key: cord-0748276-uhz5bjz2 authors: Xiao, Fei; Tang, Meiwen; Zheng, Xiaobin; Liu, Ye; Li, Xiaofeng; Shan, Hong title: Evidence for Gastrointestinal Infection of SARS-CoV-2 date: 2020-03-03 journal: Gastroenterology DOI: 10.1053/j.gastro.2020.02.055 sha: dc501e1810a41f0b41282726f1edfac1bd51ce2d doc_id: 748276 cord_uid: uhz5bjz2 nan tified in Wuhan, China, at the end of 2019, the virus has spread to 32 countries, infecting more than 80,000 people and causing more than 2600 deaths globally. The viral infection causes a series of respiratory illnesses, including severe respiratory syndrome, indicating that the virus most likely infects respiratory epithelial cells and spreads mainly via respiratory tract from human to human. However, viral target cells and organs have not been fully determined, impeding our understanding of the pathogenesis of the viral infection and viral transmission routes. According to a recent case report, SARS-CoV-2 RNA was detected in a stool specimen, 1 raising the question of viral gastrointestinal infection and a fecal-oral transmission route. It has been proven that SARS-CoV-2 uses angiotensinconverting enzyme (ACE Q8 ) 2 as a viral receptor for entry process. 2 ACE2 messenger RNA is highly expressed and stabilized by B0AT1 in gastrointestinal system, 3,4 providing a prerequisite for SARS-CoV-2 infection. To further investigate the clinical significance of SARS-CoV-2 RNA in feces, we examined the viral RNA in feces from 71 patients with SARS-CoV-2 infection during their hospitalizations. The viral RNA and viral nucleocapsid protein were examined in gastrointestinal tissues from 1 of the patients. From February 1 to 14, 2020, clinical specimens, including serum, nasopharyngeal, and oropharyngeal swabs; urine; stool; and tissues from 73 hospitalized patients infected with SARS-CoV-2 were obtained in accordance with China Q9 Disease Control and Prevention guidelines and tested for SARS-CoV-2 RNA by using the China Disease Control and Preventionstandardized quantitative polymerase chain reaction assay. 5 Zeiss MicroImaging, Oberkochen, Germany) and are shown in Figure 1 . This study was approved by the Ethics Committee of The Fifth Affiliated Hospital, Sun Yat-sen University, and all patients signed informed consent forms. From February 1 to 14, 2020, among all of the 73 hospitalized patients infected with SARS-CoV-2, 39 (53.42%), including 25 male and 14 female patients, tested positive for SARS-CoV-2 RNA in stool, as shown in Supplementary Table 1 . The age of patients with positive results for SARS-CoV-2 RNA in stool ranged from 10 months to 78 years old. The duration time of positive stool results ranged from 1 to 12 days. Furthermore Gastrointestinal endoscopy was performed on a patient as described in the Supplementary Case Clinical Information. As shown in Figure 1 , the mucous epithelium of esophagus, stomach, duodenum, and rectum showed no significant damage with H&E staining. Infiltrate of occasional lymphocytes was observed in esophageal squamous epithelium. In lamina propria of the stomach, duodenum, and rectum, numerous infiltrating plasma cells and lymphocytes with interstitial edema were seen. Importantly, viral host receptor ACE2 stained positive mainly in the cytoplasm of gastrointestinal epithelial cells ( Figure 1 ). We observed that ACE2 is rarely expressed in esophageal epithelium but is abundantly distributed in the cilia of the glandular epithelia. Staining of viral nucleocapsid protein was visualized in the cytoplasm of gastric, duodenal, and rectum glandular epithelial cell, but not in esophageal epithelium. The positive staining of ACE2 and SARS-CoV-2 *Authors share co-first authorship. Abbreviations used in this paper: ACE, angiotensin-converting enzyme; rRT-PCR, real-time reverse transcriptase polymerase chain reaction. