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1 Diagnosing common and novel respiratory virus infections in the clinical setting 常见和新型呼吸道病毒感染的临床 / 病理诊断 Prof. Nelson Lee 李礼舜教授 MD (CUHK), FRCP (Lond), FRCP (Edin) FHKCP (ID), FHKAM (Med) 香港中文大学内科及药物治疗学系感染及传染病科主管威爾斯親王醫院及香港新界東聯網醫院名譽顧問醫生香港中文大學何鴻燊防治傳染病研究中心執行委員會委員 ( 臨床 ) 香港內科醫學院感染及傳染病科主席香港醫院管理局傳染病委員會委員香港衞生福利及食物局控制傳染病研究基金評審撥款委員會委員香港傳染病醫學會理事香港醫學雜誌編輯委員會委員

2 Lecture outline 课堂内容大纲 Why test for viruses? 为什么要做病毒测试? What assays should be considered? 应考虑使用什么检测方法? Collection of samples: techniques, site/types, time from illness onset 采集样本 : 需要的技术, 部位 / 类型, 发病时间 The importance of viral load, virus isolation, and resistance testing 病毒载量, 病毒培养和耐药性检测的重要性

3 F/23, obese Fever, dyspnoea, hemoptysis (A) CXR at presentation; 6 days postsymptom onset (B) CXR 3 days after admission; respiratory failure and ARDS requiring mechanical ventilation Treated with >10 days of oseltamivir. Survived and discharged. ICU/Death: Seasonal 8.4%, Pandemic 13.5% ECMO Lee N et al. J Inf Dis Lee N et al. Thorax Hui DS, Lee N, Chan PK. Chest 2010.

4 Hospitalized adults in Hong Kong, Singapore, Beijing (N=2649) H3N2 (>45.8%), H1N1pdm09 (36.3%), B (11.1%) Complications: respiratory and non-respiratory (68.4%) pneumonia, airway; cardio/cerebro-vascular; renal/metabolic Pneumonia (40.8%) secondary bacterial infection, 10.8% Respiratory failure / hypoxemia (48.6%) requiring assisted ventilation, 11.5% Overall: 30-day mortality (5.9% 7.4%) 60-day mortality (6.9% 8.8%) Eur Resp J, 2015

5 NAI treatment can reduce risk of death in hospitalized patients (-72%) 减少住院患者的死亡风险 (adjusted hazard ratio 0.28, 95% CI ) mortality (NAI vs nil) % vs % onset 2d = best survival 3-5d = some benefit [based on both conventional and time-dependent Cox proportional Hazards models, adjusted for patient characteristics and treatment propensity score] Eur Resp J, 2015

6 NAI: yes vs no (N=29,234) NAI: early vs late Adults Pregnant women ICU patients PRIDE Consortium Investigators. Lancet Resp Med 2014

7 *(confirmed flu cases) Lancet, Jan 2015

8 Respiratory Syncytial Virus (RSV) in Adults 2 nonstructural (NS1, NS2) and 9 structural proteins (e.g. F, G) Infects 3-10% of adults annually 5-15% of CAP 9-10% of hospital admissions for acute cardio-respiratory diseases excessive deaths outbreaks in nursing homes Globally, among elderly: 7.4% ILI 7.1% CAP 12.5% ILI-hospitalization Vaccine and antiviral (e.g. fusion inhibitor) will soon become available 疫苗和抗病毒药物不久将完成试验 Syncytial formation Lee N, Qureshi ST. Crit Care Clin 2013; Walsh EE, Falsey AR. J Infect Dis 2007 & 2014; Falsey AR. NEJM 2005

9 Lee and colleagues confirm age and chronic lung disease as major risk factors for severe RSV disease and provide useful information for future research targeting groups that would most benefit from vaccines and therapeutics. (N >600) 2013;57(8): 感染 RSV 的成年住院患者 : 高并发症率及死亡率

