Tsukamurella asaccharolytica sp. nov., Tsukamurella conjunctivitidis sp. nov. and Tsukamurella sputi sp. nov., isolated from patients with bacteraemia, conjunctivitis and respiratory infection in Hong Kong.

Three bacterial strains, HKU70T, HKU71T and HKU72T, were isolated from the conjunctival swab, blood and sputum samples of three patients with conjunctivitis, bacteraemia and respiratory infection, respectively, in Hong Kong. The three strains were aerobic, Gram-stain positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from currently recognized Tsukamurella species. 16S rRNA, secA, rpoB and groEL gene sequence analyses revealed that the three strains shared 99.6-99.9, 94.5-96.8, 95.7-97.8 and 97.7-98.9 % nucleotide identities with their corresponding closest Tsukamurella species respectively. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella (26.2±2.4 to 36.8±1.2 % DNA-DNA relatedness), in line with results of in silico genome-to-genome comparison (32.2-40.9 % Genome-to-Genome Distance Calculator and 86.3-88.9 % average nucleotide identity values]. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content determined based on the genome sequence of strains HKU70T, HKU71T and HKU72T were 69.9, 70.2 and 70.5 mol%, respectively. Taken together, our results supported the proposition and description of three new species, i.e. Tsukamurella sputi HKU70T (=JCM 33387T=DSM 109106T) sp. nov., Tsukamurella asaccharolytica HKU71T (=JCM 33388T=DSM 109107T) sp. nov. and Tsukamurella conjunctivitidis HKU72T (=JCM 33389T=DSM 109108T) sp. nov.

Rare/cryptic Aspergillus species infections and importance of antifungal susceptibility testing.

BACKGROUND: The number of patients infected with Aspergillus rose dramatically in recent years. However, studies on the clinical spectrum and antifungal susceptibilities of non-classical (non-fumigatus, non-flavus, non-niger and non-terreus) pathogenic Aspergillus species are very limited. OBJECTIVES: We examined the clinical spectrum and antifungal susceptibilities of 34 non-duplicated, non-classical Aspergillus isolates collected from Hong Kong, Shenzhen and Shanghai. METHODS: The Aspergillus isolates were identified by internal transcribed spacer, partial BenA and partial CaM sequencing and phylogenetic analyses. Susceptibility testing against eight antifungals was performed following the European Committee for Antimicrobial Susceptibility Testing's methodology. RESULTS: The 34 Aspergillus isolates were identified as 14 different rare/cryptic species of four sections (Flavi [n = 8], Nidulantes [n = 8], Nigri [n = 17] and Restricti [n = 1]). Except for one patient whose clinical history could not be retrieved, 72.7% of the remaining patients had underlying conditions predisposing them to Aspergillus infections. The most common diseases were pulmonary infections (n = 15), followed by skin/nail infections (n = 6), chronic otitis externa and/or media (n = 5), wound infections (n = 2) and mastoiditis/radionecrosis (n = 1), while three were colonisations. Five patients succumbed due to the infections during the admission, and another two died 5 years later because of chronic pulmonary aspergillosis. Antifungal susceptibility testing showed that they possessed different susceptibility profiles compared to the classical Aspergillus species. The majority of isolates characterised were sensitive or wild-type to amphotericin B. The minimum effective concentrations for all the three echinocandins were also low. CONCLUSION: Susceptibility testing should be performed for infections due to these rare/cryptic Aspergillus species to guide proper patient management.

Tsukamurella ocularis sp. nov. and Tsukamurella hominis sp. nov., isolated from patients with conjunctivitis in Hong Kong.

