8.1%) were non-Candida yeast. The majority of non-Candida yeast were ENSY (146/175, 83.4%). These included Cryptococcus (109 isolates), Trichosporon (23), Rhodotorula (10) and Malassezia (4). The proportion of ENSY isolates (146/2155, 6.7%) differed between tropical (India, Thailand and Singapore; 51/593, 8.6%) and non-tropical countries/regions (China, Hong Kong and Taiwan; 95/1562, 6.1%, P = 0.038). ENSY was common in outpatient clinics (25.0%) and emergency departments (17.8%) but rare in intensive care units (4.7%) and in haematology-oncology units (2.9%). Cryptococcus accounted for the majority of the non-Candida species in emergency departments (21/24, 87.5%) and outpatient clinics (4/5, 80.0%). CONCLUSIONS: Isolation of non-Candida yeast from blood cultures was not rare, and the frequency varied among medical units and countries.

Monitoring Anti-Pythium insidiosum IgG Antibodies and (1→3)-ß-d-Glucan in Vascular Pythiosis.

Despite aggressive treatment, vascular pythiosis has a mortality rate of 40%. This is due to delays in diagnosis and a lack of effective monitoring tools. To overcome this drawback, serum beta-d-glucan (BG) and P. insidiosum-specific antibody (Pi-Ab) were examined as potential monitoring markers in vascular pythiosis. A prospective cohort study of vascular pythiosis patients was carried out from January 2010 to July 2016. Clinical information and blood samples were collected and evaluated by the BG and Pi-Ab assays. Linear mixed-effect models were used to compare BG and Pi-Ab levels. The in vitro susceptibility test was performed with all P. insidiosum isolates from culture-positive cases. A total of 50 patients were enrolled: 45 survived and 5 died during follow-up. The survivors had a significantly shorter time to medical care (P < 0.0001) and a significantly shorter waiting time to the first surgery (P < 0.0001). There were no differences in BG levels among the groups at diagnosis (P = 0.33); however, BG levels among survivors were significantly lower than those of the deceased group at 0.5 months (P < 0.0001) and became undetectable after 3 months. Survivors were able to maintain an enzyme-linked immunosorbent assay (ELISA) value (EV) of Pi-Ab above 8, whereas the EV among deceased patients was less than 4. In vitro susceptibility results revealed no synergistic effects between itraconazole and terbinafine. This study showed that BG and Pi-Ab are potentially valuable markers to monitor the disease after treatment initiation. An unchanged BG level at 2 weeks after surgery should prompt an evaluation for residual disease.

High-resolution melting analysis: A novel approach for clade differentiation in Pythium insidiosum and pythiosis.

Pythium insidiosum causes life-threatening human pythiosis. Based on phylogenetic analysis using internal transcribed spacer (ITS) region, mitochondrial cytochrome C oxidase II (COX2) gene, intergenic spacer (IGS) region and exo-1,3-ß-glucanase gene (exo1), P. insidiosum is classified into clade ATH, BTH, and CTH related to geographic distribution. At present, polymerase chain reaction in any of these specific regions with DNA sequencing is the only technique to provide clade diagnosis. In this study, P. insidiosum-specific primers targeting COX2 gene were designed and used in real-time quantitative polymerase chain reaction (qPCR) with subsequent high-resolution melting (HRM) to provide rapid identification as well as clade classification for P. insidiosum. Based on the qPCR-HRM method, 15 P. insidiosum isolates could be differentiated from 28 related organisms with 100% specificity and 1 pg limit of detection. This technique was, in addition, directly tested on clinical samples from proved human pythiosis cases: nine corneal scrapes and six arterial clots. The qPCR-HRM results of all nine corneal samples were a 100% match with the results from the conventional PCR at clade level. However, the qPCR-HRM results of arterial clot samples were only matched with the nucleotide sequencing results from the conventional PCR at species level. In conclusion, the qPCR-HRM is a simple one closed tube, inexpensive and user-friendly method to identify P. insidiosum into clade level.

Differential diagnosis for pythiosis using thermophilic helicase DNA amplification and restriction fragment length polymorphism (tHDA-RFLP).