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 BRIEF COMMUNICATIONS was also observed in gastrointestinal epithelium from other patients who tested positive for SARS-CoV-2 RNA in feces (data not shown). In this article, we provide evidence for gastrointestinal infection of SARS-CoV-2 and its possible fecal-oral transmission route. Because viruses spread from infected to uninfected cells, 6 viral-specific target cells or organs are determinants of viral transmission routes. Receptormediated viral entry into a host cell is the first step of viral infection. Our immunofluorescent data showed that ACE2 protein, which has been proven to be a cell receptor for SARS-CoV-2, is abundantly expressed in the glandular cells of gastric, duodenal, and rectal epithelia, supporting the entry of SARS-CoV-2 into the host cells. ACE2 staining is rarely seen in esophageal mucosa, probably because the esophageal epithelium is mainly composed of squamous epithelial cells, which express less ACE2 than glandular epithelial cells. Our results of SARS-CoV-2 RNA detection and intracellular staining of viral nucleocapsid protein in gastric, duodenal, and rectal epithelia demonstrate that SARS-CoV-2 infects these gastrointestinal glandular epithelial cells. Although viral RNA was also detected in esophageal mucous tissue, absence of viral nucleocapsid protein staining in esophageal mucosa indicates low viral infection in esophageal mucosa. After viral entry, virus-specific RNA and proteins are synthesized in the cytoplasm to assemble new virions, 7 which can be released to the gastrointestinal tract. The continuous positive detection of viral RNA from feces suggests that the infectious virions are secreted from the virusinfected gastrointestinal cells. Recently, we and others have isolated infectious SARS-CoV-2 from stool (unpublished Q12 data), confirming the release of the infectious virions to the gastrointestinal tract. Therefore, fecal-oral transmission could be an additional route for viral spread. Prevention of fecal-oral transmission should be taken into consideration to control the spread of the virus. Our results highlight the clinical significance of testing viral RNA in feces by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) because infectious virions released from the gastrointestinal tract can be monitored by the test. According to the current Centers for Disease Control and Prevention guidance for the disposition of patients with SARS-CoV-2, the decision to discontinue transmissionbased precautions for hospitalized patients with SARS-CoV-2 is based on negative results rRT-PCR testing for SARS-CoV-2 from at least 2 sequential respiratory tract specimens collected 24 hours apart. 8 However , in more than 20% of patients with SARS-CoV-2, we observed that the test result for viral RNA remained positive in feces, even after test results for viral RNA in the respiratory tract converted to negative, indicating that the viral gastrointestinal infection and potential fecal-oral transmission can last even after viral clearance in the respiratory tract. Therefore, we strongly recommend that rRT-PCR testing for SARS-CoV-2 from feces should be performed routinely in patients with SARS-CoV-2 and that transmission-based precautions for hospitalized patients with SARS-CoV-2 should continue if feces test results are positive by rRT-PCR testing. Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at https://doi.org/10.1053/ j.gastro.2020.02.055. The authors disclose no conflicts Q5 . This Q6 work was funded by the Q23 National Natural Science Foundation of China (grant 81870411). The funders had no involvement in study design, writing the report, or decision for publication. 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 BRIEF COMMUNICATIONS On Q14 January 17, 2020, a 78-year-old man, who, along with his wife, had come from Wuhan 6 days earlier to visit his daughter, presented to the outpatient clinic at our hospital in Zhuhai, Guangdong Province, China, with a 7-day cough and fever. He was admitted to the negative-pressure isolation room in the Department of Infectious Diseases at our hospital as a suspected case of SARS-CoV-2 infection. On admission, the physical examination showed a body temperature of 37.5 C, blood pressure of 105/56 mm Hg, pulse of 67 beats per minute, and respiratory rate of 22 breaths per minute with oxygen saturation of 97%. On physical examination, auscultation indicated rhonchi and cracks on bilateral lungs. Initial arterial blood gas analysis showed the arterial