10 Comparisons between RSV and seasonal influenza : No significant difference in outcomes Variable RSV (N=607) Influenza (N=547) P-value Age, [mean (SD), yr] 75.1 (16.4) 74.7 (16.6) Major comorbidities (%) Chronic lung disease (%) <0.001 Symptom onset [mean (SD), d] 2.6 (2.2) 2.0 (1.7) <0.001 Fever > 37.5 C (%) <0.001 Wheezy breathing, SOB (%) <0.001 LRT complications (%) <0.001 肺炎 下呼吸道并发症 Pneumonia (%) 心血管并发症 Supplementary Oxygen (%) Ventilation (invasive/non-invasive) (%) d mortality (%) d mortality (%) Length-of-stay [median (IQR), d] 7 (5 14) 6 (5 11) 0.238

11 RSV-A strain novel ON1 genotype (72-nucleotide duplication, G gene) phylogenetic analysis RSV outbreak in 2013 N=123 (RSV-A 9; RSV-B 114) RSV-B strains BA genotypes 2015

12 Diagnosis of RSV cases during the 12 week seasonal peak (Hong Kong, 2013) 1. ground-glass opacities 2. consolidations, pathcy 3. hyperinflation Wong S, Lee N, et al. ARJ Patient characteristics, manifestations and complications (n=123) Variables No. (%) Patient characteristics Age, year (mean ±SD) 78.0 ±15.0 Male sex 64 (52.0) Nursing home resident 50 (40.7) Comorbidity, major 1 99 (80.5) Chronic statin user 1 32 (26.0) Aspirin/NSAID user 1 30 (24.4) Symptoms and complications Day of illness, at presentation, median (IQR) 2 (1-3) Fever 102 (82.9) Cough 108 (87.8) Shortness of breath 92 (74.8) Pleuritic chest pain 10 (8.1) WBC count (x10 9 /L, mean ±SD) 9.6 ±3.6 Neutrophil count (x10 9 /L, mean ±SD) 7.5 ±3.3 C-reaction protein (mg/l, mean ±SD) 59.8± 52.7 Lower respiratory-tract complications (89.4) pneumonia, clinico-radiological 83 (67.5) bronchitis/bronchiolitis 15 (12.2) exacerbation of chronic airway diseases 28 (22.8) Cardiovascular complications 2 30 (24.4) Bacterial superinfection 2, community-acquired 25 (20.3) hospital-acquired 17 (13.8)

13 Viruses are common causes of pneumonia worldwide, constituting 7-36% among all cases, and nearly half in those with established etiologies 病毒是全世界造成肺炎的常见原因, 占所有病例的 7-36%, 占病原学确诊的 50% incidence vary with seasonality, geographic location, and age group; e.g. seasonal influenza viruses and RSV typically cause pneumonia in elderly/infants, pandemic viruses affect mostly children and younger adults 发病率随季节 地理位置和年龄变化 如 : 季节性流感病毒和 RSV 通常造成老人和婴儿肺炎, 流行性病毒主要影响儿童和年轻人 isolation of a bacterial pathogen does not exclude a concomitant viral etiology, as secondary infection is not infrequent (12-40%); biologically, it aggravates inflammation and severity. 分离出病原菌不应该排除病毒感染, 因为继发感染并不少见 (12-40%); 它加重了炎症 Jain S, NEJM 2015; Karhu J, CID 2014; Bautista E, NEJM 2010; Harper SA, IDSA/CID 2009; Lee N, Antiv Ther 2012

