Sporothrix schenckii sensu stricto related to zoonotic transmission in Thailand.

Two sporotrichosis cases were related to zoonotic transmission as their cats were diagnosed of feline sporotrichosis. The result of fungus culture as dimorphic fungi and flower-like arrangement of typical S. schenckii complex morphology confirms the diagnosis. The species were identified by Polymerase Chain Reaction (PCR) using species-specific primers of calmodulin gene (CAL). The result showed that etiological agents of these two cases in Northern Thailand are S. schenckii sensu stricto. One case was successfully treated with daily dose of 250 mg terbinafine, and the other was treated with daily dose of 100 mg itraconazole with an excellent outcome. 2012 Elsevier Ltd. All rights reserved.

Combined Locked Nucleic Acid Probes and High-Resolution Melting Curve Analysis for Detection of Rifampicin-Resistant Tuberculosis in Northern Thailand.

Rifampicin-resistant tuberculosis (RR-TB) has become a major threat globally. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the class-III and IV mutations of rpoB, H526D, and D516V. LNA probes showed 100% specificity in the detection of mutant targets among characterized and blinded Mycobacterium tuberculosis (Mtb) isolates. The performance of RIF-RDp was evaluated using 110 blinded clinical Mtb isolates in northern Thailand against drug-susceptibility testing (DST), DNA sequencing, and a commercial real-time PCR kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared to DNA sequencing. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing. The Cohen's Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (κ = 0.95), and RIF-RDp and DNA sequencing (κ = 0.96). Furthermore, this is the first report of the rare mutation profiles, S531W, and a triple codon deletion (510-512) in northern Thailand. According to high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future.

Comparison of Simple RNA Extraction Methods for Molecular Diagnosis of Hepatitis C Virus in Plasma.

Nucleic acid extraction from biological samples is an important step for hepatitis C virus (HCV) diagnosis. However, such extractions are mostly based on silica-based column methodologies, which may limit their application for on-site diagnosis. A simple, rapid, and field-deployable method for RNA extraction is still needed. In this study, we evaluated the efficacy of four simple RNA extraction methods for the detection of HCV in plasma samples: a silica-membrane-based method, a magnetic-beads-based method, boiling with diethyl pyrocarbonate (DEPC)-treated distilled water, and using a commercial lysis buffer. HCV RNA was detected using both real-time reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Using real-time RT-PCR, extracted RNA from the silica-membrane-based and magnetic-beads-based methods had a 100% detection rate for RNA extraction from plasma. Using RT-LAMP, extracted RNA from the silica-membrane-based method showed a 66% detection rate, while the magnetic-beads-based method had a 62% detection rate. In summary, magnetic-beads-based extraction can be used as an alternative RNA extraction method for on-site HCV detection. Boiling with DEPC-treated distilled water was not appropriate for low HCV load samples, and boiling with a lysis buffer was not recommended.

Contamination of antimicrobial-resistant bacteria on toothbrushes used with mechanically ventilated patients: A cross sectional study.

OBJECTIVE: The purpose of this study was to determine pathogenic and antimicrobial-resistant bacteria on used toothbrushes of mechanically ventilated patients. RESEARCH METHODOLOGY: A cross-sectional study was conducted by collecting toothbrushes used with mechanically ventilated patients. The total bacterial count on each toothbrush was assessed by culturing on Trypticase soy agar (TSA). Gram stain and biochemical testing were used to identify bacterial species. Antibiotic susceptibility of pathogenic bacteria was assessed by the Kirby-Bauer disk diffusion method. RESULTS: Thirty-five toothbrushes (97%) had bacterial contamination, 27 toothbrushes had at least two bacterial species, and 13 toothbrushes harboured antimicrobial-resistant bacteria. The most commonly isolated bacteria were Klebsiella spp. (21%), followed by Acinetobacter baumannii (18%). Five isolates of A. baumannii, six isolates of K. pneumoniae, and two isolates of Enterobacter cloacae were multidrug-resistant (MDR) strains. Four isolates of K. pneumoniae were identified as extended-spectrum beta-lactamase (ESBL) producing strains, and two isolates of P. aeruginosa were extensively drug-resistant (XDR). The average total bacterial count was 104-105 CFU/toothbrush head. CONCLUSIONS: Antimicrobial-resistant bacteria were detected on toothbrushes. Therefore, practice of toothbrush care should be reconsidered in associated to maintaining the oral hygiene of mechanically ventilated patients to prevent ventilator-associated pneumonia (VAP).

