Lab-scale characterization and semi-field trials of Wolbachia Strain wAlbB in a Taiwan Wolbachia introgressed Ae. aegypti strain.

Dengue fever is one of the most severe viral diseases transmitted by Aedes mosquitoes, with traditional approaches of disease control proving insufficient to prevent significant disease burden. Release of Wolbachia-transinfected mosquitoes offers a promising alternative control methodologies; Wolbachia-transinfected female Aedes aegypti demonstrate reduced dengue virus transmission, whilst Wolbachia-transinfected males cause zygotic lethality when crossed with uninfected females, providing a method for suppressing mosquito populations. Although highly promising, the delicate nature of population control strategies and differences between local species populations means that controlled releases of Wolbachia-transinfected mosquitoes cannot be performed without extensive testing on specific local Ae. aegypti populations. In order to investigate the potential for using Wolbachia to suppress local Ae. aegypti populations in Taiwan, we performed lab-based and semi-field fitness trials. We first transinfected the Wolbachia strain wAlbB into a local Ae. aegypti population (wAlbB-Tw) and found no significant changes in lifespan, fecundity and fertility when compared to controls. In the laboratory, we found that as the proportion of released male mosquitoes carrying Wolbachia was increased, population suppression could reach up to 100%. Equivalent experiments in semi-field experiments found suppression rates of up to 70%. The release of different ratios of wAlbB-Tw males in the semi-field system provided an estimate of the optimal size of male releases. Our results indicate that wAlbB-Tw has significant potential for use in vector control strategies aimed at Ae. aegypti population suppression in Taiwan. Open field release trials are now necessary to confirm that wAlbB-Tw mediated suppression is feasible in natural environments.

R432 is a key residue for the multiple functions of Ndt80p in Candida albicans.

Ndt80p is an important transcription modulator to various stress-response genes in Candida albicans, the most common human fungal pathogen in systemic infections. We found that Ndt80p directly regulated its target genes, such as YHB1, via the mid-sporulation element (MSE). Furthermore, the ndt80(R432A) allele, with a reduced capability to bind MSE, failed to complement the defects caused by null mutations of NDT80. Thus, the R432 residue in the Ndt80p DNA-binding domain is involved in all tested functions, including cell separation, drug resistance, nitric oxide inactivation, germ tube formation, hyphal growth, and virulence. Hence, the importance of the R432 residue suggests a novel approach for designing new antifungal drugs by blocking the interaction between Ndt80p and its targets.

Oropharyngeal colonization of HIV-infected outpatients in Taiwan by yeast pathogens.

Among 234 isolates comprising 26 different Candida species colonizing the oropharynx of 181 (54.3% of 399 surveyed) HIV-infected outpatients, 27 (11.7%) were fluconazole resistant. Antibacterial treatment was associated with increased rates of yeast colonization, while antiretroviral therapy and pneumococcal vaccination protected patients from yeast colonization.

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array.

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.

Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions.

Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods.

Evaluation of an impedance method for subtyping of Pseudomonas aeruginosa.

Eight isolates (no. 1 to 8) of Pseudomonas aeruginosa isolated from 8 burn patients were typed by pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (PCR) (AP-PCR), biotyping, antimicrobial susceptibility testing, and a newly developed technique-impedance method. By both PFGE and AP-PCR, isolates 1 and 2 were designated type A, while isolates 3 to 8 were designated type B. However, isolates 3 to 8 could be further divided into three distinct subtypes (B, C, and D) by the impedance method. Four antibiograms were obtained by testing the 8 isolates against six antimicrobial agents and designation of antibiogram to each of the 8 isolates was in accordance with those obtained by the impedance method. The results of biotyping did not agree with any of the above typing methods. In conclusion, the impedance technique had a high discriminatory ability to differentiate genetically related clones into subtypes. The method is simple, reproducible, and has a high typeability.