Three bacterial strains, HKU63T, HKU64 and HKU65T, were isolated from the conjunctival swabs of three patients with conjunctivitis in Hong Kong. The three strains were aerobic, Gram-stain-positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from closely related Tsukamurella species. 16S rRNA gene sequence analysis revealed that the three strains shared identical sequences with each other, being most closely related to Tsukamurella tyrosinosolvens and Tsukamurella pulmonis, sharing 99.9 % sequence identity. Sequence analysis of three additional housekeeping genes, groEL, secA and rpoB, revealed 100 % nucleotide sequence identity between HKU63T and HKU64, 94.2-97.0 % nucleotide sequence identities between HKU63T/HKU64 and HKU65T and the three strains shared 82.9-98.9 % sequence identities with other currently recognized Tsukamurella species. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella(23.0±4.2 to 50.7±3.7 % DNA-DNA relatedness), of which HKU63T and HKU64 represented the same species (≥95.2±4.8 % DNA-DNA relatedness) while HKU65T represented another species. Fatty acid, mycolic acid, cell-wall sugar and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content of strains HKU63T, HKU64 and HKU65T were 71.3±1.9, 71.3±2.0 and 71.2±2.3 mol% (mean±sd; n=3), respectively. A novel species, Tsukamurella ocularis sp. nov. is proposed to accommodate strains HKU63T and HKU64, with HKU63T (=JCM 31969T=DSM 105034T) designated as the type strain whilst another novel species, Tsukamurella hominis sp. nov., is proposed to accommodate the third strain, HKU65T, which is designated as the type strain (=JCM 31971T=DSM 105036T).

Fatal Talaromyces marneffei Infection in a Patient with Autoimmune Hepatitis.

Talaromyces marneffei, previously known as Penicillium marneffei, is the most important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. Traditionally, T. marneffei infection in human was mainly associated with acquired immunodeficiency syndrome caused by HIV infection. In recent years, there has been an increasing number of T. marneffei infections reported in non-HIV-infected patients with other immunocompromised conditions, including autoantibodies against interferon-gamma, systemic lupus erythematosis, solid organ transplantation, Job's syndrome, hematological malignancies, and use of novel targeted therapies. In this article, we describe the first case of fatal T. marneffei infection in a patient with underlying autoimmune hepatitis, presented as fever without localizing features. The diagnosis of talaromycosis was confirmed with the identification of the fungi isolated from the blood culture specimen by conventional methods and using matrix-assisted laser desorption-ionization time-of-flight mass spectrometer. This case shows the importance of a high index of suspicion, particularly for such a highly fatal but potentially treatable fungal infection.

In Vitro Activity of Posaconazole against Talaromyces marneffei by Broth Microdilution and Etest Methods and Comparison to Itraconazole, Voriconazole, and Anidulafungin.

We determined the susceptibilities of 57 Talaromyces marneffei strains to anidulafungin, itraconazole, voriconazole, and posaconazole with MICs of 2 to 8, 0.002 to 0.004, 0.016 to 0.063, and 0.001 to 0.002 µg/ml by broth microdilution and >32, ≤0.002 to 0.008, ≤0.002 to 0.008, and ≤0.002 µg/ml by Etest, respectively, at yeast phase; MICs at mycelial phase for anidulafungin and posaconazole were 1 to 2 and 0.004 to 0.063 µg/ml, respectively. The results suggest promising activities of posaconazole. Etest can be used for testing of azoles against T. marneffei.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid identification of mold and yeast cultures of Penicillium marneffei.

BACKGROUND: Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in HIV-infected and other immunocompromised patients in Southeast Asia. However, laboratory diagnosis of penicilliosis, which relies on microscopic morphology and mycelial-to-yeast conversion, is time-consuming and expertise-dependent, thus delaying diagnosis and treatment. Although matrix -assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is useful for identification of various medically important fungi, its performance for identification of P. marneffei is less clear. RESULTS: We evaluated the performance of the Bruker MALDI-TOF MS system for identification of mold and yeast cultures of 59 clinical strains and the type strain of P. marneffei using the direct transfer method, with results compared to four phylogenetically closely related species, P. brevi-compactum, P. chrysogenum, Talaromyces aurantiacus and T. stipitatus. Using the Bruker original database combined with BDAL v4.0.0.1 and Filamentous Fungi Library 1.0, MALDI-TOF MS failed to identify the 60 P. marneffei strains grown in mold and yeast phase (identified as P. funiculosum and P. purpurogenum with scores <1.7 respectively). However, when the combined database was expanded with inclusion of spectra from 21 P. marneffei strains in mold and/or yeast phase, all the remaining 39 P. marneffei strains grown in mold or phase were correctly identified to the species level with score >2.0. The MS spectra of P. marneffei exhibited significant difference to those of P. brevi-compactum, P. chrysogenum, T. aurantiacus and T. stipitatus. However, MALDI-TOF MS failed to identify these four fungi to the species level using the combined database with or without spectra from P. marneffei. CONCLUSIONS: MALDI-TOF MS is useful for rapid identification of both yeast and mold cultures of P. marneffei and differentiation from related species. However, accurate identification to the species level requires database expansion using P. marneffei strains.