In Vitro Antifungal and Antivirulence Activities of Biologically Synthesized Ethanolic Extract of Propolis-Loaded PLGA Nanoparticles against Candida albicans.

Propolis is a natural substance and consists of bioactive compounds, which gives it antioxidant and antimicrobial properties. However, the use of propolis is limited by the low solubility in aqueous solutions. Thus, nanoparticles may be likely to accomplish enhanced delivery of poorly water-soluble phytomedicine. The aim of the present study was to fabricate and evaluate the biological activity of ethanolic extract of propolis-loaded poly(lactic-co-glycolic acid) nanoparticles (EEP-NPs). The EEP-NPs were prepared using the oil-in-water (o/w) single-emulsion solvent evaporation technique. The physicochemical properties of EEP-NPs were characterized and tested on their cytotoxicity, antifungal activity, and impact on key virulence factors that contribute to pathogenesis of C. albicans. EEP-NPs were successfully synthesized and demonstrated higher antifungal activity than EEP in free form. Moreover, EEP-NPs exhibited less cytotoxicity on Vero cells and suppressed the virulence factors of C. albicans, including adhesion, hyphal germination, biofilm formation, and invasion. Importantly, EEP-NPs exhibited a statistical decrease in the expression of hyphal adhesion-related genes, ALS3 and HWP1, of C. albicans. The results of this study revealed that EEP-NPs mediates a potent anticandidal activity and key virulence factors by reducing the gene-encoding virulence-associated hyphal- adhesion proteins of C. albicans and, thereby, disrupting the morphologic presence and attenuating their virulence.

Melioidosis in Thailand: Present and Future.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

Cell wall protection by the Candida albicans class I chitin synthases.

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

Anti-Candida targets and cytotoxicity of casuarinin isolated from Plinia cauliflora leaves in a bioactivity-guided study.

In addition to the bio-guided investigation of the antifungal activity of Plinia cauliflora leaves against different Candida species, the major aim of the present study was the search for targets on the fungal cell. The most active antifungal fraction was purified by chromatography and characterized by NMR and mass spectrometry. The antifungal activity was evaluated against five Candida strains according to referenced guidelines. Cytotoxicity against fibroblast cells was determined. The likely targets of Candida albicans cells were assessed through interactions with ergosterol and cell wall composition, porosity and architecture. The chemical major component within the most active antifungal fraction of P. cauliflora leaves identified was the hydrolysable tannin casuarinin. The cytotoxic concentration was higher than the antifungal one. The first indication of plant target on cellular integrity was suggested by the antifungal activity ameliorated when using an osmotic support. The most important target for the tannin fraction studied was suggested by ultrastructural analysis of yeast cell walls revealing a denser mannan outer layer and wall porosity reduced. It is possible to imply that P. cauliflora targeted the C. albicans cell wall inducing some changes in the architecture, notably the outer glycoprotein layer, affecting the cell wall porosity without alteration of the polysaccharide or protein level.

Cell wall stress induces alternative fungal cytokinesis and septation strategies.

In fungi, as with all walled organisms, cytokinesis followed by septation marks the end of the cell cycle and is essential for cell division and viability. For yeasts, the septal cross-wall comprises a ring and primary septal plate composed of chitin, and a secondary septum thickened with ß(1,3)-glucan. In the human pathogen Candida albicans, chitin synthase enzyme Chs1 builds the primary septum that is surrounded by a chitin ring made by Chs3. Here we show that the lethal phenotype induced by repression of CHS1 was abrogated by stress-induced synthesis of alternative and novel septal types synthesized by other chitin synthase enzymes that have never before been implicated in septation. Chs2 and Chs8 formed a functional salvage septum, even in the absence of both Chs1 and Chs3. A second type of salvage septum formed by Chs2 in combination with Chs3 or Chs8 was proximally offset in the mother-bud neck. Chs3 alone or in combination with Chs8 formed a greatly thickened third type of salvage septum. Therefore, cell wall stress induced alternative forms of septation that rescued cell division in the absence of Chs1, demonstrating that fungi have previously unsuspected redundant strategies to enable septation and cell division to be maintained, even under potentially lethal environmental conditions.

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans.

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1ß in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.