Application of a multiplex suspension array for rapid and simultaneous identification of clinically important mold pathogens.

We have developed a microsphere-based suspension array (MSA) for the identification of 23 medically important mold pathogens including Aspergillus spp., Fusarium spp., Mucor spp., Rhizopus spp., Rhizomucor pusillus, Penicillium marneffei, Saksenaea vasiformis, Apophysomyces elegans, Lichtheimia corymbifer, and Syncephalastrum racemosum. Twenty-one oligonucleotide probes were designed based on the internal transcribed spacer (ITS2) region for species level identification of molds. Among the 21 probes, 2 probes are shared by more than one species due to low or absence of sequence variability, i.e. Rpam for Rhizopus azygosporus/Rhizopus microsporus and Fumop for Fusarium moniliforme/Fusarium oxysporum/Fusarium pallidoroseum. No cross reactivity was identified except for probes of Mucor racemosus (Murac) which cross react with Mucor hiemalis and Mucor ramosissimus. The sensitivity of MSA is 100 fg-1 ng. The whole procedure including DNA extraction and PCR amplification can be finished within 5 h. The MSA is simple, rapid, specific, high-throughput and capable of multiple-species detection in one reaction tube.

Cutaneous manifestations of Nocardia brasiliensis infection in Taiwan during 2002-2012-clinical studies and molecular typing of pathogen by gyrB and 16S gene sequencing.

To observe the clinicopathologic and resistance profiles of the Nocardia brasiliensis causing cutaneous nocardiosis in Taiwan, 12 N. brasiliensis isolates were prospectively collected from patients with cutaneous nocardiosis in a hospital during 2002-2012. Clinicopathologic data were obtained, and isolates were identified by biochemical methods and 16S rRNA sequencing. Susceptibilities to 14 antimicrobial compounds were tested. Isolates were further genotyped by sequencing of 16S rRNA, secA1, hsp65, and gyrB genes. The nodulopustular pyoderma associated with sporotrichoid spreading was the most common skin presentations caused by N. brasiliensis. All of the isolates were susceptible to amikacin, gentamicin, tobramycin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole and resistant to kanamycin, erythromycin, and oxacillin, while susceptibilities to imipenem, vancomycin, penicillin-G, tetracycline, clindamycin, and ciprofloxacin varied among the 12 isolates. GyrB genotyping delineated the 12 isolates into 2 major groups, which was coincident with different single nucleotide substitutions at position 160 (G versus T) of 16S rRNA, different levels of imipenem minimum inhibition concentration (4-32 versus 0.25-0.75 mg/L), and prevalence of lymphadenitis (66.7 versus 16.7%). We have noted that tiny pustular lesions can be the first sign of cutaneous nocardiosis, which we believe has not been previously emphasized. No resistance to trimethoprim and sulfamethoxazole was found; therefore, sulphonamide drugs remain effective for treatment of cutaneous nocardiosis in Taiwan.