Tsukamurella serpentis sp. nov., isolated from the oral cavity of Chinese cobras (Naja atra).

Two bacterial strains, HKU54T and HKU55, were isolated from the oral cavity of two Chinese cobras (Naja atra) in Hong Kong. 16S rRNA gene sequence analysis revealed 100 % sequence identity between HKU54T and HKU55, and the two strains shared 99.0 % sequence identities with Tsukamurella inchonensis ATCC 700082T. The two strains had unique biochemical profiles distinguishable from closely related species of the genus Tsukamurella. DNA-DNA hybridization confirmed that they belonged to the same species (≥92.1±7.9 % DNA-DNA relatedness) but were distinct from all other known species of the genus Tsukamurella (≤52.6±5.3 % DNA-DNA relatedness). Chemotaxonomic and morphological analyses of the two strains also demonstrated results consistent with their classification in the genus Tsukamurella. The DNA G+C contents of strains HKU54T and HKU55 were 69.2±1.5 mol% and 69.2±1.3 mol% (mean±sd; n=3) respectively. A novel species, Tsukamurella serpentis sp. nov., is proposed to accommodate strains HKU54T and HKU55, with HKU54T (=JCM 31017T=DSM 100915T) designated as the type strain.

Tsukamurella hongkongensis sp. nov. and Tsukamurella sinensis sp. nov., isolated from patients with keratitis, catheter-related bacteraemia and conjunctivitis.

Three bacterial strains, HKU51T, HKU52T and HKU53, were isolated from a conjunctival swab, corneal scraping and blood culture of three patients in Hong Kong with conjunctivitis, keratitis and catheter-related bacteraemia, respectively. Cells were Gram-stain-positive, aerobic, catalase-positive, non-sporulating and non-motile bacilli. The three strains had unique biochemical profiles that were distinguishable from those of closely related species of the genus Tsukamurella. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. 16S rRNA gene sequence analysis revealed 100 % sequence identity between HKU52T and HKU53, and the two strains shared 99.5 % sequence identity with Tsukamurella sunchonensis JCM 15929T and Tsukamurella pseudospumae JCM 13375T; HKU51T shared 99.6 % sequence identity with Tsukamurella pulmonis CCUG 35732T. The DNA G+C contents of strains HKU51T, HKU52T and HKU53 were 70.9 ± 2.2, 71.3 ± 2.1 and 71.2 ± 2.3 mol% (mean ± sd; n = 3), respectively. DNA-DNA hybridization confirmed that the novel strains were distinct from other known species of the genus Tsukamurella ( ≤ 50.1 ± 3.7 % DNA-DNA relatedness); two of the isolates, HKU52T and HKU53, represented the same species ( ≥ 94.6 ± 5.6 % DNA-DNA relatedness), while the third isolate, HKU51T, represented another species. The novel species Tsukamurella hongkongensis sp. nov. is proposed to accommodate strains HKU52T and HKU53, with HKU52T ( = JCM 30715T = DSM 100208T) as the type strain; whilst another novel species, Tsukamurella sinensis sp. nov., is proposed to accommodate the third isolate, HKU51T ( = JCM 30714T = DSM 100207T), which is designated the type strain.

Cutaneous hyalohyphomycosis due to Parengyodontium album gen. et comb. nov.

"Engyodontium album" is an environmental saprobic mould and an emerging opportunistic pathogen able to cause both superficial and systemic infections. In this study, we isolated a mould from the skin lesion biopsy specimen of the right shin in a patient who received renal transplantation for end-stage renal failure with prednisolone, tacrolimus, and azathioprine immunosuppressant therapy. Histology of the skin biopsy showed mild squamous hyperplasia and neutrophilic infiltrate in the epidermis, active chronic inflammation in the dermis, and fat necrosis in the subcutis, with numerous fungal elements within the serum crusts. On Sabouraud glucose agar, the fungus grew as white, cobweb-like, floccose colonies. Microscopically, conidiogenous cells were arranged in whorls of one to seven at wide angles, with zigzag-shaped terminal fertile regions and smooth, hyaline, oval, apiculate conidia. DNA sequencing showed the mould isolate belonged to "E. album" but matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) failed to identify the isolate. Phylogenetic analyses based on the internal transcribed spacer region, 28S nuclear ribosomal DNA, and ß-tubulin gene and MALDI-TOF MS coupled with hierarchical cluster analysis showed that "E. album" is distantly related to other Engyodontium species and should be transferred to a novel genus within the family Cordycipitaceae, for which the name Parengyodontium album gen. et comb. nov. is proposed. Three potential cryptic species within this species complex were also revealed. Antifungal susceptibility testing showed posaconazole and voriconazole had high activities against all clinical P. album isolates and may be better drug options for treating P. album infections.

Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review.

Fusarium species are environmental saprophytic fungi. Among the many Fusarium species, members of the Fusarium solani species complex (FSSC) are the most prevalent and virulent in causing human and animal infections. In this study, we describe the first case of fatal FSSC infection in a black spotted stingray and three concomitant infections in scalloped hammerhead sharks. In the stingray, cutaneous lesions were characterised by ulcers and haemorrhage of the ventral pectoral fin, or 'ray', especially around the head; while cutaneous lesions in the sharks were characterised by ulcers, haemorrhage, as well as white and purulent exudates at the cephalic canals of the cephalofoil and lateral line. Histological sections of the cutaneous lesions revealed slender (1-4 µm in diameter), branching, septate fungal hyphae. Internal transcribed spacer region and 28S nrDNA sequencing of the fungal isolates from the fish showed two isolates were F. keratoplasticum (FSSC 2) and the other two were FSSC 12. Environmental investigation revealed the FSSC strains isolated from water and biofilms in tanks that housed the elasmobranchs were also F. keratoplasticum and FSSC 12. Fusarium is associated with major infections in elasmobranchs and FSSC 12 is an emerging cause of infections in marine animals. DNA sequencing is so far the most reliable method for accurate identification of Fusarium species.

Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

Subcutaneous phaeohyphomycotic nodule due to Phialemoniopsis hongkongensis sp. nov.

Phialemoniopsis species are ubiquitous dematiaceous molds associated with a wide variety of superficial and systemic infections in human. In this study, we isolated a mold from the forearm nodule biopsy specimen from a patient with underlying liver cirrhosis, ankylosing spondylosis, and tuberculosis. He was treated with itraconazole, but unfortunately, he succumbed as a result of disseminated tuberculosis with multiorgan failure. The histology results of the skin biopsy showed necrotizing granulomas in which numerous fungal elements were found. On Sabouraud dextrose agar, the fungal isolate grew as white-to-cream and smooth-to-velvety colonies. Microscopically, oval-to-cylindrical conidia were observed from abundant adelophialides, which possessed barely visible parallel collarettes but no basal septa. The azole drugs voriconazole, itraconazole, and posaconazole, as well as amphotericin B, showed high activities against this fungus. Internal transcribed spacer, 28S nuclear ribosomal DNA (nrDNA), and ß-actin and ß-tubulin gene sequencing showed that this fungus is most closely related to but distinct from Phialemonium curvata. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and hierarchical cluster analysis showed that the MALDI-TOF MS spectrum of this fungus is most closely related to that of Phialemonium pluriloculosa. We propose a new species, Phialemoniopsis hongkongensis sp. nov., to describe this fungus.

Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, ß-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, ß-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. ß-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

Streptobacillus hongkongensis sp. nov., isolated from patients with quinsy and septic arthritis, and emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis.

Two bacterial strains, HKU33(T) and HKU34, were isolated in Hong Kong from the pus aspirated from the right peritonsillar abscess of a patient with quinsy and the left elbow joint fluid of another patient with tophaceous gout and left elbow septic arthritis, respectively. The bacteria were Gram-stain-negative, non-motile, non-spore-forming, non-haemolytic pleomorphic bacilli. They grew best on Columbia agar with 5 % defibrinated sheep blood in an anaerobic environment or aerobic environment with 5 % CO2. They also grew on chocolate agar but not on MacConkey agar. They were catalase- and cytochrome oxidase-negative. They showed a unique profile of enzyme activities distinguishable from their closely related species. Phylogenetic analysis of the complete 16S rRNA gene, and partial groEL, gyrB and recA gene sequences showed the two isolates formed a distinct branch within the family Leptotrichiaceae, being related most closely to Streptobacillus moniliformis. Hierarchical cluster analysis of mass spectra of whole-cell protein contents showed that strains HKU33(T) and HKU34 were closely related to each other, but were distinct from Streptobacillus moniliformis, Sneathia sanguinegens and 'Leptotrichia amnionii'. The DNA G+C content of strain HKU33(T) was 26.0±2.1 mol% (mean±sd; n = 3). DNA-DNA hybridization demonstrated ≤45.02 % DNA relatedness between the two isolates and Streptobacillus moniliformis CCUG 13453(T). A novel species, Streptobacillus hongkongensis sp. nov., is proposed to accommodate strains HKU33(T) and HKU34, with HKU33(T) ( = JCM 18691(T) = NCTC 13659(T) = DSM 26322(T)) designated the type strain. Emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis are also given.