14 Diagnosis commonly involves antigen tests, culture, serology and molecular assays, each with its own advantages and limitations 诊断通常包括抗原检测 培养 血清学和分子检测, 每项都有各自的优势和局限性 specimen type, site and time of collection with respect to illness onset, and the pretest probability (e.g. seasonal peak) may significantly affect test performance 关于疾病发生 预测疾病发生 ( 如季节性高峰 ) 的标本类型 采集地点和时间, 可能显著影响检测结果 Testing is generally recommended in hospitalized patients because specific antimicrobial treatments (e.g. neuraminidase inhibitors) are available, and diagnosis may assist effective implementation of isolation precautions 由于住院患者可进行抗菌治疗 ( 如神经氨酸酶抑制剂 ), 通常建议其做检查, 诊断能帮助隔离预防的有效实施 Clinical diagnosis is unreliable! 临床诊断是不可靠的! ( 发热 >37.8 C, 咳嗽伴 / 不伴咽喉疼痛 ): 灵敏度 21 43% 特异度 86 93% PPV 23 50% 与其它病毒 / 细菌无法区分 indistinguishable from other viruses/bacteria

15 Influenza testing Rapid Influenza Diagnostic Tests 快速流感诊断试验 (RIDT, antigen immunoassays 抗原免疫测定 ) 方便 可商品化 高度特异性 (90-95%); 主要缺点是灵敏度低 (40-80%) 因此阴性结果不能排除流感病毒感染 在发病后 48-72h 内 病毒载量显著下降之前, 应用将有更好的疗效 Immunofluorescence antigen assays 免疫荧光抗原检测 : 更敏感 (50-85%) 适用于上 下呼吸道标本 能够检测一系列呼吸道病毒 ( 如 RSV 副流感病毒病毒) if included in the panel 实验室技术和丰富的上皮细胞标本染色的质量是至关重要的

16 rapid antigen tests (RAT) or rapid influenza diagnostic tests (RIDT)

17 Hierarchical summary receiver-operating characteristic curve plot of rapid influenza diagnostic test RAT studies (n = 159). Individual studies are shown as open circles whose size is proportionate to the size of the study by American College of Physicians Chartrand C et al. Ann Intern Med doi: /

18 诊断 Diagnosis of Influenza / RSV Nasopharyngeal aspirate (NPA) Nasopharyngeal swab (NPS) Flocked swab (brush head) Immunofluorescence assay (IFA) 免疫荧光法 植绒拭子

19 Polymerase chain reaction (PCR) is now considered the test of choice 灵敏度和特异度高 检测的窗口期更长 ;TAT 短 所有甲型流感病毒的检测使用通用引物 (targeting the M- gene, same for influenza B), 或者个别亚型使用 HA- 特异性引物 (e.g. H1, H3, H5, H7) 可能有治疗意义 (e.g.pre-pandemic H1N1 was oseltamivirresistant) 暗示可能的新型或禽流感病毒株 (e.g. H1N1pdm09 and H7N9 were initially untypable using existing primers) 半定量 实时分析能监控病毒性应答 ( viral load ) CT 值用于备选评估指标 RNA 检测不能提供感染的依据!

20 Polymerase chain reaction (PCR) assay 流感性肺炎的鼻咽标本可能会出现上呼吸道结果 假阴性 (differential virokinetics changes along resp. tract) 下呼吸道样本额外检测 (e.g. TA, BAL), 因为普遍有更高的病毒载量 对已知突变株的快速直接的基因型耐药 genotypic resistance 检测是可行的 (e.g. H275Y in H1N1pdm09) 由于流感症状有别于其他感染, 多重 PCR 检测一系列常见病毒和非典型病原体正越来越多地用于临床 e.g. influenza, RSV, human metapneumovirus, parainfluenza virus, rhinovirus; M. pneumoniae, C. pneumoniae

21 Testing LRT sample in pneumonia case important Lee N, Antivir Ther 2011

22 Immunofluorescence test and Multiplex PCR for influenza and other respiratory viruses detection in the clinical setting (sample: NPA)

