High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis.

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 "ESBL-positive" isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

Severe underlying liver diseases and high mortality associated with Laribacter hongkongensis bacteremia.

We characterized a strain of Laribacter hongkongensis isolated from the blood of a patient with fatal sepsis, who had alcoholic cirrhosis with ascites and portal hypertension. L. hongkongensis bacteremia is associated with underlying liver diseases (P < 0.001) and mortality (P < 0.05), whereas L. hongkongensis gastroenteritis is associated with recent travel history (P < 0.05).

Comparative evaluation of a laboratory-developed real-time PCR assay and RealStar® Adenovirus PCR Kit for quantitative detection of human adenovirus.

BACKGROUND: Human adenoviruses are common causes of community-acquired respiratory tract and enteric infections. Severe disseminated infections with high mortality rates may be seen in immunocompromised individuals. An accurate and cost-effective quantitative assay is essential not only for laboratory diagnosis of adenoviral infections, but also for monitoring of response to antiviral treatment. The diagnostic performance of an in-house quantitative polymerase chain reaction assay was compared to a commercial system. METHODS: The analytical sensitivity, specificity, linearity, precision and accuracy of an in-house adenovirus quantitative polymerase chain reaction assay were evaluated against the RealStar® Adenovirus PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany), using 122 clinical specimens and 18 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results by the in-house assay showed the dynamic range from 2.60 to 9 log10 (plasma) and 2.94 to 9 log10 (viral transport medium) copies/mL, with the coefficient of determination (R2) of 0.996 and 0.998, respectively. A dilution series demonstrated the limits of detection and lower limits of quantification for plasma were 2.06 log10 and 2.60 log10 copies/mL and those for viral transport medium were 2.31 log10 and 2.94 log10 copies/mL respectively. The precision of the in-house assay was highly reproducible among runs with coefficients of variance ranging from 0.07 to 3.21% for plasma and 0.17% to 2.11% for viral transport medium. A comparison of 52 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house assay and the RealStar® Adenovirus PCR Kit (R2 = 0.984), with an average bias of - 0.16 log10 copies/mL. CONCLUSIONS: The in-house adenovirus assay is a sensitive and reliable assay with lower cost for the detection and quantification of adenoviral DNA when compared to the RealStar® Adenovirus PCR Kit.

Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

BACKGROUND: HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar® HHV-6 PCR Kit. METHOD: The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R2) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar® HHV-6 PCR Kit (R2=0.926; P<0.0001), with an average bias of -0.24 log10 copies/ml. CONCLUSIONS: The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar® HHV-6 PCR Kit.

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

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

Characterization of a Tsukamurella pseudo-outbreak by phenotypic tests, 16S rRNA sequencing, pulsed-field gel electrophoresis, and metabolic footprinting.

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.

Streptococcus hongkongensis sp. nov., isolated from a patient with an infected puncture wound and from a marine flatfish.

A bacterium, HKU30(T), 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 HKU30(T), 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 39954(T)). 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 HKU30(T) ( = DSM 26014(T) = CECT 8154(T)).

In silico analysis of 16S rRNA gene sequencing based methods for identification of medically important aerobic Gram-negative bacteria.

This study provides guidelines on the usefulness of full and 527 bp 16S rRNA gene sequencing and Microseq databases for identifying medically important aerobic Gram-negative bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 26.1 % and 32.6 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15.2 % and 26.1 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level. Among the major groups of aerobic Gram-negative bacteria, the methods and databases are least useful for identification of Aeromonas, Bordetella and Bartonella species. None of the Aeromonas species can be confidently or doubtfully identified, whereas only 0 % and 0-33.3 % of Bordetella species and 0-10 % and 0-10 % of Bartonella species can be confidently and doubtfully identified, respectively. On the other hand, these methods and databases are most useful for identification of members of the families Pasteurellaceae and Legionellaceae and Campylobacter species: 29.6-59.3 % and 7.4-18.5 % of members of Pasteurellaceae, 36-52 % and 12-24 % of members of Legionellaceae, and 26.7-60 % and 0-13.3 % of Campylobacter species can be confidently and doubtfully identified, respectively. Thirty-nine medically important aerobic Gram-negative bacteria that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. Twenty-three medically important aerobic Gram-negative bacteria that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. Compared with results of our previous studies on anaerobic and Gram-positive bacteria, full and 527 bp 16S rRNA gene sequencing are able to confidently identify significantly more anaerobic Gram-positive and Gram-negative bacteria than aerobic Gram-positive and Gram-negative bacteria.

Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria.

This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0-2 and 2-13 % of staphylococci, and 0 and 0-10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36-56 and 11-14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S rRNA gene sequencing but are not found in the MicroSeq databases should be included.

Surgical site abscess caused by Lactobacillus fermentum identified by 16S ribosomal RNA gene sequencing.

We report the first case of surgical site abscess caused by Lactobacillus fermentum from a 53-year-old woman with squamous cell carcinoma of the esophagus after transthoracic esophagectomy and neoadjuvant chemoirradiation. 16S rRNA gene sequencing is a useful tool to better characterize the epidemiology and clinical significance of L. fermentum.

Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada.

Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37 degrees C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences from Clostridium propionicum, Clostridium neopropionicum, and Atopobium minutum, respectively. The G+C content of strain HKU16T is 40.2% +/- 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species, Catabacter hongkongensis gen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family, Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility of C. hongkongensis. Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.