Subcutaneous phaeohyphomycosis in a patient with IgG4-related sclerosing disease caused by a novel ascomycete, Hongkongmyces pedis gen. et sp. nov.: first report of human infection associated with the family Lindgomycetaceae.

No members of the freshwater ascomycetes family Lindgomycetaceae have been associated with human infections. We isolated a mould (HKU35(T)) from the biopsy specimen of a patient with invasive foot infection and underlying immunoglobulin G4-related sclerosing disease. Histology showed florid, suppurative, granulomatous inflammation in the dermis, with central microabscess formation surrounded by epithelioid histiocytes, scattered giant cells, and a small number of lymphocytes. A Grocott stain revealed fungal elements in the center of the lesion. On Sabouraud glucose agar, HKU35(T) grew as gray and velvety colonies. Among the members of the family Lindgomycetaceae, HKU35(T) was the only strain that grew at 37°C. Microscopically, only sterile mycelia, but no fruiting bodies, were observed. HKU35(T) was susceptible to itrazonazole, voriconazole, and posaconazole, which was in line with the patient's clinical response to itraconazole treatment. Internal transcribed spacer and partial 18S nuclear rDNA (nrDNA), 28S nrDNA, ß-tubulin gene, and EF1α gene sequencing showed that HKU35(T) occupied a unique phylogenetic position, most closely related to but distinct from members of the genera Clohesyomyces and Lindgomyces. We propose a new genus and species, Hongkongmyces pedis gen. et sp. nov., to describe this fungus, which belongs to the family Lindgomycetaceae in the orderPleosporales of class Dothideomycetes. This case also represents the first report of human infection associated with the family Lindgomycetaceae.

Clinical spectrum of exophiala infections and a novel Exophiala species, Exophiala hongkongensis.

We characterized 12 Exophiala strains isolated from patients over a 15-year period to the species level using phenotypic tests and internal transcribed spacer (ITS) and Rpb1 sequencing and described the clinical spectrum of the 12 patients. Eight patients had nail or skin infections, two had invasive infections, and two had colonization of the gastrointestinal tract. ITS and Rpb1 sequencing showed that 11 of the 12 strains were known Exophiala species (E. oligosperma [n = 3], E. jeanselmei [n = 2], E. lecanii-corni [n = 2], E. bergeri [n = 1], E. cancerae [n = 1], E. dermatitidis [n = 1], and E. xenobiotica [n = 1]), which included the first reported cases of onychomycosis caused by E. bergeri and E. oligosperma. The 12th strain (HKU32(T)), isolated from the nail clipping of the right big toe of a 68-year-old female patient with onychomycosis, possessed unique morphological characteristics distinct from other Exophiala species. It grew very slowly and had a velvety colony texture after 28 days, short conidiophores of the same olivaceous color as the supporting hyphae, numerous spores, and no chlamydospore-like cells. ITS, Rpb1, ß-tubulin, and ß-actin gene sequencing unambiguously showed that HKU32(T) was clustered with but formed branches distinct from other Exophiala species in phylogenetic trees. We propose the new species Exophiala hongkongensis to describe this novel fungus.

Advantages of using matrix-assisted laser desorption ionization-time of flight mass spectrometry as a rapid diagnostic tool for identification of yeasts and mycobacteria in the clinical microbiological laboratory.

