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Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis., published_or_final_version

Aims: To identify a strain of Gram negative facultative anaerobic curved bacillus, concomitantly isolated with Escherichia coli and Streptococcus milleri, from the blood culture of a 69 year old woman with acute gangrenous appendicitis. The literature on arcobacter bacteraemia and arcobacter infections associated with appendicitis was reviewed. Methods: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacterium was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. Literature review was performed by MEDLINE search (1966-2000). Results: The bacterium grew on blood agar, chocolate agar, and MacConkey agar to sizes of 1 mm in diameter after 24 hours of incubation at 37°C in 5% CO 2. It grew at 15°C, 25°C, and 37°C; it also grew in a microaerophilic environment, and was cytochrome oxidase positive and motile, typically a member of the genus arcobacter. Furthermore, phenotypic testing showed that the biochemical profile of the isolate did not fit into the pattern of any of the known arcobacter species. 16S rRNA gene sequencing showed one to two base differences between the isolate and A butzleri, but 35 to 39 base differences between the isolate and A cryaerophilus, indicating that the isolate was a strain of A butzleri. Only three cases of arcobacter bacteraemia with detailed clinical characteristics were found in the English literature. The sources of the arcobacter species in the three cases were largely unknown, although the gastrointestinal tract is probably the portal of entry of the A butzleri isolated from the present case because the two concomitant isolates (E coli and S milleri) in the blood culture were common flora of the gastrointestinal tract. In addition, A butzleri has previously been isolated from the abdominal contents or peritoneal fluid of three patients with acute appendicitis. Conclusions: 16S rRNA gene sequencing was useful in the identification of the strain of A butzleri isolated from the blood culture of a patient with acute gangrenous appendicitis. Arcobacter bacteraemia is rare. Further studies using selective medium for the delineation of the association between A butzleri and acute appendicitis are warranted., published_or_final_version

A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO2. It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37°C. It is Voges-Proskauer test positive. It produces leucine arylamidase and β-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of Streptococcus gordonii, Streptococcus intermedius, Streptococcus constellatus, Streptococcus sanguis, and Streptococcus anginosus, respectively. The G+C content of it (mean ± standard deviation) was 53.0% ± 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of Streptococcus. For these reasons a new species, Streptococcus sinensis sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis., published_or_final_version

Hepatitis E virus (HEV) is a major cause of viral hepatitis globally. Zoonotic HEV is an important cause of chronic hepatitis in immunocompromised patients. The rapid identification of novel HEV variants and accumulating sequence information has prompted significant changes in taxonomy of the family Hepeviridae. This family includes two genera: Orthohepevirus, which infects terrestrial vertebrates, and Piscihepevirus, which infects fish. Within Orthohepevirus, there are four species, A–D, with widely differing host range. Orthohepevirus A contains the HEV variants infecting humans and its significance continues to expand with new clinical information. We now recognize eight genotypes within Orthohepevirus A: HEV1 and HEV2, restricted to humans; HEV3, which circulates among humans, swine, rabbits, deer and mongooses; HEV4, which circulates between humans and swine; HEV5 and HEV6, which are found in wild boars; and HEV7 and HEV8, which were recently identified in dromedary and Bactrian camels, respectively. HEV7 is an example of a novel genotype that was found to have significance to human health shortly after discovery. In this review, we summarize recent developments in HEV molecular taxonomy, epidemiology and evolution and describe the discovery of novel camel HEV genotypes as an illustrative example of the changes in this field.

In May 2011, an enteroaggregative verocytotoxin-producing strain of Escherichia coli O104:H4 caused an outbreak of hemolytic uremic syndrome in Germany. Although traditional culture and phenotypic tests can identify the outbreak strain, rapid molecular testing is useful for timely diagnosis. While

We report here four cases of continuous ambulatory peritoneal dialysis-related peritonitis caused by three different species of Gordonia. The portal of entry was likely through Tenckhoff catheters. 16S rRNA and secA1 gene sequencing are so far the most reliable methods for the accurate identification of Gordonia species.

Streptococcus sinensis is a recently discovered human pathogen isolated from blood cultures of patients with infective endocarditis. Its phylogenetic position, as well as those of its closely related species, remains inconclusive when single genes were used for phylogenetic analysis. For example, S. sinensis branched out from members of the anginosus, mitis, and sanguinis groups in the 16S ribosomal RNA gene phylogenetic tree, but it was clustered with members of the anginosus and sanguinis groups when groEL gene sequences used for analysis. In this study, we sequenced the draft genome of S. sinensis and used a polyphasic approach, including concatenated genes, whole genomes, and matrix-assisted laser desorption ionization-time of flight mass spectrometry to analyze the phylogeny of S. sinensis .T he size of theS. sinensis draft genome is 2.06 Mb, with GC content of 42.2%. Phylogenetic analysis using 50 concatenated genes or whole genomes revealed that S. sinensis formed a distinct cluster with Streptococcus oligofermentans and Streptococcus cristatus, and these three streptococci were clustered with the “sanguinis group.” As for phylogenetic analysis using hierarchical cluster analysis of the mass spectra of streptococci, S. sinensis also formed a distinct cluster with S. oligofermentans and S. cristatus, but these three streptococci were clustered with the “mitis group.” On the basis of the findings, we propose a novel group, named “sinensis group,” to include S. sinensis, S. oligofermentans, and S. cristatus ,i n the Streptococcus genus. Our study also illustrates the power of phylogenomic analyses for resolving ambiguities in bacterial taxonomy.

Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin biosynthesis in Penicillium marneffei, a diffusible red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate than Monascus species. We also documented that the red pigment of P. marneffei is a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin, and showed that this polyketide gene cluster and pathway are also responsible for biosynthesis of ankaflavin and citrinin, a mycotoxin with nephrotoxic activity in mammals. The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species.

Background Emerging human picornaviruses, including human parechovirus (HPeV), Aichi virus (AiV) and salivirus (SalV) were found to be associated with gastroenteritis, but their roles in enteric infections are not fully understood. In addition, no report on the circulation of these viruses in Hong Kong is available. The objective of this study was to investigate the prevalence and genetic diversity of HPeV, AiV and SalV in fecal samples from hospitalized children with gastroenteritis in Hong Kong. Methods Fecal samples from hospitalized children with gastroenteritis were subject to detection of HPeV, AiV and SalV by RT-PCR using consensus primers targeted to their 5′UTRs. Positive samples were subject to capsid and/or 3CD region analysis for genotype determination. The epidemiology of HPeV, AiV and SalV infections was analyzed. Results Among 1,708 fecal samples subjected to RT-PCR using primers targeted to 5′UTR of HPeV, AiV and SalV, viruses were detected in 55 samples, with 50 positive for HPeV only, 3 positive for AiV only, 1 positive for both HPeV and AiV, and 1 positive for both HPeV and SalV. Phylogenetic analysis of the partial VP1 gene of the 33 HPeV strains revealed the presence of genotypes of HPeV- 1, 3, 4, 5, 7, 10, among which HPeV-1 was the predominant genotype circulating in our population. The peak activity of HPeV infection was in fall. Of the 3 children with AiV infection, the 3 AiV strains were found to belong to genotype A based on the phylogenetic analysis of their partial VP1 and 3CD regions. The genotype of a SalV strain detected in this study could not be determined. Co-detection of different pathogens was observed in 24 samples (43.6%) of 55 fecal samples positive for HPeV, AiV and SalV. Conclusions HPeV, AiV and SalV were detected in fecal samples of hospitalized children with gastroenteritis in Hong Kong, with the former having the highest prevalence. HPeV-1 was the predominant genotype among HPeVs, while genotype A was the predominant genotype among AiVs in this study.

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions., Author Summary Penicillium marneffei is a significant dimorphic fungal pathogen capable of causing lethal systemic infections. It grows in a yeast-like form at mammalian body temperature and a mold-like form at ambient temperature. The thermal dimorphism of P. marneffei is closely related to its virulence. In the present study, we re-sequenced the genome of P. marneffei using Illumina and PacBio sequencing technologies, and simultaneously assembled these newly sequenced reads in different lengths with previously obtained Sanger sequences. This hybrid assembly greatly improved the quality of the genome sequences. Next, we used RNA-seq to measure the global gene expression of P. marneffei at different phases and during dimorphic phase transitions. We found that 27% of genes showed signature expression patterns, suggesting that these genes function at different stages in the life cycle of P. marneffei. Moreover, genes with same expression patterns tend to be clustered together as neighbors to each other in the genome, suggesting an orchestrated transcriptional regulation for multiple neighboring genes. Over-expression of the MADS-box transcription factor, madsA, located in one of these clusters, confirms the function of this gene in driving the yeast-to-mycelia phase transition irrespective of the temperature cues. Our data also implies diverse roles of secreted proteins and non-coding RNAs in dimorphic transition in P. marneffei.

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.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. Originally considered to be restricted to humans, it is now clear that HEV and HEV-like viruses have several animal reservoirs with complex ecology and genetic diversity, as exemplified by the recent discovery of HEV in dromedaries, a previously underestimated reservoir of zoonotic viruses prior to the emergence of Middle East Respiratory Syndrome coronavirus. Zoonotic foodborne transmission from pigs and feral animals such as wild boar is of increasing importance in the rapidly industrializing countries of the Asia Pacific region. Such zoonotic hepatitis E infection has particular relevance to the increasing population living with immunosuppression, due to the risk of chronic hepatitis E in these patients. Fortunately, major strides have been made recently in the management of chronic hepatitis E patients. Furthermore, an effective vaccine is also available that promises better control of hepatitis E burden in the near future. This review highlights these major recent developments in the epidemiology, treatment, and prevention of hepatitis E.

We report the complete mitochondrial genome sequence of Lichtheimia ramosa (syn. Lichtheimia hongkongensis ), the first complete mitochondrial DNA sequence of the genus Lichtheimia . This 31.8-kb mitochondrial genome encodes 11 subunits of respiratory chain complexes, 3 ATP synthase subunits, 25 tRNAs, and small and large rRNAs, with the gene order atp9-cox2-atp6-cox3-cox1-nad2-nad3-cob-nad1-nad6-nad5-nad4l-nad4-atp8.

Two bacterial strains, HKU33T 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 HKU33T 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 HKU33T 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 13453T. A novel species, Streptobacillus hongkongensis sp. nov., is proposed to accommodate strains HKU33T and HKU34, with HKU33T ( = JCM 18691T = NCTC 13659T = DSM 26322T) designated the type strain. Emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis are also given.

While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.

