Persistent viral infectivity after 27 days from COVID-19 symptoms onset
Persistent viral infectivity after 27 days from COVID-19 symptoms onset
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COVID-19 症状出现 27 天后病毒感染持续存在
DOI：
10.1136/jclinpath-2021-207394
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发表时间：
2021
影响因子：
3.4
通讯作者：
S. Crovella
中科院分区：
文献类型：
--
作者：
L. Zupin;F. Fontana;Libera Clemente;Pierino Boschian;M. Ruscio;S. Crovella
在新冠肺炎感染患者的咽拭子中持续携带SARSCoV-2并不少见。事实上，通过实时逆转录聚合酶链式反应(RRTPCR)测试确定的病毒载量在症状出现时达到峰值，并在1-3周内下降到无法检测到的水平。然而，很少有研究调查临床标本的潜在传染性，到目前为止，病毒培养分离在第一次出现症状几周后就失败了，这是病毒在免疫能力强的宿主体内实现体外复制的最后18天。在这里，我们报告了42天后分子检测SARSCoV-2感染阳性的病例，描述了从第一个阳性拭子中获得27天后在体外成功复制病毒的情况。一名58岁的男子在对卫生工作者进行常规筛查时，SARSCoV-2呈阳性；他最初没有症状。他接受了隔离，并接受了乳铁蛋白、维生素C和D治疗。第二天，在检测呈阳性后，他妻子和女儿的拭子样本SARSCoV-2感染呈阳性；然而，分别在15天和10天后，他们的拭子样本呈阴性。4天后，他出现发热(最高38.7摄氏度)和咳嗽，并接受了扑热息痛和左氯匹林的治疗。拭子阳性后第7天加重，出现用力呼吸困难，饱和度94%~97%，直接肺部听诊无肺部异常，开始用阿奇霉素(500 mg/d)治疗6天。经过3天的治疗，他没有发烧。23天后，他继续咳嗽，并成功地用强的松治疗(50毫克/天，4天，25毫克/天，2天，12.5毫克/天，2天)。在血液学分析中，独特的改变表现为D二聚体的增加(D二聚体=996ngmL)，这一特征常见于重症新冠肺炎患者的高凝状态。开始使用福达肝素(2.5毫克/天，连续10天)治疗。白细胞数在正常范围内，淋巴细胞轻度减少(8.82×10/L白细胞、6.56×10/L中性粒细胞、1.39×10/L淋巴细胞)。42天后，他出现呼吸急促的弥漫性喘息，并通过直接肺部听诊推断出肺阻塞的特征，并开始使用赛必可160/45(每12小时两次吸入)治疗。48天后，分子试验呈阴性。49d后，D二聚体恢复至正常水平(420 ng/mL)，白细胞减少(6.14×10/L白细胞、2.48×10/L中性粒细胞、2.83×10/L淋巴细胞)，并检测到抗SARS冠状病毒S1/S2抗体(193AU/mL)(图1)。在新冠肺炎期间共收集了7份拭子，用Allplex SARSCoV-2检测方法(韩国首尔Seegene)检测了病毒E、RDR和N基因(表1)。分析显示，所有时间的周期阈值都在35以上，这是积极的。在拭子3周呈阳性后，实验室医学部(ASUGI，的里雅斯特，意大利)决定尝试病毒培养，以确定患者是否仍有感染性分泌物。简单地说，第三个拭子(第一次阳性后27天)被过滤并转移到Vero E6细胞的单层，然后监测7天。RT-PCR对SARSCoV-2的分子检测显示，在病毒接种后第2、5和7天，周期阈值降低，病毒载量相对增加(从E+4拷贝/毫升到E+10拷贝/毫升，图2)，并在第7天出现明显的细胞病变效应(图2)。由于患者在随后的拭子检测中仍呈阳性，进一步尝试了病毒分离，但未获成功。同时，用化学发光微粒子免疫分析(CMIA，SARSCoV-2免疫分析，美国伊利诺伊州芝加哥，雅培)进行的血清学试验显示，抗核衣壳抗体的信号截断比为7.74。因此，该血清在体外对SARSCoV-2进行了测试，显示即使在1：100稀释时也能中和病毒(图3)。此时，患者仍出现呼吸道症状，但免疫系统现在可能部分抵消了感染，因为没有实现病毒分离。然而，第6次拭子(42天)出现了新的阳性，具有较高的Ct，但病毒的体外复制再次失败。最后，第七次拭子(48天)在分子测试中呈阴性，患者被解除隔离。世卫组织患者出院标准建议结束隔离10封函件
The persistence of SARSCoV-2 in pharyngeal swabs belonging to patients affected by COVID-19 is not a rare occurrence. Indeed, the viral load determined through realtime reversetranscriptase PCR (rRTPCR) test peaks at the onset of symptoms and decreases to undetectable level within 1–3 weeks. Nevertheless, few studies investigated the potential infectivity of the clinical specimens, and to date viral culture isolation failed after some weeks from the first symptoms, being the 18 day the last timing in which virus replication in vitro was achieved in immunocompetent host. Here we report the case of SARSCoV-2 infection positivity at the molecular test after 42 days, describing successful viral replication in vitro obtained after 27 days from the first positive swab. A 58yearold man resulted positive to SARSCoV-2 during routine screening for health workers; he was initially asymptomatic. He underwent isolation and was treated with lactoferrin, vitamin C and D. The subsequent day after testing positive, swab specimens from his wife and daughter yielded positive for SARSCoV-2 infection; however, they returned negative after 15 and 10 days, respectively. After 4 days he developed fever (max 38.7°C) and cough and was treated with paracetamol and levocloperastine. At the seventh day after the swab positivity he aggravated and presented