【病毒外文文獻】2003 Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia_ a prospective

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For personal use Only reproduce with permission from The Lancet Publishing Group ARTICLES THE LANCET Vol 361 May 24 2003 1767 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 Lancet 2003 361 1767 72 Published online May 9 2003 Members listed at end of paper Departments of Microbiology and Medicine Queen Mary Hospital University of Hong Kong Hong Kong Special Administrative Region China Prof J S M Peiris DPhil V C C Cheng MRCP I F N Hung MRCP L L M Poon DPhil B S F Tang MB K H Chan PhD Y Guan PhD B J Zheng PhD Prof K Y Yuen MD Department of Medicine Intensive Care Radiology and Pathology United Christian Hospital Hong Kong C M Chu MRCP K S Chan FRCP K I Law MRCP T Y W Hon FRCP C S Chan FRCP J S C Ng MB W L Ng MRCP R W M Lai FRCPA Correspondence to Prof K Y Yuen Department of Microbiology University of Hong Kong Queen Mary Hospital Pokfulam Road Hong Kong Special Administrative Region China e mail kyyuen hkucc hku hk Introduction Severe acute respiratory syndrome SARS is a new emerging disease that has affected many countries with more than 3500 cases reported A novel virus the SARS associated coronavirus has been identified as the causal agent 1 4 The clinical radiological and other investigative findings at initial presentation have been previously described 5 7 but the temporal progression is unclear The routes and duration of viral shedding and the best clinical samples for diagnosis at different stages of the illness are also largely unknown From March 24 2003 a major outbreak of SARS involving 321 patients occurred in Amoy Gardens a high rise housing estate in Hong Kong Epidemiological investigations suggested a point source outbreak which was linked to a faulty sewage system initially contaminated by the excreta of the index case who visited the housing block on March 14 and 19 8 The first 75 adult patients belonging to 57 households were admitted with a clinical diagnosis of SARS to one hospital We did a prospective study on the clinical haematological radiological and microbiological findings of these 75 patients over a period of 24 days and correlated these findings with treatment 9 10 and with viral load in the nasopharyngeal aspirate to elucidate the pathogenesis and the impact of treatment We report the usefulness of different clinical samples for virological diagnosis and its importance in transmission Patients and methods Patients Between March 24 and 28 2003 we included 75 patients admitted to the United Christian Hospital from the Amoy Gardens housing estate who fulfilled the modified WHO definition of SARS 3 Briefly the case definition is fever 38 C or higher cough or shortness of breath new pulmonary infiltrates on chest radiography or high resolution CT in the absence of an alternative diagnosis to explain the clinical presentation Patients were nursed in an isolation ward with other SARS patients Methods The study was approved by the ethics committee of the United Christian Hospital Hong Kong We entered on a predesigned database the daily clinical findings based on history and physical examination oximetric measure ment and haematological biochemical radiological and microbiological investigations We collected nasopharyngeal aspirates and clotted blood for virological studies from all patients at presentation and on day 14 after the onset of illnesses Anteroposterior chest radiography was done daily for each patient All chest radiographs were jointly reported by specialist radiologists and respiratory physicians We did high resolution CT of the thorax for patients who had apparently normal equivocal or atypical chest radiographs For patients who developed acute respiratory distress syndrome ARDS with partial arterial pressure of oxygen PaO 2 to fraction of inspired oxygen FiO 2 less than 26 6 kPa 11 and recorded acute physiology and chronic health evaluation II APACHE II scores 12 Clinical progression and viral load in a community outbreak of coronavirus associated SARS pneumonia a prospective study J S M Peiris C M Chu V C C Cheng K S Chan I F N Hung L L M Poon K I Law B S F Tang T Y W Hon C S Chan K H Chan J S C Ng B J Zheng W L Ng R W M Lai Y Guan K Y Yuen and members of the HKU UCH SARS Study Group For personal use Only reproduce with permission from The Lancet Publishing Group From 20 patients in whom SARS associated coronavirus RNA was detected by reverse transcriptase RT PCR in the initial nasopharyngeal aspirate samples we collected further nasopharyngeal aspirates urine