Laribacter hongkongensis is a gram-negative, facultative anaerobic, motile, S-shaped, asaccharolytic, urease-positive bacillus in the Neisseriaceae family of β-proteobacteria. To date, all patients with L. hongkongensis infection have survived, including the two patients with L. hongkongensis bacteremia and patients with L. hongkongensis gastroenteritis. In this study, we describe the clinical, microbiological and molecular characterization of the first fatal case associated with L. hongkongensis bacteremia in a patient with colonic carcinoma that metastasized to the liver. The identity of the isolate was confirmed via phenotypic tests and 16S rRNA gene sequencing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), using the Bruker database extended with L. hongkongensis reference strains, also identified the isolate as L. hongkongensis, with a top match score of 2.473. Multilocus sequence typing revealed a new sequence type (ST), and phylogenetic analysis and eBURST demonstrated unambiguously that the ST of the isolate was clustered with two other STs found exclusively in human patients, consistent with the theory that some clones of L. hongkongensis could be more virulent than others. Underlying liver diseases and ascites potentially represent distinct risk factors for invasive L. hongkongensis infection. More widespread use of MALDI-TOF MS for identification and improvements of selective media should facilitate the identification of more cases of L. hongkongensis infection.

BACKGROUND: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model. RESULTS: HLHK9ureA, HLHK9ureC, HLHK9ureD and HLHK9ureE all exhibited no urease activity. HLHK9arcA1 and HLHK9arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9ureA/arcA1/arcA2 and HLHK9arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly reduced (p < 0.01). CONCLUSIONS: In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis., published_or_final_version

We report a fatal case of Schizophyllum commune empyema thoracis with cross-reactive cryptococcal antigenemia. In vitro testing confirmed the ability of the fungus to cause a positive cryptococcal antigen latex agglutination system (CALAS) test result. Such a result may lead to delay in diagnosis and treatment, as most strains of S. commune are resistant to fluconazole. Copyright © 2014, American Society for Microbiology. All Rights Reserved., link_to_OA_fulltext

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 β-actin and β-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.

Beginning in July 2011, 31 green anaconda ( Eunectes murinus ) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 × 10 4 to 1.05 × 10 8 copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical “herringbone” appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus , named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3′-N-U-P/V/I-M-F-HN-L-5′. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission.

A bacterium was isolated from the blood and empyema of a cirrhotic patient. The cells were facultatively anaerobic, nonsporulating, gram-negative, seagull shaped or spiral rods. The bacterium grows on sheep blood agar as nonhemolytic, gray colonies 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs on MacConkey agar and at 25 and 42°C but not at 4, 44, and 50°C. The bacterium can grow in 1 or 2% but not 3, 4, or 5% NaCl. No enhancement of growth is observed with 5% CO2. The organism is aflagellated and nonmotile at both 25 and 37°C. It is oxidase, catalase, urease, and arginine dihydrolase positive, and it reduces nitrate. It does not ferment, oxidize, or assimilate any sugar tested. 16S rRNA gene sequencing showed that there are 91 base differences (6.2%), 112 base differences (7.7%), and 116 base differences (8.2%) between the bacterium and Microvirgula aerodenitrificans, Vogesella indigofera, and Chromobacterium species, respectively. The G+C content (mean and standard deviation) is 68.0% ± 2.43%, and the genomic size is about 3 Mb. Based on phylogenetic affiliation, the bacterium belongs to the Neisseriaceae family of the β-subclass of Proteobacteria. For these reasons, a new genus and species, Laribacter hongkongensis gen. nov., sp. nov., is proposed, for which HKU1 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging pathogen., published_or_final_version

Little is known about the relative importance of the four species of Lancefield group G beta-hemolytic streptococci in causing bacteremia and the factors that determine the outcome for patients with group G beta-hemolytic streptococcal bacteremia. From 1997 to 2000, 75 group G beta-hemolytic streptococcal strains were isolated from the blood cultures of 66 patients. Sequencing of the 16S rRNA genes of the group G beta-hemolytic streptococci showed that all 75 isolates were Streptococcus dysgalactiae subspecies equisimilis. The API system (20 STREP) and Vitek system (GPI) successfully identified 65 (98.5%) and 62 (93.9%) isolates, respectively, as S. dysgalactiae subspecies equisimilis with >95% confidence, whereas the ATB Expression system (ID32 STREP) only successfully identified 49 isolates (74.2%) as S. dysgalactiae subspecies equisimilis with >95% confidence. The median age of the patients was 76 years (range, 33 to 99 years). Fifty-six patients (85%) were over 60 years old. All patients had underlying diseases. No source of the bacteremia was identified (primary bacteremia) in 34 patients (52%), whereas 17 (26%) had cellulitis and 8 (12%) had bed sore or wound infections. Fifty-eight patients (88%) had community-acquired group G streptococcal bacteremia. Sixty-two patients (94%) had group G Streptococcus recovered in one blood culture, whereas 4 patients (6%) had it recovered in multiple blood cultures. Fifty-nine patients (89%) had group G Streptococcus as the only bacterium recovered in their blood cultures, whereas in 7 patients other bacteria were recovered concomitantly with the group G Streptococcus in the blood cultures (Staphylococcus aureus in 3, Clostridium perfringens in 2, Citrobacter freundii in 1, and Bacteroides fragilis in 1). Overall, 10 patients (15%) died. Male sex, diagnosis other than cellulitis, hospital-acquired bacteremia, and multiple positive blood cultures were associated with mortality {P < 0.005 (relative risk [RR] = 7.6), P < 0.05 (RR = 3.7), P < 0.005 (RR = 5.6), and P < 0.05 (RR = 5.6), respectively}. Unlike group C beta-hemolytic streptococcal bacteremia, group G beta-hemolytic streptococcal bacteremia is not a zoonotic infection in Hong Kong., published_or_final_version

Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in Southeast Asia.1, 2, 3 About 8% of AIDS patients in Hong Kong are infected with P. marneffei. Clinically, penicilliosis manifests as a systemic febrile illness, which results from intracellular infection of the reticuloendothelial cells by the yeast phase of the fungus. Besides HIV-positive patients, P. marneffei infections have been reported in other immunocompromised patients, such as renal transplant recipients, patients with systemic lupus erythematosus and patients who are receiving corticosteroid therapy.4, 5 Here, we describe the first case of P. marneffei infection in a bone marrow transplant recipient.

Although the revolutionary matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated for identification of various groups of bacteria, its application in bacteria that are 'difficult-to-identify' by phenotypic tests has been less well studied. We aim to evaluate the usefulness of MALDI-TOF MS for identification of 'difficult-to-identify' bacterial isolates.We evaluated the performance of the Bruker MALDI-TOF MS system for a collection of 67 diverse clinically important bacterial isolates that were less commonly encountered, possessed ambiguous biochemical profiles or belonged to newly discovered species. The results were compared with 16S rRNA gene sequencing as a reference method for species identification.Using 16S rRNA gene sequencing as the reference method, 30 (45%) isolates were identified correctly to species level (score ≥2.0), 20 (30%) were only identified to genus level (score ≥1.7), four (6%) were misidentified (incorrect species with score ≥2.0 or incorrect genus with score ≥1.7) and 13 (19%) showed 'no identification' (score1.7). Aerobic Gram-positive bacteria showed the highest percentage of correct species identification, followed by aerobic Gram-negative, anaerobic Gram-positive and anaerobic Gram-negative bacteria. Sixteen isolates identified to genus level actually showed the correct species but with scores below the threshold for species identification. Most isolates which showed 'no identification' were due to the absence of the corresponding species in the Bruker database.Expansion of commercial databases to include reference spectra of less commonly encountered and newly discovered species and to increase available spectra for each species is required to improve the accuracy of MALDI-TOF MS for identifying 'difficult-to-identify' bacteria.

The high mortality associated with the novel Middle East respiratory syndrome coronavirus (MERS-CoV) has raised questions about the possible role of a cytokine storm in its pathogenesis. Although recent studies showed that MERS-CoV infection is associated with an attenuated IFN response, no induction of inflammatory cytokines was demonstrated during the early phase of infection. To study both early and late cytokine responses associated with MERS-CoV infection, we measured the mRNA levels of eight cytokine genes [TNF-α, IL-1β, IL-6, IL-8, IFN-β, monocyte chemotactic protein-1, transforming growth factor-β and IFN-γ-induced protein (IP)-10] in cell lysates of polarized airway epithelial Calu-3 cells infected with MERS-CoV or severe acute respiratory syndrome (SARS)-CoV up to 30 h post-infection. Among the eight cytokine genes, IL-1β, IL-6 and IL-8 induced by MERS-CoV were markedly higher than those induced by SARS-CoV at 30 h, whilst TNF-α, IFN-β and IP-10 induced by SARS-CoV were markedly higher than those induced by MERS-CoV at 24 and 30 h in infected Calu-3 cells. The activation of IL-8 and attenuated IFN-β response by MERS-CoV were also confirmed by protein measurements in the culture supernatant when compared with SARS-CoV and Sendai virus. To further confirm the attenuated antiviral response, cytokine response was compared with human HCoV-229E in embryonal lung fibroblast HFL cells, which also revealed higher IFN-β and IP-10 levels induced by HCoV-229E than MERS-CoV at 24 and 30 h. Whilst our data supported recent findings that MERS-CoV elicits attenuated innate immunity, this represents the first report to demonstrate delayed proinflammatory cytokine induction by MERS-CoV. Our results provide insights into the pathogenesis and treatment of MERS-CoV infections.

We report the first complete genome sequence of a feline astrovirus (FAstV), FAstV2 strain 1637F, identified from a domestic cat. The genome is 6,779 nucleotides (nt) in length and consists of three overlapping open reading frames (ORF1a-ORF1b-ORF2). Sequence analysis suggests that FAstV2 represents a new FAstV genotype that is closely related to human astroviruses.

We describe the discovery and characterization of a novel paramyxovirus, porcine parainfluenza virus 1 (PPIV-1), from swine. The virus was detected in 12 (3.1 %) of 386 nasopharyngeal and two (0.7 %) of 303 rectal swab samples from 386 deceased pigs by reverse transcription-PCR, with viral loads of up to 106 copies ml−1. Complete genome sequencing and phylogenetic analysis showed that PPIV-1 represented a novel paramyxovirus within the genus Respirovirus, being most closely related to human parainfluenza virus 1 (HPIV-1) and Sendai virus (SeV). In contrast to HPIV-1, PPIV-1 possessed a mRNA editing function in the phosphoprotein gene. Moreover, PPIV-1 was unique among respiroviruses in having two G residues instead of three to five G residues following the A6 run at the editing site. Nevertheless, PPIV-1, HPIV-1 and SeV share common genomic features and may belong to a separate group within the genus Respirovirus. The presence of PPIV-1 in mainly respiratory samples suggests a possible association with respiratory disease, similar to HPIV-1 and SeV.

Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicillium marneffei remain largely unknown. By comparing the extracellular proteomes of P. marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P. marneffei–host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P. marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P. marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P. marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

Interpretation of 16S rRNA sequences is a difficult problem faced by clinical microbiologists and technicians. In this study, we evaluated the updated 16SpathDB 2.0 database, using 689 16S rRNA sequences from 689 complete genomes of medically important bacteria. Among these 689 16S rRNA sequences, none was wrongly identified, with 35.8% reported as a single bacterial species having >98% identity with the query sequence (category 1), 63.9% reported as more than 1 bacterial species having >98% identity with the query sequence (category 2), 0.3% reported to the genus level (category 3), and none reported as no match (category 4). For the 16S rRNA sequences of non-duplicated bacterial species reported as category 1 or 2, the percentage of bacterial species reported as category 1 was significantly higher for anaerobic Gram-positive/Gram-negative bacteria than aerobic/facultative anaerobic Gram-positive/Gram-negative bacteria. 16SpathDB 2.0 is a user-friendly and accurate database for 16S rRNA sequence interpretation in clinical laboratories.

Background Enterovirus 71 (EV71) is a common etiological agent of hand, foot and mouth disease (HFMD) in children. EV71 epidemics have been reported in Hong Kong in recent years, and yet the genetic information of EV71 strains circulating in our locality is limited. The objective of this study was to investigate the genetic evolution of these EV71 isolates in Hong Kong over a 7-year period. Methods Twenty-two EV71 isolates from Hong Kong during 2004–2010 were included for phylogenetic analysis using partial VP2-VP3, 2C and 3D regions. Eight EV71 strains were selected for complete genome sequencing and recombination analysis. Results Among the 22 EV71 isolates, 20 belonged to subgenotype C4 and 2 belonged to subgenotype C2 based on the phylogenetic analysis of partial VP2-VP3, 2C and 3D gene regions. Phylogenetic, similarity plot and bootscan analyses using complete genome sequences of seven EV71 isolates of subgenotype C4 supported that the “double-recombinant” strains of subgenotype C4 persistently circulating in Hong Kong should belong to a newly proposed genotype D. Further analysis revealed two clusters, subgenotypes C4b and C4a (proposed genotypes D1a and D1b respectively), with “genotype D1b” strains being predominant in recent years in Hong Kong. A distinct lineage of EV71 subgenotype C2 has emerged in Hong Kong in 2008. The evolutionary rate of EV71 was 3.1 × 10-3 nucleotide substitutions per site per year similar to that of other enterovirus, such as EV68, but was relatively lower than those of echovirus 30 and poliovirus. Molecular clock analysis using VP1 gene dated the time to the most recent common ancestor of all EV71 genotypes to 1900s, while the EV71 “double-recombinant” strains of “genotype D” were detected as early as 1998. Conclusions This study provides the molecular basis for proposing a new “genotype D” of EV71 and assigning a discrete lineage of subgenotype C2. EV71 strains of “genotype D” have been circulating in Hong Kong for over 7 years, with “genotype D1b” being predominant.

Laribacter hongkongensis is a Gram-negative, facultative anaerobic, motile, S-shaped, urease-positive bacillus associated with invasive infections in liver cirrhosis patients and community-acquired gastroenteritis. Most cases of L hongkongensis infections occur in eastern countries. Information is lacking on the usefulness of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI–TOF MS) for the identification of bacteria important in eastern countries. Using the Bruker database extended with 21 L hongkongensis reference strains, all 240 L hongkongensis isolates recovered from patients, fish, frogs and water were correctly identified, with 224 (93.3%) strains having top match scores ≥2.0. Notably, the strain of Chromobacterium violaceum was not reliably identified although it is included in the database. MALDI–TOF MS is useful for the accurate routine identification of L hongkongensis after adding reference L hongkongensis main spectra to the database. The number of strains for each species in MALDI–TOF MS databases should be expanded to cover intraspecies variability.

While the novel Middle East respiratory syndrome coronavirus (MERS-CoV) is closely related to Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV HKU5) in bats from Hong Kong, and other potential lineage C betacoronaviruses in bats from Africa, Europe, and America, its animal origin remains obscure. To better understand the role of bats in its origin, we examined the molecular epidemiology and evolution of lineage C betacoronaviruses among bats. Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were detected in 29% and 25% of alimentary samples from lesser bamboo bat ( Tylonycteris pachypus ) and Japanese pipistrelle ( Pipistrellus abramus ), respectively. Sequencing of their RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes revealed that MERS-CoV is more closely related to Pi-BatCoV HKU5 in RdRp (92.1% to 92.3% amino acid [aa] identity) but is more closely related to Ty-BatCoV HKU4 in S (66.8% to 67.4% aa identity) and N (71.9% to 72.3% aa identity). Although both viruses were under purifying selection, the S of Pi-BatCoV HKU5 displayed marked sequence polymorphisms and more positively selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecological niches along with its host in diverse habitats. Molecular clock analysis showed that they diverged from a common ancestor with MERS-CoV at least several centuries ago. Although MERS-CoV may have diverged from potential lineage C betacoronaviruses in European bats more recently, these bat viruses were unlikely to be the direct ancestor of MERS-CoV. Intensive surveillance for lineage C betaCoVs in Pipistrellus and related bats with diverse habitats and other animals in the Middle East may fill the evolutionary gap.