23 CDC US: FDA-cleared Molecular Assays for Influenza and Other Viruses Products esensor Respiratory Viral Panel (RVP) FilmArray Respiratory Panel Manufacturer (s) GenMark Diagnostics, Inc. BioFire Diagnostics, LLC Influenza Virus Type Detected Influenza A and B Influenza A and B Influenza Virus Subtype(s) Differentiated A/HI, A/H3, A/2009 H1 A/HI, A/H3, A/2009 H1 Other Respiratory Viruses Differentiated RSV-A, RSV-B, Parainfluenza 1,2,3 virus, hmpv, Adenovirus B/E/C, Rhinovirus RSV, Parainfluenza 1,2,3,4 virus, hmpv, Rhinovirus/ Enterovirus, Adenovirus, Coronavirus HKU1, Coronavirus NL63 Acceptable Specimens Nasopharyngeal swabs (in VTM 5 ) Nasopharyngeal swabs (in VTM 5 ) Test Time/ Complexity ~8 h/ High 1.0 h/ Moderate

24 Products Verigene Respiratory Pathogen Nucleic Acid Test (RP Flex) x-tag Respiratory Viral Panel (RVP) x-tag Respiratory Viral Panel Fast (RVP FAST) Manufacturer (s) Nanosphere, Inc Luminex Molecular Diagnostics Inc. Luminex Molecular Diagnostics Inc. Influenza Virus Type Detected Influenza A and B Influenza Virus Subtype(s) Differentiated A/H1 (inc. H1 and 2009 H1), and A/H3 Influenza A and B A/H1, A/H3 Influenza A and B A/H1, A/H3 Other Respiratory Viruses Differentiated RSV A/B, Parainfluenza 1,2,3,4 virus, hmpv, Adenovirus, Rhinovirus RSV-A/B, Parainfluenza 1,2, 3 virus, hmpv, Rhinovirus, Adenovirus RSV, hmpv, Rhinovirus, Adenovirus Acceptable Specimens Nasopharyngeal swabs (in VTM 5 ) Nasopharyngeal swabs (in VTM 5 ) Nasopharyngeal swabs (in VTM 5 ) Test Time/ Complexity 3.5 h/ Moderate ~8 h/ High ~6 h/ High

25 Point-of-Care tests (POCT) 基于分子的 POCT 检测目前可用于流感病毒和其他病毒的检测 [e.g. loop mediated isothermal nucleic acid amplification (LAMP), real-time PCR], 准确性可及传统的实验室 PCR 检测 进一步必要的研究在于它们对临床结果的影响和成本高效益 一般来说, 血清学和培养不能帮助病人的治疗, 因为它们为回顾性检测或回报结果慢, 但对监测和临床实验更有用 (e.g. strain identification, phenotypic resistance testing)

26 Testing Method Advantages Disadvantages Influenza Rapid Influenza Diagnostic Tests (RIDT) [immunoassay for antigens] 1 Immunofluorescence, Direct (DFA) or Indirect (IFA) Antibody Staining Viral cell culture [conventional or shell vial] Reverse-transcription polymerase chain reaction (RT-PCR) results available in <30 minutes applicable to a range of upper respiratory samples a may differentiate between influenza A and B high specificity (90-95%) results available within hours applicable to upper and lower respiratory samples may detect influenza A, B, and other viruses simultaneously (e.g. RSV) if included in the panel high specificity (90-95%) high specificity b allows virus subtyping, strain identification, titre assay/quantification, and resistance testing b provides information on infectiousness results available within hours c high sensitivity and specificity ( gold standard ) applicable to a wider range of specimen types able to distinguish virus subtypes (e.g. H1, H3, H5, H7), and detect genotypic resistance (e.g. H275Y) d multiplex PCR methods may allow simultaneous detection of other respiratory pathogens d low sensitivity (40-70%); performance affected by virus subtype/strain, sampling time, specimen type, and pre-test probability a unable to distinguish virus subtypes moderate sensitivity (50-85%) a quality specimen containing adequate epithelial cells (e.g. NP aspirate, flocked swab) a and laboratory expertise are essential unable to distinguish virus subtypes moderate sensitivity (7-20% lower than PCR) required optimal sampling (e.g. prior to antiviral use) and post-sampling processing conditions b results too slow to guide patient care (conventional, >3-10 days; shell-vial, >2-3 days) accessibility, technical demands and cost are the practical concerns c unable to distinguish non-viable from viable viruses false-negative upper tract samples ( 10%) may result from site differential viral kinetic changes in pneumonia cases

