Tak-Lun Que

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.

Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus

BACKGROUND Human infection with an avian influenza A virus (subtype H5N1) was reported recently in Hong Kong. We describe the clinical presentation of the first 12 patients and options for rapid viral diagnosis. METHODS Case notes of 12 patients with virus-culture-confirmed influenza A H5N1 infection were analysed. The clinical presentation and risk factors associated with severe disease were defined and the results of methods for rapid virus diagnosis were compared. FINDINGS Patients ranged from 1 to 60 years of age. Clinical presentation was that of an influenza-like illness with evidence of pneumonia in seven patients. All seven patients older than 13 years had severe disease (four deaths), whereas children 5 years or younger had mild symptoms with the exception of one who died with Reyes syndrome associated with intake of aspirin. Gastrointestinal manifestations, raised liver enzymes, renal failure unrelated to rhabdomyolysis, and pancytopenia were unusually prominent. Factors associated with severe disease included older age, delay in hospitalisation, lower-respiratory-tract involvement, and a low total peripheral white blood cell count or lymphopenia at admission. An H5-specific reverse-transcription PCR assay (RT-PCR) was useful for rapid detection of virus directly in respiratory specimens. A commercially available enzyme immunoassay was more sensitive than direct immunofluorescence for rapid viral diagnosis. Direct immunofluorescence with an H5-specific monoclonal antibody pool was useful for rapid exclusion of H5-subtype infection. INTERPRETATION Avian Influenza A H5N1 virus causes human influenza-like illness with a high rate of complications in adults admitted to hospital. Rapid H5-subtype-specific laboratory diagnosis can be made by RT-PCR applied directly to clinical specimens.

Emergence of Fluoroquinolone Resistance among Multiply Resistant Strains of Streptococcus pneumoniae in Hong Kong

ABSTRACT The MICs of 17 antimicrobial agents for 181 Streptococcus pneumoniae strains were determined by the E-test. Overall, 69.1% were penicillin resistant (MIC > 0.06 μg/ml). Resistance to ciprofloxacin (MIC > 2 μg/ml), levofloxacin (MIC > 2 μg/ml), or trovafloxacin (MIC > 1 μg/ml) was found in 12.1, 5.5, or 2.2% of the strains, respectively. These high rates of resistance raise concerns for the future.

Clinical and Molecular Epidemiology of Human Bocavirus in Respiratory and Fecal Samples from Children in Hong Kong

Abstract Background. Human bocavirus (HBoV) is a recently discovered parvovirus associated with respiratory tract infections in children. We conducted the first systematic prospective clinical and molecular study using nasopharyngeal aspirates (NPAs) and fecal samples. Methods. NPAs negative for influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, and coronavirus and fecal samples from patients with acute gastroenteritis were included. On the basis of results from a pilot study using 400 NPAs from all age groups, a prospective 12-month study was conducted to detect HBoV in 1200 NPAs and 1435 fecal samples from patients <18 years old by polymerase chain reaction. The complete genome sequences of HBoVs from 12 NPAs and 12 fecal samples were determined. Results. Of the 400 NPAs collected in the pilot study, 20 (5.0%) were found to contain HBoV, all from children <5 years old. In the subsequent prospective study of pediatric patients, HBoV was detected in 83 (6.9%) of 1200 NPAs. Upper and lower respiratory tract infections were equally common. HBoV was detected in 30 (2.1%) of 1435 fecal samples. Fever and watery diarrhea were the most common symptoms. The seasonality of HBoV in NPAs and fecal samples was similar. Codetection with other pathogens occurred in 33% and 56% of NPAs and fecal samples, respectively, from patients with HBoV infection. Genomes of HBoVs from NPAs and fecal samples displayed minimal sequence variations. Conclusions. HBoV was detected in fecal specimens in children with acute gastroenteritis. A single lineage of HBoV was associated with both respiratory tract and enteric infections.

Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles

ABSTRACT Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.

Infection of immunocompromised patients by avian H9N2 influenza A virus

Avian influenza A (H9N2) virus is transmitted sporadically from avian species to human causing mild diseases in immunocompetent person. We report two cases of human infection in immunocompromised patients in Hong Kong between 2008 and 2009. One patient had uneventful recovery with viral shedding at day 10 after symptom onset despite her underlying acute lymphoblastic leukaemia. The other patient with post-bone marrow transplant chronic graft-versus-host disease and bronhioltis obliterans went into respiratory failure. Genetic analysis revealed that these cases were caused by different genetic variants which are circulating in poultry in this region. Review of literature identified another 9 human cases reported in Southern China since 1988. It is possible that human infection with H9N2 is more common than what has been recognized. Continuous surveillance of H9N2 influenza virus infection in human is warranted.

Community-associated methicillin-resistant and methicillin-sensitive Staphylococcus aureus: skin and soft tissue infections in Hong Kong

This prospective study assessed the epidemiology of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among patients with purulent skin and soft tissue infections (SSTIs) in Hong Kong. Among 298 patients with SSTIs, 10.4% (13/125) of all S. aureus isolates and 5% (12/241) of all abscesses were attributed to pvl-positive CA-MRSA. Overall, 77% and 69.9% of CA-MRSA and methicillin-sensitive S. aureus (MSSA) were susceptible to erythromycin, 77% and 74.8% to clindamycin, 100% and 97.1% to minocycline, and 100% and 98.1% to rifampin, respectively. Filipino ethnicity was the only clinical and epidemiologic factor significantly associated with CA-MRSA infection (odds ratio, 14.8; 95% confidence interval, 3.3-70.0; P < 0.001). Pulsed-field gel electrophoresis analysis showed that 6 CA-MRSA isolates belonged to the ST30-HKU100 clone, 5 belonged to the ST59-HKU200 clone, and 1 was singleton. Features of HKU100 isolates include SCCmec type IV, agr3, spa t019, and pan-susceptibility to non-beta-lactam antibiotics. In contrast, HKU200 isolates are characterized by having SCCmec type IV or V, agr4, spa t437, and variable non-beta-lactam susceptibility profiles. The major CA-MRSA spa types were shared by a minority of the MSSA.