A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans.Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests.Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses.Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC.

A bacterium, HKU30T, was isolated from the infected tissue of a patient with wound infection after puncture by a fish fin. Cells are facultative anaerobic, non-spore-forming, non-motile, Gram-positive cocci arranged in chains. Colonies were non-haemolytic. The strain was catalase, oxidase, urease and Voges–Proskauer test negative. It reacted with Lancefield’s group G antisera and was resistant to optochin. It grew on bile aesculin agar and in 5 % NaCl. It was unidentified by three commercial identification systems. 16S rRNA gene sequence analysis indicated that the bacterium shared 98.2, 97.7, 97.4 and 97.1 % nucleotide identities with Streptococcus iniae , Streptococcus pseudoporcinus , Streptococcus parauberis and Streptococcus uberis , respectively. The DNA G+C content was 35.6±0.9 mol% (mean±sd). In view of the occupational exposure of the patient, an epidemiological study was performed to isolate the bacterium from marine fish. Two strains, with similar phenotypic and genotypic characteristics to those of HKU30T, were isolated from a three-lined tongue sole (Cynoglossus abbreviatus) and an olive flounder (Paralichthys olivaceus) respectively. Phylogenetic analysis of four additional housekeeping genes, groEL, gyrB, sodA and rpoB, showed that the three isolates formed a distinct branch among known species of the genus Streptococcus , being most closely related to S. parauberis (CCUG 39954T). DNA–DNA hybridization demonstrated ≤53.8 % DNA relatedness between the three isolates and related species of the genus Streptococcus . A novel species, Streptococcus hongkongensis sp. nov., is proposed. The type strain is HKU30T ( = DSM 26014T = CECT 8154T).

While novel picornaviruses are being discovered in rodents, their host range and pathogenicity are largely unknown. We identified two novel picornaviruses, rosavirus B from the street rat, Norway rat, and rosavirus C from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) in China. Analysis of 13 complete genome sequences showed that “Rosavirus B” and “Rosavirus C” represent two potentially novel picornavirus species infecting different rodents. Though being most closely related to rosavirus A, rosavirus B and C possessed distinct protease cleavage sites and variations in Yn-Xm-AUG sequence in 5’UTR and myristylation site in VP4. Anti-rosavirus B VP1 antibodies were detected in Norway rats, whereas anti-rosavirus C VP1 and neutralizing antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats. While the highest prevalence was observed in Coxing's white-bellied rats by RT-PCR, the detection of rosavirus C from different rat species suggests potential interspecies transmission. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with high viral loads and positive viral antigen expression in organs of infected mice after oral or intracerebral inoculation. Histological examination revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lungs, and hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation in liver sections of sacrificed mice. Since rosavirus A2 has been detected in fecal samples of children, further studies should elucidate the pathogenicity and emergence potential of different rosaviruses., Author Summary We identified two novel picornaviruses, rosavirus B and C, infecting street and wild rats respectively in China. While rosavirus B was detected from Norway rats, rosavirus C was detected from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) by RT-PCR. Anti-rosavirus B antibodies were detected in Norway rats, whereas anti-rosavirus C antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats, supporting potential interspecies transmission of rosavirus C. Genome analysis supported the classification of rosavirus B and C as two novel picornavirus species, with genome features distinct from rosavirus A. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with viruses and pathologies detected in various organs of infected mice after oral or intracerebral inoculation. Our results extend our knowledge on the host range and pathogenicity of rodent picornaviruses.

Background Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. Methodology/Principal Findings We examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P, Author Summary Talaromyces (Penicillium) marneffei is an opportunistic thermal dimorphic fungus most prevalent in Southeast Asia. Our team has previously shown that Mp1p, a protein encoded by the MP1 gene, is an immunogenic surface and secretory protein of T. marneffei. In this study, we showed that mice challenged with T. marneffei with the MP1 gene died but those challenged with T. marneffei without the MP1 gene did not die. There was also significantly higher fungal load and more necrosis in organs of mice challenged with T. marneffei with the MP1 gene than T. marneffei without the MP1 gene. Furthermore, T. marneffei with the MP1 gene survived better in macrophages than T. marneffei without the MP1 gene and Pichia pastoris with the MP1 gene survived in mice better than P. pastoris without the MP1 gene. Our data support that Mp1p is a key virulence factor of T. marneffei and Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.

Talaromyces (Penicillium) marneffei is an important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. The clinical significance of T. marneffei became evident when the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome epidemic arrived in Southeast Asia in 1988. Subsequently, a decline in the incidence of T. marneffei infection among HIV-infected patients was seen in regions with access to highly active antiretroviral therapy and other control measures for HIV. Since the 1990s, an increasing number of T. marneffei infections have been reported among non-HIV-infected patients with impaired cell-mediated immunity. Their comorbidities included primary adult-onset immunodeficiency due to anti-interferon-gamma autoantibodies and secondary immunosuppressive conditions including other autoimmune diseases, solid organ and hematopoietic stem cell transplantations, T-lymphocyte-depleting immunsuppressive drugs and novel anti-cancer targeted therapies such as anti-CD20 monoclonal antibodies and kinase inhibitors. Moreover, improved immunological diagnostics identified more primary immunodeficiency syndromes associated with T. marneffei infection in children. The higher case-fatality rate of T. marneffei infection in non-HIV-infected than HIV-infected patients might be related to delayed diagnosis due to the lack of clinical suspicion. Correction of the underlying immune defects and early use of antifungals are important treatment strategies. Clinicians should be familiar with the changing epidemiology and clinical management of T. marneffei infection among non-HIV-infected patients.