27 Culture vs PCR (n=550) (n=121) culture positivity and RNA level (Lee N. Clin Inf Dis 2013) RNA, culture, and symptoms H3, H1 Suess T. PLoS One 2012; Lau LL. JID 2010

28 RSV testing 由于抗原检测的敏感性低, 诊断困难 (20-60%, usually higher in children) 培养缓慢且技术上有挑战 更敏感的 PCR 成为一种选择 (singleplex, multiplex) esp. in adult cases where viral loads are generally lower 在 RSV 肺炎中, 包括痰标本在内的下呼吸道标本应考虑在内 Other viruses (e.g. parainfluenza, coronavirus, adenovirus) 越来越认识到其在 CAP 住院 ICU 的重要作用 广谱 / 病毒特异性药物的最新临床试验 e.g. nitazoxanide, favipiravir, DAS181 实验室谨慎评估通过培养和分子技术, 如 PCR NGS 等未明确诊断的肺炎 (e.g. SARS/MERS-coronavirus, H1N1pdm09, H7N9)

29 Testing Method Advantages Disadvantages RSV Rapid Diagnostic Tests and Immunofluorescence assays similar to influenza Viral cell culture similar to influenza Reverse-transcription polymerase chain reaction (RT-PCR) results available within hours c high sensitivity and specificity ( gold standard ) applicable to a wider range of specimen types e able to distinguish virus subtypes (RSV-A, RSV-B) multiplex PCR methods may allow simultaneous detection of other respiratory pathogens d low sensitivity (20-60% among adults) e performance affected by specimen type and quality, and pre-test probability low sensitivity technically demanding and required optimal sampling and post-sampling processing conditions (e.g. thermolability of RSV) results too slow to guide patient care accessibility, technical demands and cost are the practical concerns c unable to distinguish non-viable from viable viruses concomitant, paired serology testing may further improve diagnostic yield e Note: (a) Most RIDTs are applicable to nasopharygngeal (NP) swabs, nasal and/or throat swabs, and NP aspirates/washes. Performance is best if applied within hours before drop in viral load. (b) Culture detects viable viruses, including those contained in the live-attenuated influenza vaccines. Isolates can be subjected to phenotypic resistance assays (e.g. neuraminidase enzyme inhibition assay). Viral load, specimen quality, transport, storage, and processing techniques may affect test performance

30 Other respiratory viruses in Hong Kong Year (N=19,405): influenza A (39.4%), RSV (24.9%), influenza B (10.2%), parainfluenza virus (14.5%), adenovirus (10.0%) Chan PKS, Lee N. et al. MEDICINE 2015 Substantial morbidity and mortality with non-flu viruses!

31 Hospitalized patients > outpatients [ 1.5 log 10 copies/ml; P<0.01] Correlation with symptom scores and complications [Spearman s ρ, +0.22; P=0.01] Slow clearance in compromised host = longer window for treatment [β +0.77, P=0.032] Antiviral treatment improves viral clearance

32 Longer duration in the lower respiratory tract and lung Relapse possible if antiviral stopped prematurely Cited by WHO. NEJM 2010 H1N1pdm09 influenza pneumonia Viral replication continues beyond 5 days of treatment Lee N et al. Antiv Ther 2011