Pythiosis is caused by Pythium insidiosum, a fungus-like microbe belonging to the kingdom Stramenopila. Its diagnosis is challenging due to clinical and histopathological similarities with the fungal microbes that cause mucormycosis and entomophthoramycosis. In addition, the proper identification of P. insidiosum in the clinical laboratory is difficult. We have developed a rapid and accurate, species-specific identification method using a thermophilic helicase DNA amplification (tHDA) technique, to differentiate this pathogen from closely related pathogenic fungi. Sixty-seven fungal isolates, including 39 of P. insidiosum, were evaluated. A 91 base-pair (bp) DNA fragment was consistently amplified using a COX2 primer. The limiting concentrations of the one- and two-step tHDA protocols were 100 picograms (1.74 × 102 copies) and 100 femtograms (1.74 × 10-1 copies), respectively. The CviKI-1 enzyme in restriction fragment length polymorphism (RFLP) with the 91 bp amplicons accurately separated P. insidiosum from other fungal species. The data suggest that this tHDA-RFLP assay is a rapid and accurate test for the identification of P. insidiosum. The potential use of the assay directly in clinical samples is also discussed.

Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: An Asia Fungal Working Group (AFWG) initiative.

An online survey of mycology laboratories in seven Asian countries was conducted to assess the status, competence, and services available. Country representatives from the Asia Fungal Working Group (AFWG) contacted as many laboratories performing mycology diagnosis as possible in their respective countries, requesting that the laboratory heads complete the online survey. In total, 241 laboratories responded, including 71 in China, 104 in India, 11 in Indonesia, 26 in the Philippines, four in Singapore, 18 in Taiwan, and seven in Thailand. Overall, 129/241 (53.5%) surveyed mycology laboratories operate as separate designated mycology laboratories, 75/241 (31.1%) conduct regular formal staff training, 103/241 (42.7%) are accredited, and 88/157 (56.1%) participate in external quality assurance scheme (EQAS) programs. Microscopy and culture methods are available in nearly all laboratories, although few perform DNA sequencing (37/219; 16.9%) or use matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (27/219; 12.3%) for isolate identification. Antifungal susceptibility testing is performed in 142/241 (58.9%) laboratories, mainly for yeasts. The most commonly performed nonculture diagnostic is cryptococcal antigen testing (66 laboratories), followed by galactomannan testing (55), polymerase chain reaction (PCR) diagnosis (37), and beta-D-glucan testing (24). Therapeutic drug monitoring is conducted in 21 laboratories. There is almost no access to advanced diagnostic tests, like galactomannan, ß-D-glucan, and PCR, in the surveyed laboratories in Indonesia, the Philippines, and Thailand. These results highlight the need for development of quality laboratories, accreditation and training of manpower in existing laboratories, and access to advanced non-culture-based diagnostic tests to facilitate the diagnosis of fungal infections in Asia.

Diversity and Antifungal Drug Susceptibility of Cryptococcus Isolates in Thailand.

Yeasts of the Cryptococcus species complex are the causative agent of cryptococcosis, especially in human immunodeficiency virus (HIV) positive individuals. Cerebral or disseminated cryptococcosis has a very high mortality rate worldwide, including in Thailand. Additionally, an increasing rate of antifungal drug resistant cryptococcal isolates has been reported in several neighboring countries, complicating therapeutic approaches. To understand the situation of this infection in Thailand, we retrospectively investigated the molecular epidemiology and antifungal drug resistance in a collection of 74 clinical, 52 environmental and two veterinary isolates using the URA5-RFLP for typing and the EUCAST guideline for susceptibility testing. Where no EUCAST breakpoints (AMB and 5FC) were available, CLSI epidemiologic cutoff values were used for interpretation. Cryptococcal molecular type diversity showed most isolates were C. grubii, molecular type VNI. One clinical isolate was C. deuterogattii (mol. type VGII) and another C. grubii (mol. type VNII). One strain from environment was classified as C. grubii (mol. type VNII). No resistant strains were detected in this retrospective study for either of the antimycotics tested; however, monitoring of the epidemiology of Cryptococcus species in infected patients in Thailand needs to be continued to detect emergence of resistance.

Prevalence of azole-resistant Aspergillus fumigatus in the environment of Thailand.