We report a pseudo-outbreak of Tsukamurella due to improperly wrapped scissors used for processing of tissue specimens. A polyphasic approach, involving biochemical, genetic, and metabolomic techniques, was used in the laboratory investigation. This report highlights that early recognition of pseudo-outbreaks is important in preventing unnecessary and incorrect treatment of patients.

We report two genome sequences of novel noroviruses isolated from fecal swab specimens of brown rats in Hong Kong. The complete genome is approximately 7.5 kb in length and consists of 3 overlapping open reading frames encoding ORF1 polyprotein, VP1, and VP2, respectively. Sequence analysis suggested that these noroviruses should be classified in genogroup V, but they are distinct from other known rodent noroviruses and represent a novel cluster within the genogroup.

The recent outbreak of severe respiratory infections associated with a novel group C betacoronavirus (HCoV-EMC) from Saudi Arabia has drawn global attention to another highly probable “SARS-like” animal-to-human interspecies jumping event in coronavirus (CoV). The genome of HCoV-EMC is most closely related to Tylonycteris bat coronavirus HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat coronavirus HKU5 (Pi-BatCoV HKU5) we discovered in 2006. Phylogenetically, HCoV-EMC is clustered with Ty-BatCoV HKU4/Pi-BatCoV HKU5 with high bootstrap supports, indicating that HCoV-EMC is a group C betaCoV. The major difference between HCoV-EMC and Ty-BatCoV HKU4/Pi-BatCoV HKU5 is in the region between S and E, where HCoV-EMC possesses five ORFs (NS3a-NS3e) instead of four, with low (31%–62%) amino acid identities to Ty-BatCoV HKU4/Pi-BatCoV HKU5. Comparison of the seven conserved replicase domains for species demarcation shows that HCoV-EMC is a novel CoV species. More intensive surveillance studies in bats and other animals may reveal the natural host of HCoV-EMC.

Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of β-galactosidase activity. This is commonly tested using o -nitrophenyl-β- d -galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent β-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative β-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) should be used for testing staphylococci.

Pneumocystis colonization has been associated with airway inflammation and obstruction. We conducted a retrospective cohort study to investigate the clinical significance of Pneumocystis in the airway of patients with active tuberculosis. Of the 108 respiratory specimens tested positive for M. tuberculosis by polymerase chain reaction (PCR), 11 (10.2%) were also positive for Pneumocystis by PCR. Compared with patients tested negative for Pneumocystis, those with Pneumocystis had a higher serum alanine transaminase level, a greater likelihood of requiring oxygen supplementation, and a worse 30-day mortality. The proportion of patients with chronic obstructive pulmonary disease was not significantly different between the 2 groups, but lung malignancy was more prevalent among patients with Pneumocystis. Multivariate analysis showed that Pneumocystis was independently associated with oxygen supplementation. Our study has shown an association between the detection of Pneumocystis in lower respiratory tract specimens and greater impairment of pulmonary function among patients with active tuberculosis.

Although coronaviruses are known to infect various animals by adapting to new hosts, interspecies transmission events are still poorly understood. During a surveillance study from 2005 to 2010, a novel alphacoronavirus, BatCoV HKU10, was detected in two very different bat species, Ro-BatCoV HKU10 in Leschenault's rousettes ( Rousettus leschenaulti ) (fruit bats in the suborder Megachiroptera) in Guangdong and Hi-BatCoV HKU10 in Pomona leaf-nosed bats ( Hipposideros pomona ) (insectivorous bats in the suborder Microchiroptera) in Hong Kong. Although infected bats appeared to be healthy, Pomona leaf-nosed bats carrying Hi-BatCoV HKU10 had lower body weights than uninfected bats. To investigate possible interspecies transmission between the two bat species, the complete genomes of two Ro-BatCoV HKU10 and six Hi-BatCoV HKU10 strains were sequenced. Genome and phylogenetic analyses showed that Ro-BatCoV HKU10 and Hi-BatCoV HKU10 represented a novel alphacoronavirus species, sharing highly similar genomes except in the genes encoding spike proteins, which had only 60.5% amino acid identities. Evolution of the spike protein was also rapid in Hi-BatCoV HKU10 strains from 2005 to 2006 but stabilized thereafter. Molecular-clock analysis dated the most recent common ancestor of all BatCoV HKU10 strains to 1959 (highest posterior density regions at 95% [HPDs], 1886 to 2002) and that of Hi-BatCoV HKU10 to 1986 (HPDs, 1956 to 2004). The data suggested recent interspecies transmission from Leschenault's rousettes to Pomona leaf-nosed bats in southern China. Notably, the rapid adaptive genetic change in BatCoV HKU10 spike protein by ∼40% amino acid divergence after recent interspecies transmission was even greater than the ∼20% amino acid divergence between spike proteins of severe acute respiratory syndrome-related Rhinolophus bat coronavirus (SARSr-CoV) in bats and civets. This study provided the first evidence for interspecies transmission of coronavirus between bats of different suborders.

