Pcy Woo

Identification of a Novel Coronavirus in Bats

ABSTRACT Exotic wildlife can act as reservoirs of diseases that are endemic in the area or can be the source of new emerging diseases through interspecies transmission. The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlights the importance of virus surveillance in wild animals. Here, we report the identification of a novel bat coronavirus through surveillance of coronaviruses in wildlife. Analyses of the RNA sequence from the ORF1b and S-gene regions indicated that the virus is a group 1 coronavirus. The virus was detected in fecal and respiratory samples from three bat species (Miniopterus spp.). In particular, 63% (12 of 19) of fecal samples from Miniopterus pusillus were positive for the virus. These findings suggest that this virus might be commonly circulating in M. pusillus in Hong Kong.

Clinical Spectrum of Paradoxical Deterioration During Antituberculosis Therapy in Non-HIV-Infected Patients

Abstract.Paradoxical deterioration during antituberculosis therapy, defined as the clinical or radiological worsening of pre-existing tuberculous lesions or the development of new lesions in a patient who initially improves, remains a diagnostic dilemma. Although different clinical presentations of paradoxical response have been described, a systematic analysis of the entity in non-HIV-infected patients is lacking. Reported here are two cases of paradoxical deterioration in which sequential changes in lymphocyte counts and tuberculin skin test results are emphasized. In addition, 120 episodes of paradoxical response after antituberculosis treatment were reviewed. Of the total 122 episodes, 101 (82.8%) were associated with extrapulmonary tuberculosis. The median time from commencement of treatment to paradoxical deterioration was 60 days. The median time to onset of central nervous system manifestations (63 days) was longer than the time to onset of manifestations at other sites (56 days) (P=0.02). Development of new lesions in anatomical sites other than those observed at initial presentation was observed in 31 (25.4%) episodes. A surge in the lymphocyte count, accompanied by an exaggerated tuberculin skin reaction, was observed in our patients during the paradoxical deterioration, analogous to the findings in HIV-positive patients. Treatment of the paradoxical response included surgical intervention (60.7%) and administration of steroids (39.3%). The use of steroids appeared to be safe in this series, as 95% of the Mycobacterium tuberculosis isolates were susceptible to first-line antituberculosis therapy.

Clinical evaluation of the polymerase chain reaction for the rapid diagnosis of tuberculosis

Aims: Use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis (TB PCR) as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. Methods: A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. TB PCR was performed on pulmonary and extrapulmonary specimens from these patients. The sensitivity of TB PCR was analysed. Results: Of the 155 patients, 144 fitted the clinical diagnosis of tuberculosis, and 112 of them were culture positive for M tuberculosis. Sixty (58.3%) patients with clinical features suggestive of tuberculosis received antituberculosis treatment based on positive TB PCR alone. Of 224 clinical specimens (138 pulmonary and 86 extrapulmonary) sent for TB PCR, 148 (99 pulmonary and 49 extrapulmonary) were positive in 117 patients. Of the 690 clinical specimens sent for culture, 279 were positive for M tuberculosis in 112 patients. The diagnostic sensitivity of TB PCR was 75.9% (85 of 112) and 81.3% (117 of 144) in patients with culture confirmed and clinically diagnosed tuberculosis, respectively. Using culture as the gold standard, the overall sensitivity of TB PCR was 78.3%, and for pulmonary and extrapulmonary specimens it was 82.3% and 72.0%, respectively. Conclusions: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.

Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis

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% CO2. 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.

Fatal Interaction between Clarithromycin and Colchicine in Patients with Renal Insufficiency: A Retrospective Study

BACKGROUND Clarithromycin is frequently used to treat community-acquired pneumonia in elderly persons. Like erythromycin, it may interact with other drugs by interfering with metabolism by cytochrome P450 enzymes and with the P-glycoprotein transporter system. Colchicine, used for treatment of acute gout and for prophylaxis, may cause bone marrow toxicity. It is metabolized by CYP3A4 and is transported by P-glycoprotein. Initial case reports suggested potentially fatal interactions between clarithromycin and colchicine. METHODS A retrospective study was conducted with 116 patients who were prescribed clarithromycin and colchicine during the same clinical admission. Case-control comparisons were made between patients who received concomitant therapy with the 2 drugs and patients who received sequential therapy. We assessed the clinical presentations and outcomes of the 2 patient groups and analyzed the risk factors associated with fatal outcomes. RESULTS Nine (10.2%) of the 88 patients who received the 2 drugs concomitantly died. Only 1 (3.6%) of the 28 patients who received the drugs sequentially died. Multivariate analysis of the 88 patients who received concomitant therapy showed that longer overlapped therapy (relative risk [RR], 2.16; 95% confidence interval [CI], 1.41-3.31; P< or =.01), the presence of baseline renal impairment (RR, 9.1; 95% CI, 1.75-47.06; P<.001), and the development of pancytopenia (RR, 23.4; 95% CI, 4.48-122.7; P<.001) were independently associated with death. CONCLUSIONS Clarithromycin increases the risk of fatal colchicine toxicity, especially for patients with renal insufficiency. Since there are other drugs for treatment of pneumonia and gout, these 2 drugs should not be coprescribed, because of the risk of fatality.