and faecal samples every 3 days In 14 of these patients quantitative PCR was done on the nasopharyngeal aspirates collected on days 5 10 and 15 after onset of symptoms The nasopharyngeal aspirates on admission were assessed by rapid immunofluorescent antigen detection for influenza A and B parainfluenza types 1 2 and 3 respiratory syncytial virus and adenovirus and were cultured for conventional respiratory pathogens on Mardin Darby Canine Kidney LLC Mk2 RDE Hep 2 MRC 5 and fetal rhesus kidney FRhK 4 cell lines RT PCR for SARS coronavirus was done directly on all clinical samples Briefly total RNA from clinical samples was reverse transcribed with random hexamers and cDNA was amplified with primers 5H11032TACACACCTCAGCGTTG 3H11032 and 5H11032 CACGAAC GTGACGAAT 3H11032 3 For real time quantitative PCR assays cDNA was amplified in an SYBR Green I fluorescence reactions Roche Mannheim Germany as described 13 Briefly 20 H9262L reaction mixtures containing 2 H9262L cDNA 3 5 mmol L magnesium chloride and 0 25 H9262mol L of the same forward and reversed primers as the reaction mixtures were thermal cycled by a Light cycler Roche Mannheim 95 C 10 min followed by 50 cycles of 95 C 10 min 57 C 5 s 72 C 9 s Plasmids with the target sequence were used to generate the standard curve At the end of the assay PCR products 182 bp were subjected to a melting curve analysis 65 95 C 0 1 C s to find out the specificity of the assay The acute and convalescent sera were tested in parallel for SARS associated coronavirus IgG with SARS associated coronavirus infected Vero cells fixed in acetone in an indirect immunofluorescent format ARTICLES 1768 THE LANCET Vol 361 May 24 2003 Temperature average over 3 days Onset of diarrhoea Onset of desaturation ARDS requiring intubation Temperature C 36 0 36 5 37 5 37 0 38 0 38 5 39 0 39 5 40 0 Number of new patients 30 20 25 15 10 5 0 1 3 4 6 7 9 10 12 13 15 19 21 16 18 22 24 Time after onset of symptoms days Figure 1 Temporal clinical profiles in 75 patients with SARS Mean SD are presented Figure 2 Chest radiographs and high resolution CT scans from two SARS patients A Man aged 34 years admitted for high fever and cough A Consolidation seen in left upper and middle zones which progressed maximally at day 7 B At day 20 resolution of consolidation in the left upper and middle zones but new widespread air space opacities noted those in left lung base were confluent Man aged 32 years presented with fever chills rigors and myalgia with clear chest radiograph at admission C High resolution CT of thorax shows peripheral subpleural consolidation in medial basal segment of left lower lobe D Resolution of original left lower lobe consolidation at day 18 E Disease complicated by spontaneous pneumomediastinum For personal use Only reproduce with permission from The Lancet Publishing Group We investigated blood sputum or endotracheal aspirates and urine bacteriologically as clinically indicated All patients had sepsis work up on admission and if fever recurred Stools were sent for routine bacteriological culture Clostridium difficile cytotoxin assay and examined for parasites in selected patients All patients were treated with 1 2 g intravenous amoxicillin clavulanate every 8 h and 500 mg oral azithromycin daily In patients with a known penicillin allergy we administered 500 mg oral levofloxacin every 24 h As soon as the diagnosis of SARS was established 8 mg kg intravenous ribavirin every 8 h for 14 days and a tailing regimen of hydrocortisone starting dose 200 mg intravenously every 8 h over 10 days followed by oral prednisolone for 11 days 1 mg kg for 5 days 0 5 mg kg for 3 days and 0 25 mg kg for 3 days were given We used pulses of methylprednisolone 500 mg intravenously daily for two or three doses if patients worsened with increasing shortness of breath oxygen desaturation and radiological worsening All HBsAg positive patients were given 100 mg oral lamivudine daily while taking corticosteroids Statistical analysis We compared risk factors associated with the development of ARDS by Fisher s exact test for categorical variables and Student s t test for continuous variables Significant risk factors identified on univariate analyses were further analysed by multiple logistic regressions to identify independent risk factors associated with the development of ARDS We took p 0 05 to be significant We used SPSS version 11 0 for all analyses Role of the funding source The sponsors of the study had no role in the study design data collection data analysis data interpretation or in the writing of the report Results Of the 75 patients 71 