33 RSV viral load shown according to presence (grey box-plot) or absence (white box-plot) of underlying major comorbidities Lee N, et al. J Infect Dis th percentile 1. underlying comorbidities (7.36±1.40 vs 6.57±1.83 log 10 copies/ml, P=0.021) Median (IQR) RNA concentration, 7.66 ( ) log 10 copies/ml 2. bronchoconstriction and hypoxemia (7.53±1.36 vs 6.84±1.60 log 10 copies/ml, P=0.011) 3. death (7.79±1.34 vs 7.14±1.53 log 10 copies/ml, P=0.163) High viral load even beyond 2 days of illness window for antiviral treatment

34

35 (A/H1N1) H275Y mutant (A/H1N1) readily transmissible can cause severe diseases IC fold oseltamivir peramivir zanamivir Primary resistance: <3% Secondary resistance: children immunosuppressed ICU half-dose prophylaxis Dharan NJ, JAMA 2009; Gooskens J, JAMA 2009; Hurt AC et al. NEJM 2011, JID 2012, and Lancet ID 2014; Lee N, Viruses 2015

36 Antiviral resistance ( strain) 1-3% (H275Y)* (E119V, R292K) A/H7N9 100%? (R292K) Detection could be difficult due to a mixed of wild-type and resistant mutants * (have caused nosocomial and community outbreaks) Ison MG & Lee N, CCJM Storms AD, EID Whitley RJ, CID 2013

37 R292K mutation detected in 2 patients receiving corticosteroids and ECMO associated with viral rebound & clinical failure increases resistance to oseltamivir by 100-fold and zanamivir by 30-fold H7N9, Arg292Lys mutation, viral rebound ECMO continuous monitoring and surveillance are required MV Pneumonia no ECMO/MV

38 Li TC, Chan MC, Lee N. Viruses 2015

39 Note: Analyze samples collected before antiviral treatment and subsequent serial samples will determine whether the resistant strain (exists in whole or as a subpopulation) has caused the initial infection, or emerged de novo. There are phenotypic and genotypic assays for resistance testing, which are complementary. Li TC, Chan MC, Lee N. Viruses 2015

40 表型分析提供药敏结果 (e.g., IC50 and IC90 values), 但对 耐药 的定义还在研究中 可检测未知或罕见株的耐药 基因型分析能快速且更容易检测耐药, 如果突变已知且表型分析和临床耐药性的敏感度降低 (e.g., H275Y) 这种快速报告可以指导临床 焦磷酸测序能提供野生型和多重耐药突变的相对比例 能直接应用于临床标本 二代测序 (NGS; massively parallel sequencing) : 优于之前方 法的测序深度 能同时检测有低频率 (<1%) 耐药突变的病毒准物种

41 Use of biomarkers 生物标记, e.g. C-reactive protein, procalcitonin (PCT), proadrenomedullin (ProADM), myxoma resistance protein-1 (MxA), and combinations 能阐明病毒 / 细菌病原学 严重程度和需要的抗菌药治疗 PCT 是由细菌毒素和相关细胞因子调节反应所上调 (e.g. interleukin/il-1b, IL-6, TNF-α), 但是在病毒感染中减弱 (e.g. interferon-γ) 相较于细菌性和病毒细菌混合性肺炎, 流感性肺炎有更低的水平 阴性预测价值高 (>70-80%) 最佳的 cut-off? 重复测量的作用? 最近的一项试验表明, 使用 cut-off 值 < 0.25µg /ml,pct 和病毒测试可以减少下呼吸道感染的住院患者的抗生素使用

42 Use of biomarkers 宿主反应有希望辅助诊断 使用转录 ( 芯片 ) 分析 炎症和中性粒细胞基因的过度表达, 和干扰素基因, 分别被发现在细菌和病毒 ( 多数流感和 RSV) 呼吸道感染 它们比 PCT 的识别能力更优越 ( 敏感性和特异性 > 90%) 在另一项关于儿童的研究中,RSV 转录标志的特点是先天免疫过度和适应性免疫抑制, 有与其他病毒病因学不同的独特的模式 ; 提出了预后价值的基因组评分

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