Clinical and laboratory findings of SARS in Singapore.

INTRODUCTION: Singapore was one of 29 countries worldwide affected by severe acute respiratory syndrome (SARS) in 2003. MATERIALS AND METHODS: There were 238 cases identified during the outbreak. We performed a retrospective analysis of the clinical and laboratory data of 234 patients admitted to Tan Tock Seng Hospital and Singapore General Hospital. RESULTS: The mean age of patients was 21 years, 31.6% of patients were males and 41.8% were healthcare workers. At presentation, the common symptoms were fever, myalgia, cough and headache; rhinorrhoea was uncommon. On admission, 21% had leukopenia, 18% had thrombocytopaenia, 29% had hyponatraemia, 31% had hypokalaemia, 21% had transaminitis. Polymerase chain reaction (PCR) testing of respiratory and stool samples provided the best yield at the end of the first week of illness. Thirty-two patients were initially not recognised as probable SARS and were reclassified when the serology test results were available. The chief reasons for not identifying these patients early were persistently normal chest X-rays (68.8%), very mild presentation (43.8%) and the presence of a concomitant illness (12.5%). Overall, 12% of the patients were probable SARS with atypical presentations. Overall mortality was 11.8%. CONCLUSION: Patients infected with the SARS coronavirus had a wide clinical presentation with non-specific symptoms.

Influenza-associated deaths in tropical Singapore.

We used a regression model to examine the impact of influenza on death rates in tropical Singapore for the period 1996-2003. Influenza A (H3N2) was the predominant circulating influenza virus subtype, with consistently significant and robust effect on mortality rates. Influenza was associated with an annual death rate from all causes, from underlying pneumonia and influenza, and from underlying circulatory and respiratory conditions of 14.8 (95% confidence interval 9.8-19.8), 2.9 (1.0-5.0), and 11.9 (8.3-15.7) per 100,000 person-years, respectively. These results are comparable with observations in the United States and subtropical Hong Kong. An estimated 6.5% of underlying pneumonia and influenza deaths were attributable to influenza. The proportion of influenza-associated deaths was 11.3 times higher in persons age >65 years than in the general population. Our findings support the need for influenza surveillance and annual influenza vaccination for at-risk populations in tropical countries.

Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus.

A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99 x 10(2) TCID(50)/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100.

SARS transmission pattern in Singapore reassessed by viral sequence variation analysis.

BACKGROUND: Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS)-based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS) outbreak in Singapore. METHODS AND FINDINGS: The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV) sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host. CONCLUSION: This study has further demonstrated the importance of improving clinical and epidemiological studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order to allow the most effective control.

Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus.

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.

Virus-specific RNA and antibody from convalescent-phase SARS patients discharged from hospital.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). In a longitudinal cross-sectional study, we determined the prevalence of virus in bodily excretions and time of seroconversion in discharged patients with SARS. Conjunctival, throat, stool, and urine specimens were collected weekly from 64 patients and tested for SARS-CoV RNA by real-time polymerase chain reaction; serum samples were collected weekly and tested for SARS-CoV antibody with indirect enzyme immunoassay and immunofluorescence assay. In total, 126 conjunctival, 124 throat swab, 116 stool, and 124 urine specimens were analyzed. Five patients had positive stool samples, collected in weeks 5-9. Two patients seroconverted in weeks 7 and 8; the others were seropositive at the first serum sample collection. In this study, 5 (7.8%) of 64 patients continued to shed viral RNA in stool samples only, for up to week 8 of illness. Most seroconversions occurred by week 6 of illness.

Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003.

BACKGROUND: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. METHODS: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. RESULTS: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 x 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. CONCLUSIONS: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3'--most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

Inhibition of SARS coronavirus infection in vitro with clinically approved antiviral drugs.

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a newly identified human coronavirus (SARS-CoV). Currently, no effective drug exists to treat SARS-CoV infection. In this study, we investigated whether a panel of commercially available antiviral drugs exhibit in vitro anti-SARS-CoV activity. A drug-screening assay that scores for virus-induced cytopathic effects on cultured cells was used. Tested were 19 clinically approved compounds from several major antiviral pharmacologic classes: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors, and neuraminidase inhibitors. Complete inhibition of cytopathic effects of SARS-CoV in culture was observed for interferon subtypes, b-1b, a-n1, a-n3, and human leukocyte interferon a. These findings support clinical testing of approved interferons for the treatment of SARS.

Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus.

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.

Atypical SARS and Escherichia coli bacteremia.

