Samson S. Y. Wong

The mitochondrial genome of the thermal dimorphic fungus Penicillium marneffei is more closely related to those of molds than yeasts.

We report the complete sequence of the mitochondrial genome of Penicillium marneffei, the first complete mitochondrial DNA sequence of a thermal dimorphic fungus. This 35 kb mitochondrial genome contains the genes encoding ATP synthase subunits 6, 8, and 9 (atp6, atp8, and atp9), cytochrome oxidase subunits I, II, and III (cox1, cox2, and cox3), apocytochrome b (cob), reduced nicotinamide adenine dinucleotide ubiquinone oxireductase subunits (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), ribosomal protein of the small ribosomal subunit (rps), 28 tRNAs, and small and large ribosomal RNAs. Analysis of gene contents, gene orders, and gene sequences revealed that the mitochondrial genome of P. marneffei is more closely related to those of molds than yeasts.

Biotyping of Penicillium marneffei Reveals Concentration- Dependent Growth Inhibition by Galactose

ABSTRACT Thirty-two isolates of the dimorphic fungus Penicillium marneffei were studied for their biochemical properties. All isolates possessed the enzyme urease and were inhibited by 500 mg of cycloheximide per liter. No strain fermented glucose, and thus no strain fermented any of the other five sugars tested. All assimilated glucose, maltose, and cellobiose; only one of the isolates did not assimilate salicin. Totals of 65.6, 84.4, and 71.9% of the isolates assimilated trehalose, xylose, and nitrate, respectively. Twelve strains possessed the enzyme β-galactosidase. Overall, 17 different biotypes were recognized, but no association was found between the human immunodeficiency virus status of the patients and the biotype. A novel finding of concentration-dependent growth inhibition of P. marneffei by galactose is described. Inhibition of growth occurred at a low concentration of galactose (0.015 to 0.25%) when galactose was the sole carbon source in the medium. Morphological changes of the fungal cells were observed in the presence of galactose.

Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia

ABSTRACT Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 × 106 copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 × 106 copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.

Delayed Clearance of Viral Load and Marked Cytokine Activation in Severe Cases of Pandemic H1N1 2009 Influenza Virus Infection

Abstract Background. Infections caused by the pandemic H1N1 2009 influenza virus range from mild upper respiratory tract syndromes to fatal diseases. However, studies comparing virological and immunological profile of different clinical severity are lacking. Methods. We conducted a retrospective cohort study of 74 patients with pandemic H1N1 infection, including 23 patients who either developed acute respiratory distress syndrome (ARDS) or died (ARDS-death group), 14 patients with desaturation requiring oxygen supplementation and who survived without ARDS (survived-without-ARDS group), and 37 patients with mild disease without desaturation (mild-disease group). We compared their pattern of clinical disease, viral load, and immunological profile. Results. Patients with severe disease were older, more likely to be obese or having underlying diseases, and had lower respiratory tract symptoms, especially dyspnea at presentation. The ARDS-death group had a slower decline in nasopharyngeal viral loads, had higher plasma levels of proinflammatory cytokines and chemokines, and were more likely to have bacterial coinfections (30.4%), myocarditis (21.7%), or viremia (13.0%) than patients in the survived-without-ARDS or the mild-disease groups. Reactive hemophagocytosis, thrombotic phenomena, lymphoid atrophy, diffuse alveolar damage, and multiorgan dysfunction similar to fatal avian influenza A H5N1 infection were found at postmortem examinations. Conclusions. The slower control of viral load and immunodysregulation in severe cases mandate the search for more effective antiviral and immunomodulatory regimens to stop the excessive cytokine activation resulting in ARDS and death.

Avian Influenza Virus Infections in Humans

Seroepidemiologic and virologic studies since 1889 suggested that human influenza pandemics were caused by H1, H2, and H3 subtypes of influenza A viruses. If not for the 1997 avian A/H5N1 outbreak in Hong Kong of China, subtype H2 is the likely candidate for the next pandemic. However, unlike previous poultry outbreaks of highly pathogenic avian influenza due to H5 that were controlled by depopulation with or without vaccination, the presently circulating A/H5N1 genotype Z virus has since been spreading from Southern China to other parts of the world. Migratory birds and, less likely, bird trafficking are believed to be globalizing the avian influenza A/H5N1 epidemic in poultry. More than 200 human cases of avian influenza virus infection due to A/H5, A/H7, and A/H9 subtypes mainly as a result of poultry-to-human transmission have been reported with a > 50% case fatality rate for A/H5N1 infections. A mutant or reassortant virus capable of efficient human-to-human transmission could trigger another influenza pandemic. The recent isolation of this virus in extrapulmonary sites of human diseases suggests that the high fatality of this infection may be more than just the result of a cytokine storm triggered by the pulmonary disease. The emergence of resistance to adamantanes (amantadine and rimantadine) and recently oseltamivir while H5N1 vaccines are still at the developmental stage of phase I clinical trial are causes for grave concern. Moreover, the to-be pandemic strain may have little cross immunogenicity to the presently tested vaccine strain. The relative importance and usefulness of airborne, droplet, or contact precautions in infection control are still uncertain. Laboratory-acquired avian influenza H7N7 has been reported, and the laboratory strains of human influenza H2N2 could also be the cause of another pandemic. The control of this impending disaster requires more research in addition to national and international preparedness at various levels. The epidemiology, virology, clinical features, laboratory diagnosis, management, and hospital infection control measures are reviewed from a clinical perspective.

Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus

The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P = 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4+ and CD8+ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.

Bats as a continuing source of emerging infections in humans

Amongst the 60 viral species reported to be associated with bats, 59 are RNA viruses, which are potentially important in the generation of emerging and re‐emerging infections in humans. The prime examples of these are the lyssaviruses and Henipavirus. The transmission of Nipah, Hendra and perhaps SARS coronavirus and Ebola virus to humans may involve intermediate amplification hosts such as pigs, horses, civets and primates, respectively. Understanding of the natural reservoir or introductory host, the amplifying host, the epidemic centre and at‐risk human populations are crucial in the control of emerging zoonosis. The association between the bat coronaviruses and certain lyssaviruses with particular bat species implies co‐evolution between specific viruses and bat hosts. Cross‐infection between the huge number of bat species may generate new viruses which are able to jump the trans‐mammalian species barrier more efficiently. The currently known viruses that have been found in bats are reviewed and the risks of transmission to humans are highlighted. Certain families of bats including the Pteropodidae, Molossidae, Phyllostomidae, and Vespertilionidae are most frequently associated with known human pathogens. A systematic survey of bats is warranted to better understand the ecology of these viruses. Copyright

Bacteremia Caused by Staphylococci with Inducible Vancomycin Heteroresistance

The clinical significance of bacteremia due to vancomycin-heteroresistant staphylococci and a rapid laboratory screening method were examined; 203 strains of staphylococci isolated from patients with clinically significant bacteremia were screened by the disk-agar method with use of vancomycin-salt agar to demonstrate satellitism around an aztreonam disk as well as by conventional population screening. Eighteen isolates (three Staphylococcus aureus and 15 coagulase-negative staphylococci) were shown to be heteroresistant to vancomycin. A case-control clinical study showed that the interval between admission and bacteremia, admission to the intensive care unit, prior use of vancomycin and/or beta-lactams, and isolation of methicillin-resistant staphylococci were significantly more common among patients with bacteremia due to staphylococci with heteroresistance to vancomycin; these patients had an overall mortality of 44.4%. The use of vancomycin and admission to the intensive care unit were independently significant risk factors on multivariate analysis. Vancomycin heteroresistance is inducible by salt and beta-lactams. Indiscriminate sequential use of beta-lactams and glycopeptides may facilitate the emergence of glycopeptide resistance.

Invasive Streptococcus iniae Infections Outside North America

ABSTRACT Streptococcus iniae, a fish pathogen causing infections in aquaculture farms worldwide, has only been reported to cause human infections in North America. In this article, we report the first two cases of invasive S. iniae infections in two Chinese patients outside North America. While the first patient presented with bacteremic cellulitis, which is the most common presentation in previous cases, the second patient represents the first recognized case of S. iniae osteomyelitis. Both S. iniae strains isolated from the two patients were either misidentified or unidentified by three commercial systems and were only identified by 16S rRNA gene sequencing. Since no currently available commercial system for bacterial identification includes S. iniae in its database, 16S rRNA gene sequencing is the most practical and reliable method to identify the bacterium at the moment. In contrast to the distinct genetic profile described previously in clinical isolates from Canada, the present two isolates and a clinical isolate from a Canadian patient were found to be genetically unrelated, as demonstrated by pulsed-field gel electrophoresis. Morphologically, colonies of both isolates were also larger, more beta-hemolytic and mucoid, which differ from the usual morphotype described for S. iniae. Owing to their habit of cooking and eating fresh fish, the Asian population is strongly associated with S. iniae infections. As a result of the difficulty in making microbiological diagnosis in patients with cellulitis and the problem of identification in most clinical microbiology laboratories, the prevalence of S. iniae infections, especially in the Asian population, may have been under-estimated.

Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia

Summary Background Although the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. Methods We cloned and purified the nucleocapsid protein and spike polypeptide of SARS-CoV and examined their immunogenicity with serum from patients with SARS-CoV pneumonia. An ELISA based on recombinant nucleocapsid protein for IgG detection was tested with serum from 149 healthy blood donors who donated 3 years previously and with serum positive for antibodies against SARS-CoV (by indirect immunofluorescence assay) from 106 patients with SARS-CoV pneumonia. The seroprevalence of SARS-CoV was studied with the ELISA in healthy blood donors who donated during the SARS outbreak in Hong Kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. All positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). Findings Western-blot analysis showed that the nucleocapsid protein and spike polypeptide of SARS-CoV are highly immunogenic. The specificity of the IgG antibody test (ELISA with positive samples confirmed by the two western-blot assays) was 100%, and the sensitivity was 94·3%. Three of 400 healthy blood donors who donated during the SARS outbreak and one of 131 non-pneumonic paediatric inpatients were positive for IgG antibodies, confirmed by the two western-blot assays (total, 0·48% of our study population). Interpretation Our findings support the existence of subclinical or non-pneumonic SARS-CoV infections. Such infections are more common than SARS-CoV pneumonia in our locality.