Clinical isolates of Streptococcus iniae from Asia are more mucoid and beta-hemolytic than those from North America.

Streptococcus iniae, a widely distributed fish pathogen, is known to cause rare cases of human infection. We describe 2 cases of invasive S. iniae infection, one with septic arthritis complicating chronic gout and the other with bacteremic cellulitis. Both patients were Chinese and have been regularly handling fresh fish for cooking. Both isolates were unidentified or misidentified by 3 commercially available identification system and were only identified by 16S rRNA gene sequencing. When compared with a clinical isolate of S. iniae from Canada, their colonies were larger, more beta-hemolytic, and microcoid. Although bacteremic cellulitis has been described as the most common infection associated with S. iniae, the bacterium has not been reported to cause exacerbations of gouty arthritis previously. Clinical laboratories should be aware of the possibility of different colony morphology of S. iniae from Asia. More accurate identification of nongroupable beta-hemolytic streptococci, especially from patients with epidemiologic linkage to fresh fish, may uncover more cases of S. iniae infection. The Asian population and handlers of fresh fish should be informed of the risk of acquiring S. iniae infection.

Life-threatening invasive Helcococcus kunzii infections in intravenous-drug users and ermA-mediated erythromycin resistance.

We report the first two cases of life-threatening invasive Helcococcus kunzii infection, with primary bacteremia and empyema thoracis, respectively. Gram smears of both H. kunzii isolates showed a mixture of gram-positive and gram-negative cocci. The isolate from the first patient, resistant to erythromycin and clindamycin, possessed an ermA gene.

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia.

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.

Bacteremia due to Clostridium hathewayi in a patient with acute appendicitis.

Clostridium hathewayi is a newly described Clostridium species isolated from the feces of healthy human individuals, but its clinical significance is not known. We describe a case of human infection associated with C. hathewayi. The bacterium (strain HKU18) was isolated from the blood culture of a 39-year-old patient with acute gangrenous appendicitis complicated by septic shock. The cells were strictly anaerobic, nonmotile rods that stained gram negative. Conventional phenotypic tests and commercial identification systems failed to identify HKU18 to the species level. 16S rRNA gene analysis showed 1.4% nucleotide difference between the sequence of HKU18 and that of C. hathewayi, indicating that HKU18 was a strain of C. hathewayi. The patient responded to appendectomy and antibiotic treatment. 16S rRNA gene sequencing would be useful in further characterizing the clinical disease spectrum of C. hathewayi.

Eggerthella hongkongensis sp. nov. and eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia.

Eggerthella, one of the human gut flora, was rarely reported to cause bacteremia in the literature. We describe the application of 16S ribosomal RNA gene sequencing in defining the epidemiology and clinical significance of Eggerthella bacteremia during a 4-year period. Among 55 clinically significant blood culture isolates of anaerobic Gram-positive bacilli, 5 were identified as E. lenta and 5 belonged to 2 novel Eggerthella species, proposed as E. hongkongensis and E. sinensis, respectively. The 10 patients with Eggerthella bacteremia were adults, and 9 had underlying diseases. In all cases, the source of the bacteremia was likely from endogenous flora. Septic shock was a complication in 4 patients, and 3 patients died. The present study suggests that Eggerthella bacteremia is much more common than expected and is associated with significant morbidity and mortality. Moreover, the 2 novel species account for half of the cases of Eggerthella bacteremia.

Phenotypic and molecular characterization of erythromycin resistance in four isolates of Streptococcus-like gram-positive cocci causing bacteremia.

Among nine patients with bacteremia caused by Granulicatella or Gemella in a 6-year period (July 1995 to June 2001), three had bacteremia caused by erythromycin-resistant Granulicatella adiacens and one had bacteremia caused by erythromycin-resistant Gemella haemolysans. All four isolates possessed mef genes, whereas none possessed ermT, ermTR, or ermB genes.

Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia.

We report the evaluation of recombinant severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) nucleocapsid protein enzyme-linked immunosorbent assay (ELISA)-based antibody tests for serodiagnosis of SARS-CoV pneumonia and compare the sensitivities and specificities of this ELISA for detection of immunoglobulin G (IgG), IgM, IgA, and their combinations with serum samples from 149 healthy blood donors who donated blood 3 years ago as controls and 106 SARS-CoV pneumonia patients in Hong Kong. The specificities of the ELISA for IgG, IgM, and IgA detection were 95.3, 96.6, and 96.6%, respectively, with corresponding sensitivities of 94.3, 59.4, and 60.4%, respectively. The present ELISA appears to be a sensitive test for serodiagnosis of SARS-CoV pneumonia, is much more economical and less labor-intensive than the indirect immunofluorescence assay, and does not require cultivation of SARS-CoV.

Enterococcus cecorum empyema thoracis successfully treated with cefotaxime.

We report the first case of Enterococcus cecorum empyema thoracis and spontaneous bacterial peritonitis in a 44-year-old man with underlying cirrhosis. The patient responded to cefotaxime (MIC, 0.25 microg/ml) treatment and drainage of the empyema. Susceptibility of E. cecorum to expanded-spectrum cephalosporins could be due to its production of types of penicillin-binding proteins similar to those produced by Streptococcus species rather than to those produced by Enterococcus species (as predicted by phylogenetic analysis of the 16S rRNA gene sequences).