We describe a patient with severe acute respiratory syndrome (SARS) whose clinical symptoms were masked by Escherichia coli bacteremia. SARS developed in a cluster of healthcare workers who had contact with this patient. SARS was diagnosed when a chest infiltrate developed and when the patient's brother was hospitalized with acute respiratory failure. We highlight problems in atypical cases and offer infection control suggestions.

Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers.

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.

Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.

Mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in Singapore.

BACKGROUND: Severe acute respiratory syndrome (SARS) is a newly recognized infectious disease that has recently emerged in East Asia and North America. Although the clinical features of acute infection have been well described, mildly symptomatic or asymptomatic infections have not been well characterized. OBJECTIVE: To assess the spectrum of illness in health-care workers (HCWs). METHODS: A prospective seroepidemiologic cohort study was conducted on 372 HCWs in a large teaching hospital in Singapore who were both exposed and not exposed to patients with SARS. Participating HCWs completed a questionnaire and provided paired serum samples, which were analyzed by 2 different laboratories blinded to clinical data, by use of an enzyme-linked immunosorbent assay based on a protocol developed by the Centers for Disease Control and Prevention and a dot-blot immunoassay, with confirmation by a viral neutralization assay. RESULTS: A total of 21 patients with SARS were treated at our hospital. They were associated with transmission to 14 staff members, patients, and visitors in our hospital. Of the 372 HCWs participating in the present study, 8 were found to have positive antibodies to the SARS coronavirus in both samples by use of both test methods, and 6 had pneumonia and had been hospitalized for either probable or suspected SARS infection, whereas 2 had fever but did not have changes on chest radiographs. All seropositive HCWs had been exposed either directly or indirectly to patients with SARS. No asymptomatic, nonexposed staff members were found to be seropositive. There was a trend towards protection for HCWs who, while fully protected, had had contact with patients with SARS. CONCLUSIONS: Although the majority of cases of SARS are associated with pneumonia, a small number of mildly symptomatic individuals do seroconvert. HCWs who are exposed to patients with SARS can be infected with SARS, regardless of the intensity of exposure. This has implications for surveillance and infection control planning, in the event that SARS returns next winter.

Detection of severe acute respiratory syndrome coronavirus in blood of infected patients.

Severe acute respiratory syndrome (SARS) has caused major outbreaks worldwide, resulting in an urgent need to obtain sensitive and accurate diagnosis of this disease. PCR-based detection methods were developed for use on a variety of samples, including blood. Eighteen subjects were investigated, and results indicated that blood samples contain sufficient virus for detection by using quantitative real-time PCR.

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.

BACKGROUND: The worldwide outbreak of severe acute respiratory syndrome (SARS) is associated with a newly discovered coronavirus, SARS-associated coronavirus (SARS-CoV). We did clinical and experimental studies to assess the role of this virus in the cause of SARS. METHODS: We tested clinical and postmortem samples from 436 SARS patients in six countries for infection with SARS-CoV, human metapneumovirus, and other respiratory pathogens. We infected four cynomolgus macaques (Macaca fascicularis) with SARS-CoV in an attempt to replicate SARS and did necropsies on day 6 after infection. FINDINGS: SARS-CoV infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of SARS; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. SARS-CoV was, therefore, the most likely causal agent of SARS. The four SARS-CoV-infected macaques excreted SARS-CoV from nose, mouth, and pharynx from 2 days after infection. Three of four macaques developed diffuse alveolar damage, similar to that in SARS patients, and characterised by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. SARS-CoV was detected in pneumonic areas by virus isolation and RT-PCR, and was localised to alveolar epithelial cells and syncytia by immunohistochemistry and transmission electron microscopy. INTERPRETATION: Replication in SARS-CoV-infected macaques of pneumonia similar to that in human beings with SARS, combined with the high prevalence of SARS-CoV infection in SARS patients, fulfill the criteria required to prove that SARS-CoV is the primary cause of SARS.

A novel coronavirus associated with severe acute respiratory syndrome.

BACKGROUND: A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. METHODS: We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. RESULTS: None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. CONCLUSIONS: A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.

Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore.

Singapore experienced a large epidemic of hand, foot and mouth disease (HFMD) in 2000. After reviewing HFMD notifications from doctors and child-care centers, we found that the incidence of HFMD rose in September and declined at the end of October. During this period, 3,790 cases were reported. We performed enteroviral cultures on 311 and 157 specimens from 175 HFMD patients and 107 non-HFMD patients, respectively; human enterovirus 71 (HEV71) was the most frequently isolated virus from both groups. Most of the HFMD patients were