We report the first case of spontaneous intrapartum Atopobium vaginae bacteremia identified by 16S rRNA gene sequencing. The bacterium was misidentified by RapID ANA II, API Rapid ID 32A, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The likely source of bacteremia was the female genital tract. In invasive infections caused by A. vaginae, β-lactams and clindamycin are the antibiotics of choice, as most strains are resistant to metronidazole. Copyright © 2012, American Society for Microbiology. All Rights Reserved., link_to_OA_fulltext

Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination., link_to_OA_fulltext

Catabacter hongkongensis is a recently described catalase-positive, motile, anaerobic, nonsporulating, Gram-positive coccobacillus that was first isolated from blood cultures of four patients from Hong Kong and Canada. Although DNA sequences representing C. hongkongensis have been detected in environmental sources, only one additional case of human infection has been reported, in France. We describe five cases of C. hongkongensis bacteremia in Hong Kong, two presenting with sepsis, one with acute gangrenous perforated appendicitis, one with acute calculous cholecystitis, and one with infected carcinoma of colon. Three patients, with gastrointestinal malignancy, died during admission. All five isolates were catalase positive, motile, and negative for indole production and nitrate reduction and produced acid from arabinose, glucose, mannose, and xylose. They were unambiguously identified as C. hongkongensis by 16S rRNA gene analysis. Of the total of 10 reported cases of C. hongkongensis bacteremia in the literature and this study, most patients had underlying diseases, while two cases occurred in healthy young individuals with acute appendicitis. Six patients presented with infections associated with either the gastrointestinal or biliary tract, supporting the gastrointestinal tract as the source of bacteremia. C. hongkongensis bacteremia is associated with a poor prognosis, with a high mortality of 50% among reported cases, especially in patients with advanced malignancies. All reported isolates were susceptible to metronidazole. Identification of more C. hongkongensis isolates by 16S rRNA gene sequencing will help better define its epidemiology and pathogenesis., link_to_OA_fulltext

We report the identification and genome characterization of a novel bocavirus, feline bocavirus (FBoV), and novel bocaviruses closely related to canine bocavirus (CBoV) strain Con-161 in stray cats and dogs in Hong Kong, respectively. FBoV was detected by PCR in 7.2, 0.3, 1.6, 2.0 and 0.8 % of faecal, nasal, urine, kidney and blood samples, respectively, from 364 cats, while CBoV was detected in 4.6, 5.1, 6.3 and 0.3 % of faecal, nasal, urine and blood samples, respectively, from 351 dogs. Three FBoV genomes sequenced revealed the presence of three ORFs characteristic of bocaviruses. Phylogenetic analysis showed that FBoVs were related only distantly to other bocaviruses, forming a distinct cluster within the genus, with ≤59.7 % nucleotide identities to the genome of minute virus of canines. The four CBoV genomes sequenced shared 87.4–89.2 % nucleotide identities with that of CBoV strain Con-161. In addition to the three bocavirus ORFs, they encoded an additional ORF, ORF4, immediately downstream of the ORF for non-structural protein 1 (NS1), which was not found in other bocaviruses including CBoV strain Con-161. They also possessed a putative second exon encoding the C-terminal region of NS1 and conserved RNA-splicing signals, previously described in human bocaviruses. Partial VP1/VP2 sequence analysis of 23 FBoV and 25 CBoV strains demonstrated inter-host genetic diversity, with two potential genetic groups of FBoV and a novel CBoV group, CBoV-HK, distinct from the three groups, CBoV-A to -C, found in the USA. Although the pathogenicity of FBoV and CBoV remains to be determined, their presence in different host tissues suggested wide tissue tropism.

Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection—galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis.

We describe the discovery and isolation of a paramyxovirus, feline morbillivirus (FmoPV), from domestic cat (Felis catus). FmoPV RNA was detected in 56 (12.3%) of 457 stray cats (53 urine, four rectal swabs, and one blood sample) by RT-PCR. Complete genome sequencing of three FmoPV strains showed genome sizes of 16,050 bases, the largest among morbilliviruses, because of unusually long 5′ trailer sequences of 400 nt. FmoPV possesses identical gene contents (3′-N-P/V/C-M-F-H-L-5′) and is phylogenetically clustered with other morbilliviruses. IgG against FmoPV N protein was positive in 49 sera (76.7%) of 56 RT-PCR-positive cats, but 78 (19.4%) of 401 RT-PCR-negative cats (P < 0.0001) byWestern blot. FmoPV was isolated from CRFK feline kidney cells, causing cytopathic effects with cell rounding, detachment, lysis, and syncytia formation. FmoPV could also replicate in subsequent passages in primate Vero E6 cells. Infected cell lines exhibited finely granular and diffuse cytoplasmic fluorescence on immunostaining for FmoPV N protein. Electron microscopy showed enveloped virus with typical "herringbone" appearance of helical N in paramyxoviruses. Histological examination of necropsy tissues in two FmoPV-positive cats revealed interstitial inflammatory infiltrate and tubular degeneration/necrosis in kidneys, with decreased cauxin expression in degenerated tubular epithelial cells, compatible with tubulointerstitial nephritis (TIN). Immunohistochemical staining revealed FmoPV N protein-positive renal tubular cells and mononuclear cells in lymph nodes. A case-control study showed the presence of TIN in seven of 12 cats with FmoPV infection, but only two of 15 cats without FmoPV infection (P < 0.05), suggesting an association between FmoPV and TIN., link_to_OA_fulltext