Kh Chan

Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study

Summary Background We investigated the temporal progression of the clinical, radiological, and virological changes in a community outbreak of severe acute respiratory syndrome (SARS). Methods We followed up 75 patients for 3 weeks managed with a standard treatment protocol of ribavirin and corticosteroids, and assessed the pattern of clinical disease, viral load, risk factors for poor clinical outcome, and the usefulness of virological diagnostic methods. Findings Fever and pneumonia initially improved but 64 (85%) patients developed recurrent fever after a mean of 8.9 (SD 3.1) days, 55 (73%) had watery diarrhoea after 7.5 (2.3) days, 60 (80%) had radiological worsening after 7.4 (2.2) days, and respiratory symptoms worsened in 34 (45%) after 8.6 (3.0) days. In 34 (45%) patients, improvement of initial pulmonary lesions was associated with appearance of new radiological lesions at other sites. Nine (12%) patients developed spontaneous pneumomediastinum and 15 (20%) developed acute respiratory distress syndrome (ARDS) in week 3. Quantitative reverse-transcriptase (RT) PCR of nasopharyngeal aspirates in 14 patients (four with ARDS) showed peak viral load at day 10, and at day 15 a load lower than at admission. Age and chronic hepatitis B virus infection treated with lamivudine were independent significant risk factors for progression to ARDS (p=0.001). SARS-associated coronavirus in faeces was seen on RT-PCR in 65 (97%) of 67 patients at day 14. The mean time to seroconversion was 20 days. Interpretation The consistent clinical progression, shifting radiological infiltrates, and an inverted V viral-load profile suggest that worsening in week 2 is unrelated to uncontrolled viral replication but may be related to immunopathological damage. Published online May 9, 2003 http://image.thelancet.com/extras/03art4432web.pdf

Human infection with influenza H9N2

We report the clinical features of two cases of human infection with influenza A virus subtype H9N2 in Hong Kong, and show that serum samples from blood donors in Hong Kong had neutralising antibody suggestive of prior infection with influenza H9N2.

Re-emergence of fatal human influenza A subtype H5N1 disease

Summary Human disease associated with influenza A subtype H5N1 reemerged in January, 2003, for the first time since an outbreak in Hong Kong in 1997. Patients with H5N1 disease had unusually high serum concentrations of chemokines (eg, interferon induced protein-10 [IP-10] and monokine induced by interferon γ [MIG]). Taken together with a previous report that H5N1 influenza viruses induce large amounts of proinflam-matory cytokines from macrophage cultures in vitro, our findings suggest that cytokine dysfunction contributes to the pathogenesis of H5N1 disease. Development of vaccines against influenza A (H5N1) virus should be made a priority.

Children with Respiratory Disease Associated with Metapneumovirus in Hong Kong

Human metapneumovirus (HMPV) is a newly discovered pathogen thought to be associated with respiratory disease. We report the results of a study of 587 children hospitalized with respiratory infection over a 13-month period. HMPV was detected in the nasopharyngeal aspirates from 32 (5.5%) children by reverse transcription-polymerase chain reaction. HMPV infection was associated with clinical diagnoses of pneumonia (36%), asthma exacerbation (23%), or acute bronchiolitis (10%). When compared to those with respiratory syncytial virus infection, children with HMPV infection were older, and wheezing was more likely to represent asthma exacerbation rather than acute bronchiolitis. HMPV viral activity peaked during the spring-summer period in Hong Kong. Phylogenetically, all HMPV virus strains from Hong Kong belonged to one of the two genetic lineages previously described. HMPV contributed to 441.6 hospital admissions per 100,000 population <6 years of age.

Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003

Summary Background An epidemic of severe acute respiratory syndrome (SARS) has been associated with an outbreak of atypical pneumonia originating in Guangdong Province, Peoples Republic of China. We aimed to identify the causative agent in the Guangdong outbreak and describe the emergence and spread of the disease within the province. Methods We analysed epidemiological information and collected serum and nasopharyngeal aspirates from patients with SARS in Guangdong in mid-February, 2003. We did virus isolation, serological tests, and molecular assays to identify the causative agent. Findings SARS had been circulating in other cities of Guangdong Province for about 2 months before causing a major outbreak in Guangzhou, the provinces capital. A novel coronavirus, SARS coronavirus (CoV), was isolated from specimens from three patients with SARS. Viral antigens were also directly detected in nasopharyngeal aspirates from these patients. 48 of 55 (87%) patients had antibodies to SARS CoV in their convalescent sera. Genetic analysis showed that the SARS CoV isolates from Guangzhou shared the same origin with those in other countries, and had a phylogenetic pathway that matched the spread of SARS to the other parts of the world. Interpretation SARS CoV is the infectious agent responsible for the epidemic outbreak of SARS in Guangdong. The virus isolated from patients in Guangdong is the prototype of the SARS CoV in other regions and countries.