95 were ethnic Chinese and the remainder were Filipino The male to female ratio was one to 0 92 and mean age was 39 8 SD 12 2 years Five patients were smokers Underlying diseases were identified in 13 patients including nine patients positive for HBsAg with no stigmata of chronic liver disease and normal liver function tests The clinical symptoms on admission included fever in 75 100 patients chills in 49 65 rigors in 42 56 myalgia in 51 68 cough in 22 29 sore throat in eight 11 shortness of breath in three 4 headache in 11 15 dizziness in three 4 and diarrhoea in one 1 Initial chest radiograph was abnormal in 53 71 patients involvement was confined to one lung zone in 37 49 and was multizonal in 16 21 Lower zone infiltrates or consolidation occurred in 45 60 patients Initial high resolution CT was done in 33 44 patients in whom the initial chest radiographs were normal equivocal or atypical Of these 33 patients 18 55 had abnormalities confined to one lobe Multilobar involvement was seen in 15 46 patients Focal ground glass opacification was the only type of abnormality in eight 24 patients 12 36 patients had consolidation only and 13 39 had both types of infiltrates On haematological and biochemical investigation anaemia was detected in six 8 leucopenia in five 7 severe lymphopenia of less than 1H1100310 9 L in 56 75 and thrombocytopenia in 28 37 Raised values were seen for alanine aminotransferase 42 343 U L aspartate aminotransferase 39 302 U L and creatinine kinase 176 1466 U L in 22 29 24 32 and 27 36 patients respectively The median lymphocyte counts decreased from 0 8H1100310 9 L IQR 0 6 1 1 on admission to 0 6H1100310 9 L 0 5 0 9 0 4H1100310 9 L 0 3 0 5 and 0 4H1100310 9 L 0 3 0 6 at days 7 14 and 21 respectively In terms of disease progression all except one patient became afebrile within 48 h with the standard treatment protocol but the fever recurred in 64 85 patients at a mean of 8 9 days SD 3 1 Only ten of these patients had positive findings on sepsis work up three had Stenotrophomonas maltophilia bacteraemia four had clinical evidence of catheter related sepsis and three had nosocomial pneumonia due to Klebsiella pneumoniae in two and Escherichia coli one All these septic episodes responded to the appropriate antimicrobial treatment and removal of infected catheters In the remaining 54 patients fever recurred after a mean of 8 0 2 1 days which was unlikely to be caused by hospital acquired infection Between days 13 and 15 17 23 patients developed another episode of fever figure 1 Watery diarrhoea developed in 55 73 patients with onset at a mean of 7 5 2 3 days The rate of diarrhoea peaked at a mean of 8 7 2 3 days with a maximum frequency of 6 3 3 5 times daily figure 1 Work up for diarrhoeal pathogens including C difficile cytotoxin was negative in all these patients The mean duration of diarrhoea was 3 9 2 3 days Simultaneous occurrence of fever and diarrhoea occurred in 24 32 patients Concomitant fever diarrhoea and radiological worsening occurred in 16 21 patients Improvement of diarrhoea occurred in all patients by day 13 Radiological worsening was noted in 60 80 patients at a mean of 7 4 2 2 days 34 45 developed shifting of radiological lesions evidenced by improvement of original lesions followed by the appearance of new lesions figure 2 worsening of original lesions with or without ARTICLES THE LANCET Vol 361 May 24 2003 1769 ARDS No ARDS p n 15 n 60 Mean SD age years 48 5 12 6 37 3 11 3 0 002 Male female ratio 11 4 25 35 0 042 Underlying illnesses 9 60 4 7 0 001 Chronic hepatitis B virus infection 6 40 3 5 0 001 Mean SD duration of symptoms 2 6 1 1 2 38 1 2 0 51 to admission days Mean SD initial haemoglobin 135 21 134 12 0 79 concentration g L Mean SD initial total peripheral 7 1 2 1 6 1 2 1 0 09 white blood cell count H1100310 9 L Mean SD initial lymphocyte 1 1 0 9 0 9 0 4 0 19 count H1100310 9 L Mean SD initial platelet 163 56 167 41 0 77 count H1100310 9 L Mean SD initial creatinine 94 5 11 1 86 1 14 6 0 004 H9262mol L Mean SD initial ALT U L 47 8 27 5 35 9 45 2 0 33 Mean SD initial CPK 327 1 367 7 161 5 125 9 0 11 Mean SD initial LDH 482 6 242 9 384 8 119 8 0 25 NPA RT PCR positive at diagnosis 5 33 19 31 7 0 77 Mean SD day of antibody 20 0 5 5 19 9 4 9 0 94 seroconversion after onset of symptoms Apparently normal chest 4 27 18 30 1 0 radiograph on admission Multilobar involvement on chest 5 33 11 18 0 29 radiograph on admission Diarrhoea 12 80 43 72 0 75 Recurrent fever 13 87 41 68 0 21 ALT alanine aminotransferase CPK creatinine phosphokinase LDH lactic dehydrogenase NPA nasopharyngeal aspirate Three patients