Identification by 16s ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient

Aims—To ascertain the clinical relevance of a strain of Enterobacteriaceae isolated from the stool of a bone marrow transplant recipient with diarrhoea. The isolate could not be identified to the genus level by conventional phenotypic methods and required 16S ribosomal RNA (rRNA) gene sequencing for full identification. Methods—The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GNI+) and API (20E) systems. Genotypically, the 16S bacterial rRNA gene 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 database by multiple sequence alignment. Results—Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The isolate was identified as Salmonella arizonae (73%) and Escherichia coli (76%) by the Vitek (GNI+) and API (20E) systems, respectively. 16S rRNA sequencing showed that there was only one base difference between the isolate and E coli K-12, but 48 and 47 base differences between the isolate and S typhimurium (NCTC 8391) and S typhi (St111), respectively, showing that it was an E coli strain. The patient did not require any specific treatment and the diarrhoea subsided spontaneously. Conclusions—16S rRNA gene sequencing was useful in ascertaining the clinical relevance of the strain of Enterobacteriaceae isolated from the stool of the bone marrow transplant recipient with diarrhoea.

Gemella bacteraemia characterised by 16S ribosomal RNA gene sequencing.

Aims: To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. Methods: All α haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with ≥ 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. Results: Of the 302 α haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin’s lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. Conclusions: Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.

Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing

Aims—To ascertain the clinical importance of a strain of slide coagulase positive but tube coagulase negative staphylococcus species isolated from the blood culture of a 43 year old patient with refractory anaemia with excessive blasts in transformation who had neutropenic fever. Methods—The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GPI) and API (Staph) systems. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacteria 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. Results—Conventional biochemical tests did not reveal a pattern resembling a known staphylococcus species. The Vitek system (GPI) showed that it was 94% S simulans and 3% S haemolyticus, whereas the API system (Staph) showed that it was 86.8% S aureus and 5.1% S warneri. 16S rRNA gene sequencing showed that there was a 0 base difference between the isolate and S aureus, 28 base difference between the isolate and S lugdunensis, 39 base difference between the isolate and S schleiferi, 21 base difference between the isolate and S haemolyticus, 41 base difference between the isolate and S simulans, and 23 base difference between the isolate and S warneri, indicating that the isolate was a strain of S aureus. Vancomycin was subsequently prescribed and blood cultures taken four days after the start of treatment were negative. Conclusions—16S rRNA gene sequencing was useful in ascertaining the clinical importance of the strain of slide coagulase positive but tube coagulase negative staphylococcus species isolated from blood culture and allowing appropriate management.

Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov.

Aims: To characterise a strain of Gram negative aerobic straight or slightly curved rods (HKU3) isolated from the blood culture of a 9 year old Chinese boy with neutropenic fever and pseudobacteraemia. Methods: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests, scanning electron microscopy, and transmission electron microscopy. Genotypically, the 16S 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. The G + C content was determined by thermal denaturation. A phylogenetic tree was constructed by the PileUp method. Results: The cells of the bacterial strain were aerobic, sporulating, Gram negative straight or slight curved rods. The bacterium grew on horse blood agar as non-haemolytic, grey colonies of 1 mm in diameter after 24 hours of incubation at 37°C in ambient air. No enhancement of growth was seen in 5% CO2. It grew at 50°C as pinpoint colonies after 72 hours of incubation, but did not grow at 65°C or on MacConkey agar. It was non-motile. It produced catalase (weakly positive) and cytochrome oxidase. It reduced nitrate, produced β galactosidase, hydrolysed esculin, and utilised sodium acetate. A scanning electron micrograph of the bacterium showed straight or slightly curved rods. A transmission electron micrograph of the cell wall of the bacterium revealed multiple electron dense layers, including the outer membrane, middle murein layer, and inner cytoplasmic membrane, compatible with its Gram smear appearance. 16S rRNA gene sequencing showed that there were 7.7%, 8.0%, 8.2%, and 8.6% differences between the 16S rRNA gene sequence of the bacterium and those of Paenibacillus macerans, Paenibacillus borealis, Bacillus ehimensis, and Paenibacillus amylolyticus, respectively. The mean (SD) G + C content of the bacterium was 47.6 (2.1) mol%. Phylogenetically, it belongs to the genus paenibacillus (previously called group 3 bacillus). Conclusions: A bacterium that exhibited phenotypic and genotypic characteristics that are very different from closely related members of paenibacillus was the cause of pseudobacteraemia in a patient with neutropenic fever. A new species, Paenibacillus hongkongensis sp. nov. is proposed, for which HKU3 is the type strain.

Clostridium bacteraemia characterised by 16S ribosomal RNA gene sequencing

Background: Owing to problems in accurate species identification of the diverse genus clostridium, the epidemiology and pathogenicity of many species are not fully understood. Moreover, previous studies on clostridium bacteraemia have been limited and relied only on phenotypic species identification. Aims: To characterise the epidemiology, disease spectrum, and outcome of clostridium bacteraemia using 16S ribosomal RNA (rRNA) gene sequencing. Method: During a four year period (1998–2001), all cases of clostridium bacteraemia were prospectively studied and all “non-perfringens” clostridium isolates identified to the species level by 16S rRNA gene sequencing. Results: Fifty one blood culture isolates were identified as Clostridium perfringens and 17 belonged to 11 other clostridium species. The first case of C disporicum infection and two cases of clostridium bacteraemia in children with intussusception were also described. Of the 68 clostridium isolates from 68 different patients, 38 were associated with clinically relevant bacteraemia. The gastrointestinal and hepatobiliary tracts were common sites of both underlying disease and portal of entry in these patients. Clostridium perfringens accounted for 79% of all clinically relevant bacteraemia, with the remainder caused by a diversity of species. The attributable mortality rate of clinically relevant clostridium bacteraemia was 29%. Younger age and underlying gastrointestinal/hepatobiliary tract disease were associated with mortality (p < 0.05). Conclusions: Patients with clinically relevant clostridium bacteraemia should be investigated for the presence of underlying disease processes in the gastrointestinal or hepatobiliary tracts. 16S rRNA gene analysis will continue to be useful in further understanding the pathogenicity of various clostridium species.