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.

Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings

Background: The clinical response of patients with severe acute respiratory syndrome (SARS) to a combination of lopinavir/ritonavir and ribavirin was examined after establishing the in vitro antiviral susceptibility of the SARS associated coronavirus to a panel of antiviral agents. Methods: The in vitro susceptibility of the prototype of SARS associated coronavirus to a panel of nucleoside analogues and protease inhibitors currently licensed for clinical use was studied. Forty one patients with SARS followed for 3 weeks were treated with a combination of lopinavir/ritonavir and ribavirin. The clinical progress and virological outcomes were monitored and compared with 111 patients treated with ribavirin only who served as historical controls. Results: In vitro antiviral activity against SARS associated coronavirus was demonstrated for lopinavir and ribavirin at concentrations of 4 µg/ml and 50 µg/ml, respectively, only at 48 hours. The adverse clinical outcome (ARDS or death) was significantly lower in the treatment group than in the historical controls (2.4% v 28.8%, p<0.001) at day 21 after the onset of symptoms. The adverse outcome remained significantly lower in the treatment group than in the controls—both those diagnosed early (p<0.001) and those diagnosed later in the course of the epidemic (p = 0.002)—but there was no significant difference in adverse outcome rates between the two time periods (p = 0.548). No time related difference in outcome was observed in the control groups. A reduction in steroid usage and nosocomial infections was seen in patients initially treated with lopinavir/ritonavir, and these patients had a decreasing viral load and rising peripheral lymphocyte count. Multivariate analysis showed that age, hepatitis B carrier status, and lack of treatment with this antiviral combination were independent predictors of an adverse outcome. Lopinavir/ritonavir treatment was associated with a better outcome even when adjusted for baseline lactate dehydrogenase level. Conclusions: The apparent favourable clinical response with lopinavir/ritonavir and ribavirin supports further randomised placebo controlled trials in patients with SARS.

Molecular Detection of a Novel Human Influenza (H1N1) of Pandemic Potential by Conventional and Real-Time Quantitative RT-PCR Assays

Abstract Background: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed. Methods: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses. Results: All of the assays had detection limits for the positive control in the range of 1.0 × 10−4 to 2.0 × 10−3 of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses. Conclusions: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children

BACKGROUND The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.

Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus

ABSTRACT The reverse transcription (RT)-PCR protocols of two World Health Organization (WHO) severe acute respiratory syndrome (SARS) network laboratories (WHO SARS network laboratories at The University of Hong Kong [WHO-HKU] and at the Bernhard-Nocht Institute in Hamburg, Germany [WHO-Hamburg]) were evaluated for rapid diagnosis of a novel coronavirus (CoV) associated with SARS in Hong Kong. A total of 303 clinical specimens were collected from 163 patients suspected to have SARS. The end point of both WHO-HKU and WHO-Hamburg RT-PCR assays was determined to be 0.1 50% tissue culture infective dose. Using seroconversion to CoV as the “gold standard” for SARS CoV diagnosis, WHO-HKU and WHO-Hamburg RT-PCR assays exhibited diagnostic sensitivities of 61 and 68% (nasopharyngeal aspirate specimens), 65 and 72% (throat swab specimens), 50 and 54% (urine specimens), and 58 and 63% (stool specimens), respectively, with an overall specificity of 100%. For patients confirmed to have SARS CoV and from whom two or more respiratory specimens were collected, testing the second specimen increased the sensitivity from 64 and 71% to 75 and 79% for the WHO-HKU and WHO-Hamburg RT-PCR assays, respectively. Testing more than one respiratory specimen will maximize the sensitivity of PCR assays for SARS CoV.