partial pressure of oxygen (PaO 2 )/fraction of inspiration oxygen (FiO 2 ) was 176. Nasopharyngeal and oropharyngeal swab specimens tested positive by rRT-PCR for SARS-CoV-2. Chest computed tomography Q15 presented with multiple ground-glass opacities, coinciding with previous reports, 1,2 showing evidence of pneumonia in both the left and right lungs. The patient's wife and daughter tested positive for SARS-CoV-2 RNA and were admitted to the hospital on January 18, 2020. On hospital days 1 through 3, the patient remained febrile Q16 , with stable vital signs. The oxygen saturation remained above 95% with high-flow oxygen therapy. Empiric antimicrobials with oseltamivir and moxifloxacin was given during this period of time. On hospital day 4, the patient developed severe respiratory distress, with the PaO 2 /FiO 2 decreasing to 130 and was immediately transferred to the intensive care unit, receiving an intubation and mechanical ventilation. Along with sedation, proneposition mechanical ventilation was applied for 12 hours per day, and low tidal volume was set. The PaO 2 /FiO 2 increased to 350 immediately after intubation but decreased gradually again in the following several days to the lowest level of 70 at 10 days after admission. Meanwhile, the chest radiograph showed extensive bilateral consolidation; emergent veno-venous extracorporeal membrane oxygenation was applied at the same day. On day 10, coffee ground gastric contents were observed from the gastric drainage tube and fecal occult blood tested positive, indicating upper gastrointestinal bleed. Gastrointestinal endoscopy was performed to determine the exact location of bleeding. Mucosa damage in the esophagus was observed under endoscopy. Biopsy samples were taken from esophagus, gastric, duodenum, and colon for histopathologic and immunofluorescent staining. One day after treatment with octreotide and esomeprazole, gastrointestinal bleeding stopped. As of February 12, 2020, the patient remained hospitalized. The vital signs were stable with mechanical ventilation, veno-venous extracorporeal membrane oxygenation, and low-dose vasopressors. There is no obvious evidence of other organ dysfunction. Histopathologic and Immunofluorescent Staining. Esophageal, gastric, duodenal, and rectal tissues were obtained using endoscopy on day 10. Samples were embedded with paraffin and then stained with H&E. For immunofluorescent staining, 3-mm-thick sections were dewaxed in xylene, rehydrated in alcohol, and washed in distilled water 3 times before microwave repair. After washing 3 times in phosphate-buffered saline with Tween (PBST), sections were incubated with 10% goat serum in PBST for 1 hour at room temperature and then incubated overnight at 4 C with primary antibodies (anti-ACE2, Sino Biological, Beijing, China, 10108-T56, 1:500; antinucleoprotein, Sino Biological, 40143-T62, 1:500). The slides were incubated with secondary antibodies (Alexa Fluor 647-conjugated goat anti-rabbit IgG, bs-0296G-AF647, 1:100; Bioss, London, UK) for 1 hour at room temperature followed by washing 3 times with PBST. Nuclei were then counterstained with 4 0 ,6-diamidino-2phenylindole after washing 3 times with PBST. Slides were imaged by using a laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging). 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 Supplementary 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 Q4 e-mail: shanhong@mail.sysu.edu.cn; or Xiaofeng Li, MD, PhD, Department of Gastroenterology, the Fifth Affiliated Hospital e-mail: zdwylxf@163.com; or Ye Liu, MD, PhD, Department of Pathology, the Fifth Affiliated Hospital, Sun Yat-sen University, 52 East Meihua Road Chest CT Findings in 2019 Novel Coronavirus (2019-nCoV) Infections from Wuhan, China: Key Points for the Radiologist Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Supplementary Table 1.Clinical Characteristics of the 73 Hospitalized Patients With SARS-CoV-2 Q21 R, respiratory specimens; wRþSþ, remained positive in both R and S until the date of writing the manuscript on February 14, 2020; wR Sþ, tested negative in R but remained positive in S until the date of writing the manuscript on February 14