Effect of Clinical and Virological Parameters on the Level of Neutralizing Antibody against Pandemic Influenza A Virus H1N1 2009

BACKGROUND Little is known about the antibody response in natural infection by the novel 2009 influenza A (H1N1) virus and its relationship with clinical and virological parameters. The relative lack of background neutralizing antibody against this novel virus provides a unique opportunity for understanding this issue. METHODS Case patients presenting with influenza-like illness who were positive for the pandemic H1 gene by reverse transcription polymerase chain reaction were identified. The serum antibody response was assayed by neutralizing antibody titer (NAT) against the virus in 881 convalescent donors. We retrospectively analyzed clinical parameters and viral load. RESULTS Ninety percent of the 881 convalescent donors had seroprotective titer of 1:40 or greater. The geometric mean titer of donors with convalescent NAT measured between day 21 and 42 was 1:101.1. Multivariate analysis by ordinal regression showed that pneumonia (odds ratio, 3.39; 95% confidence interval, 1.49-7.61; P = .004) and sputum production (odds ratio, 1.75; 95% CI, 1.01-3.01; P = .046) were the 2 independent factors associated with a higher level of convalescent NAT. Being afebrile on influenza presentation was associated with subsequent poor NAT (<1:40) response (P = .04). A positive correlation between the nasopharyngeal viral load on presentation and the convalescent NAT was demonstrated (Spearman correlation rho, 0.238; P = .026). CONCLUSIONS About 10% of these convalescent patients do not have a seroprotective NAT and may benefit from vaccination to prevent reinfection. The convalescent NAT correlated well with the initial viral load and was independently associated with severity of the viral illness, including pneumonia. The findings provide both the clinical and virological markers for identifying potential convalescent plasma donors with high serum NAT, which can be used to produce hyperimmune intravenous immunoglobulin in a randomized treatment trial for patients with severe pandemic H1N1 infection.

Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing

Traditional ways of identifying slow growing bacteria is slow and often difficult. In this study, a small, Gram-negative, facultative anaerobic, slow growing bacillus was isolated from the blood culture of a 7-year old traffic accident victim. The bacterium was non-hemolytic, catalase and oxidase positive. An attempt to use the Vitek system (GNI+) and the API system (20NE) to identify the strain was unsuccessful as the growth controls showed negative results. 16S ribosomal RNA gene sequencing showed that there was 1 base difference between the isolate and Arcobacter cryaerophilus (GenBank Accession no. U25805), 1 base difference between the isolate and A. cryaerophilus (GenBank Accession no. U34387), 10 base differences between the isolate and A. cryaerophilus (GenBank Accession no. L14624), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34386), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34387), and 38 base differences between the isolate and A. butzleri (GenBank Accession no. L14626), indicating that the isolate most closely resembled a strain of A. cryaerophilus. Identification of the isolate in our case by conventional methods was difficult, as the absence of a curved morphology has made it confused with other Gram-negative non-fermentative bacteria, and the slow growth rate has made it unidentifiable by both the Vitek and API systems. Although the exact source of infection and route of transmission in our case remains elusive, we speculate that the bacteria were transmitted through the respiratory tract while the boy was suffocated in the mud. The present report represents an example of showing the usefulness of 16S rRNA gene sequencing for identification of slow growing bacteria.

Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish : a multicentre case-control study

BACKGROUND Laribacter hongkongensis has been recovered from several patients with gastroenteritis. However, the causative role of this organism in human gastroenteritis is still unproven, and sources of the bacterium are unknown. We undertook a multicentre case-control study to investigate the association of L hongkongensis with gastroenteritis. METHODS Faecal samples from patients with community-acquired gastroenteritis and controls were cultured for L hongkongensis. Targeted food surveillance was done to identify potential sources of this bacterium. All isolates of this organism from patients and food items were characterised by pulsed-field gel electrophoresis and ribotyping. FINDINGS During a 4-month period, L hongkongensis was recovered from 17 of 3788 patients with community-acquired gastroenteritis, but was absent in 1894 controls (p=0.001). Those who were culture-positive for this bacterium had a recent history of travel (ten [59%] patients vs two [6%] of 34 matched controls, p<0.0001), of fish consumption (16 [94%] vs 19 [56%], p=0.009), and of eating minced freshwater fish meat (five [29%] vs one [3%], p=0.012). We recovered 25 L hongkongensis isolates from intestinal samples of freshwater fish and two from minced freshwater fish meat. Bacteria with the same pulsed-field gel electrophoretic pattern and ribotype were recovered from one patient and a sample of minced freshwater fish meat, which was from the same retail market recently visited by the patient. We did not see this particular combination of electrophoretic pattern and ribotype in any other isolates. INTERPRETATION L hongkongensis is associated with community-acquired gastroenteritis and travellers diarrhoea. However, its causative role has not been shown. Freshwater fish is one source of this bacterium.