Yeast and mycobacteria can cause infections in immunocompromised patients and normal hosts. The rapid identification of these organisms can significantly improve patient care. There has been an increasing number of studies on using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid yeast and mycobacterial identifications. However, studies on direct comparisons between the Bruker Biotyper and bioMérieux Vitek MS systems for the identification of yeast and mycobacteria have been limited. This study compared the performance of the two systems in their identification of 98 yeast and 102 mycobacteria isolates. Among the 98 yeast isolates, both systems generated species-level identifications in >70% of the specimens, of which Candida albicans was the most commonly cultured species. At a genus-level identification, the Biotyper system identified more isolates than the Vitek MS system for Candida (75/78 [96.2%]versus 68/78 [87.2%], respectively; P = 0.0426) and non-Candida yeasts (18/20 [90.0%]versus 7/20 [35.0%], respectively; P = 0.0008). For mycobacterial identification, the Biotyper system generated reliable identifications for 89 (87.3%) and 64 (62.8%) clinical isolates at the genus and species levels, respectively, from solid culture media, whereas the Vitek MS system did not generate any reliable identification. The MS method differentiated 12/21 clinical species, despite the fact that no differentiation between Mycobacterium abscessus and Mycobacterium chelonae was found by using 16S rRNA gene sequencing. In summary, the MALDI-TOF MS method provides short turnaround times and a standardized working protocol for the identification of yeast and mycobacteria. Our study demonstrates that MALDI-TOF MS is suitable as a first-line test for the identification of yeast and mycobacteria in clinical laboratories.

A novel Dirofilaria species causing human and canine infections in Hong Kong.

Dirofilariasis is globally the commonest manifestation of zoonotic filariasis. We report the detection of a novel canine species causing human and canine dirofilariasis in Hong Kong. Three human cases occurring over 10 months were identified, one presenting with cervical lymphadenopathy, one with an abdominal subcutaneous mass, and one with a subconjunctival nodule. Transected worms recovered from the resected abdominal subcutaneous mass were morphologically compatible with Dirofilaria. The cox1 gene sequences of the three human isolates were identical; however, they were only 96.2% and 89.3% identical to the cox1 gene of Dirofilaria repens and Dirofilaria immitis, respectively. Sequencing of the 18S-ITS1-5.8S gene cluster was successful in the intact worm, and the nucleotide sequences were 94.0% and 94.9% identical to those of D. repens and D. immitis, respectively. Screening of the blood samples from 200 dogs and 100 cats showed the presence of the novel Dirofilaria species in 3% (6/200) of the dogs' but none of the cats' blood samples. Nucleotide sequences of the cox1 gene and 18S-ITS1-5.8S gene clusters of the dogs' samples were identical to those in the human samples. The sera of canines infected by this novel Dirofilaria species were negative when tested with the SNAP 4Dx D. immitis detection kit, except in the case of dogs with a mixed infection with D. immitis as detected by PCR. The results from this study suggest that this novel Dirofilaria species is a cause of filarial infection in humans and dogs in Hong Kong. We propose to name this Dirofilaria species "Candidatus Dirofilaria hongkongensis."

First report of disseminated Mycobacterium skin infections in two liver transplant recipients and rapid diagnosis by hsp65 gene sequencing.

We present here the first report of disseminated skin Mycobacterium infections in two liver transplant recipients, in which hsp65 gene sequencing was used for rapid species identification. Both patients had hepatitis B virus-related cirrhosis and diabetes mellitus and presented with progressive generalized, nodular skin lesions. In one patient, a 50-year-old woman who had frequent contact with marine fish, an acid-fast bacillus was isolated from skin biopsy tissue after 2 months of culture. While awaiting phenotypic identification results, hsp65 gene sequencing showed that it was most closely related to that of Mycobacterium marinum with 100% nucleotide identity. The patient was treated with oral rifampin, ethambutol, and moxifloxacin. In the other patient, a 59-year-old woman, direct PCR for Mycobacterium using hsp65 gene from skin biopsy tissue was positive, with the sequence most closely related to that of M. haemophilum with 100% nucleotide identity. Based on PCR results, the patient was treated with clarithromycin, ethambutol, moxifloxacin, and amikacin. A strain of M. haemophilum was only isolated after 3 months. Skin lesions of both patients resolved after 1 year of antimycobacterial therapy. Nontuberculous Mycobacterium infections should be considered in liver transplant recipients presenting with chronic, nodular skin lesions. This report highlights the crucial role of hsp65 gene PCR and sequencing on both cultured isolates and direct clinical specimens for rapid diagnosis of slow-growing Mycobacterium infection.