had chronic active hepatitis B one had uterine fibroid SARS established by clinical features plus high resolution CT findings Six patients had chronic active hepatitis B infection one had carcinoma of ovary one had diabetes mellitus and one had asthma Table 1 Risk factors associated with development of ARDS requiring ventilatory support and intensive care For personal use Only reproduce with permission from The Lancet Publishing Group development of new lesions was noted in the remaining 26 35 patients Overall 46 61 patients subsequently improved 11 15 had remained static at the time of writing and 18 24 further progressed into a diffuse ground glass appearance at a mean of 12 0 4 4 days Of the 18 patients with diffuse ground glass changes 15 developed ARDS Among all 75 patients nine 12 developed spontaneous pneumomediastinum during follow up The characteristic shifting of radiological changes is illustrated in figure 2 33 44 patients developed arterial oxygen desaturation of less than 90 at room air at a mean of 9 1 4 2 days after onset of symptoms figure 1 24 32 patients required intensive care at a mean of 11 0 6 4 days among whom 19 had to be intubated at a mean of 12 9 6 4 days 15 20 patients progressed to ARDS and required mechanical ventilation On day 1 of mechanical ventilation for ARDS the mean PaO 2 to FiO 2 ratio was 14 7 kPa 10 8 and the mean APACHE II score was 22 3 5 8 Time to occurrence of ARDS showed a bimodal pattern with one peak at 11 0 days and another peak at 20 0 days figure 1 Seven patients developed hospital acquired infection during their stay in the intensive care unit including hospital acquired pneumonia in three meticillin resistant Staphylococcus epidermidis bacteraemia in one and clinical sepsis in three without positive cultures On univariate analysis the risk factors associated with ARDS requiring ventilatory support and intensive care were age male sex chronic hepatitis B virus carriage raised creatinine and recurrence of fever table 1 Lymphocyte counts of ARDS patients on days 7 14 and 21 did not differ significantly from those in non ARDS patients On multivariate analysis only age and chronic hepatitis B virus infection were significant risk factors table 2 The mean length of stay for 75 patients was 22 1 3 1 days whereas for the 15 patients who developed ARDS the mean length of stay was 26 8 days at the time of writing Five patients died during the study period of whom two aged 79 and 64 years had succumbed to acute myocardial infarction on days 13 and 17 respectively One patient died of clinical sepsis on day 23 Another two patients died of clinical sepsis and ARDS on days 24 and 25 respectively 27 36 of patients were discharged home or transferred to a rehabilitation facility One patient was transferred to a specialised obstetric unit for urgent delivery of a baby at 32 weeks gestation For the 42 patients who remained in the hospital 13 were treated in intensive care for ARDS table 3 IgG seroconversion was documented in 70 93 patients at mean of 20 5 1 days figure 3 SARS associated coronavirus RNA was detected in nasopharyngeal aspirates by RT PCR in 24 32 of 75 patients at initial presentation mean 3 2 1 3 days after onset and in 51 68 at day 14 In stool samples collected later in the illness a mean of 14 2 2 2 days after onset viral RNA was detected in 65 97 of 67 Similarly viral RNA was detected in 31 42 of 74 urine samples collected at a mean of 15 2 1 7 days after onset of symptoms The 20 patients initially documented to have SARS associated coronavirus RNA in the nasopharyngeal aspirates on RT PCR were serially followed up with sequential samples the virological profile is shown in table 4 Quantitative RT PCR of the nasopharyngeal aspirates showed an inverted V pattern with mean geometric viral loads of 2 3H1100310 5 copies per mL and 1 9H1100310 7 copies per mL and 9 8H1100310 4 copies per mL on days 5 10 and 15 respectively after onset of symptoms figure 4 Discussion Studies on SARS have generally been retrospective or limited to the description of the initial clinical haematological radiological and microbiological findings The patients we studied however were residents of a housing estate placed under closed surveillance by the Department of Health soon after identification of the outbreak All residents underwent frequent health checks and symptomatic patients were admitted to hospital soon ARTICLES 1770 THE LANCET Vol 361 May 24 2003 Cumulative proportion of patients with seroconversion 0 02468 10 20 30 50 40 60 70 80 90 100 3028262422201816141210 Time after onset of symptoms days n 75