dyspnoea exertional, although with 94%–97% saturation and no pulmonary abnormalities evidenced by direct lung auscultation; treatment with azithromycin (500 mg/day) for 6 days was started. After 3 days of treatment, he did not show fever. After 23 days he continued to have a cough that was successfully treated with prednisone (50 mg/day for 4 days, 25 mg/ day for 2 days, 12.5 mg/day for 2 days). At the haematologic analysis the unique alteration regarded an increment of Ddimer (Ddimer=996 ng/mL), a characteristic associated with a hypercoagulative status often found in severe cases of COVID-19. Treatment with fodaparinux (2.5 mg/day for 10 days) was initiated. The leucocytes number were in the normal level range with a slight decrement of lymphocytes (8.82 × 10/L leucocytes, 6.56 × 10/L neutrophils, 1.39 × 10/L lymphocytes). After 42 days he showed diffuse respiratory wheezing in forced expiration with pulmonary obstructive features deduced through direct lung auscultation, and therapy with symbicort 160/45 (two inhalations every 12 hours) was started. After 48 days, the molecular test was negative. After 49 days Ddimer returned to normal level (420 ng/mL), leucocytes decremented (6.14 × 10/L leucocytes, 2.48 × 10/L neutrophils, 2.83 × 10/L lymphocytes) and antibody antiSARSCoV-2 S1/ S2 (193 AU/mL) were detected (figure 1). A total of seven swabs were collected during the course of the COVID-19 and tested for the viral E, RdR and N genes with the Allplex SARSCoV-2 assay (Seegene, Seoul, South Korea) (table 1). The analysis showed a positivity with a cycle threshold above 35 for all the timings. After 3 weeks of swab positivity, the Division of Laboratory Medicine (ASUGI, Trieste, Italy) decided to attempt the viral culture in order to unravel if the patient still presented infectious secretions. Briefly, the third swab (27 days after the first positivity) was filtered and transferred to a monolayer of Vero E6 cells and then monitored for 7 days. The molecular detection of SARSCoV-2 by RTPCR showed a decrement of cycle threshold and a relative increment of the viral load at days 2, 5 and 7 after the virus inoculation (from E+4 copies/mL to E+10 copies/mL, figure 2), as well as an evident cytopathic effect at the seventh day (figure 2). Since the patient remained positive at the subsequent swab, the viral isolation was further tried but unsuccessfully. Concomitantly, the serologic test, performed with a chemiluminescent microparticle immunoassay (CMIA, SARSCoV-2 IgG assay, Abbott, Chicago, Illinois, USA) displayed a signal to cutoff ratio of 7.74 for antinucleocapsid IgG. Therefore, the serum was tested against SARSCoV-2 in vitro, showing the viral neutralisation even at a dilution of 1:100 (figure 3). At this time the patient still presented respiratory symptoms, but probably the immune system was now able to partially counteract the infection, since the viral isolation was not achieved. Nevertheless, the sixth swab (42 days) resulted in a new positivity, with a high Ct, however the virus replication in vitro failed again. Finally, the seventh swab (48 days) resulted as negative at the molecular test and the patient was released from isolation. WHO criteria for patient discharge recommended the ending of isolation 10 Correspondence