Occurrence of azole-resistant Aspergillus fumigatus (ARAF) in the environment is an emerging problem worldwide, likely impacting on patient treatment. Several resistance mutations are thought to have initially arisen through triazole-based fungicide use in agriculture and subsequently being propagated in a similar manner. Here we investigated the prevalence of ARAF in the environment of Thailand and characterized their susceptibility profiles toward clinically used azole compounds along with underlying resistance mutations. Three hundred and eight soil samples were collected and analyzed, out of which 3.25% (n = 10) were positive for ARAF. All isolates obtained were resistant to itraconazole (MIC ≥ 8 µg/ml), two showed additional increased MIC values toward posaconazole (MIC = 0.5 µg/ml), and one other toward voriconazole (MIC = 2 µg/ml). Sequencing of the respective cyp51A genes revealed that eight of the isolates carried the TR34/L98H allele and those two with elevated MIC values to posaconazole the G54R substitution. Although a clear correlation between the use of triazole-based fungicides and isolation of ARAF strains from agricultural lands could not be established for Thailand, but this study clearly demonstrates the spread of globally observed ARAF strains to the environment of South East Asia.

Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.

Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.

OBJECTIVES: Human pythiosis is a life-threatening disease for which no standard treatment protocols with proven efficacy exist. We present the results of our institutional pythiosis treatment protocol, composed of surgery, antifungal agents, iron chelator (only vascular cases) and immunotherapy. METHODS: We retrospectively analysed patients with proven vascular and ocular pythiosis in King Chulalongkorn Memorial Hospital from April 2003 to May 2013. Fisher's exact test and Wilcoxon's rank-sum test were used. The MICs of seven antifungal agents and combination drugs were investigated in eight clinical Pythium insidiosum strains. RESULTS: Eighteen patients were evaluated. Disease-free surgical margins were obtained in all surviving patients with vascular pythiosis (P = 0.08). Patients who underwent eye enucleation were significantly older than those who did not (P < 0.05). Patients with vascular or ocular pythiosis did not differ significantly in the median time from disease onset to first surgery or in the relationship between the type of P. insidiosum antigen and treatment outcomes. In vitro susceptibility profiles of all isolates demonstrated that no single agent or combination treatment was substantially more effective than the others. The highest MIC was detected for amphotericin B, followed in order by voriconazole, fluconazole, anidulafungin, caspofungin, itraconazole and terbinafine. No synergistic effects of the combination drug treatments were found. CONCLUSIONS: Surgery with adequate surgical margins is a crucial determinant of survival in patients with vascular pythiosis. Itraconazole and terbinafine do not have synergistic effects on Thai P. insidiosum strains. The role of immunotherapy remains inconclusive for both vascular and ocular pythiosis.

(1→3)-ß-D-glucan and galactomannan testing for the diagnosis of fungal peritonitis in peritoneal dialysis patients, a pilot study.

Fungal peritonitis is an uncommon but serious complication of peritoneal dialysis (PD) due to the fact that routine culture to recovered the etiologic agents are time consuming and KOH staining has very low sensitivity. Peritoneal (1→3)-ß-D-glucan (BG) or galactomannan (GM), both fungal cell wall components, are candidate biomarkers of fungal peritonitis. Hence, a comparative cross-sectional analysis of peritoneal dialysis fluid (PDF) BG (Fungitell, Cape Cod, MA, USA) and GM (Platelia Aspergillus Ag kits, Bio-rad, France) from all PD patients with and without fungal peritonitis (13 cases, identified by culture), over a 1 year period, was performed. PDF of the fungal peritonitis group showed very high BG (494 ± 19 pg/ml) and high GM (3.41 ± 1.24) similar results were noted in specimens from cases of peritonitis with other causes, especially gram negative bacterial peritonitis. A BG cut-off value at 240 pg/ml and GM at 0.5 showed sensitivity/ specificity at 100%/ 83% and 77%/ 58%, respectively. A concomitantly positive GM reduced the false positive rate of BG from nonfungal peritonitis. In conclusion, BG and GM in peritoneal fluid with provisional cut-off values were applicable as surrogate biomarkers for the diagnosis of fungal peritonitis in PD patients.

Invasive pulmonary infection caused by Chrysosporium articulatum: the first case report.

Chrysosporium species, saprobic soil fungi, comprise more than 60 species. There is some confusion regarding the taxonomy and nomenclature between Chrysosporium and Emmonsia since the causative agents of adiaspiromycosis, the development of big thick-walled spores (adiaspores) in humans or animals, were previously thought to be Chrysosporium. Chrysosporium articulatum has never been reported to cause invasive infection in humans. We report herein the first case of invasive pulmonary infection caused by Chrysosporium articulatum in a 16-year-old man with acute T-cell lymphoblastic leukaemia. He was successfully treated with voriconazole.