A Case of Prolonged Shedding of SARS-Cov-2 with Rapid Decay of IgG Antibody

Background Coronavirus disease 2019 (COVID-19) epidemic is still spreading rapidly around the world. Recent cases with prolonged detection of SARS-CoV-2 RNA have been successively reported and the phenomenon of false-negative real-time polymerase chain reaction (RT-PCR) results of SARS-CoV-2 RNA or “repositive” was also described in patients with COVID-19.


Background
Coronavirus disease 2019 (COVID-19) epidemic is still spreading rapidly around the world.
As of December 29, 2020, severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection has caused over 79 million cases and over 1.7 million deaths worldwide and the epidemic continues to worsen in most countries (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports).As is known to all, testing positive for SARS-CoV-2 RNA is the gold standard for the diagnose of COVID-19 [1] and two consecutive negative for SARS-CoV-2 RNA test results are one of the criteria for discharge. 1A retrospective study of 301 patients with COVID-19 in China showed that the median duration of viral shedding was 20 days from illness onset, whereas prolong virus replication (＞28 days) was found in 25 patients and the longest duration was 42 days [2].
Additionally, recent reports have also showed false-negative real-time polymerase chain reaction (RT-PCR) results of SARS-CoV-2 nucleic acid or "repositive" phenomenon in patients with COVID-19 [2,[7][8][9].Herein, we report a case of recovered COVID-19 patient with repeating recurrence of positive SARS-CoV-2 RNA for at least146 days (the last positive test result on illness day 146 and the first true-negative test result on illness day 151) and the gradual disappearance of IgG against SARS-CoV-2 before the virus removal.

Case description
A 69-year-old Chinese female travelled from New York, USA to Fuzhou Changle International Airport, China on March 22 th , 2020, and her throat swab specimen was positive for SARS-CoV-2 RNA based on RT-PCR testing on the same day during routine screening.She just felt slight fatigue and loss of appetite without cough, fever, dyspnea or other infectious sympotoms.The patient with a history of hypertension and total hysterectomy for hysteromyoma was subsequently admitted to the negative pressure isolation room in Fuzhou Pulmonary Hospital of Fujian Province which was the designated hospital of COVID-19 treatment the next day.The main laboratory examinations throughout the course of the disease after onset are showed in table 1-3 and Fig. 1.Throat swab was tested again based on qualitative RT-PCR assay which was performed using a COVID-19 nucleic acid detection kit (Da An Gene Co.,Ltd. of Sun Yat-sen University, China) and the result was definitely positive for SARS-CoV-2 RNA after hospitalization.The results of cardiac-associated enzymes [creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and myohemoglobin (MYO)] and Interleukin-6 (IL-6) showed increased on admission.Leukopenia, thrombocytopenia and lymphopenia with obviously decreased T lymphocyte subgroups including CD3 + , CD4 + , and CD8 + T cells in peripheral blood were also noted.Arterial blood gas analysis showed a partial pressure of oxygen (PaO2) of 80.6 mmHg, a partial pressure of carbon dioxide (PaCO2) of 36.9 mmHg and arterial oxygen saturation (SaO2) 94.6% in the air.C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and lactic dehydrogenase (LDH) levels gradually increased during hospitalization.A human immunodeficiency virus (HIV) antibody test was negative and procalcitonin (PCT) kept normal.Chest CT findings showed as follow: (1) multiple patchy ground-glass opacities (GGOs) in both lungs indicating viral pneumonia; (2) a nodular consolidation shadow (1.0 cm×0.7 cm in size) with lobulated sign and spiculation in the dorsal lobe of the left lower lung (LLL-S 6 ) indicating suspected isolated lung tumor (Fig. 2A).A bedside echocardiography showed aortic sclerosis, aortic valve thickening, normal left ventricular systolic function, tricuspid regurgitation with mildly elevated pulmonary arterial pressure.Color doppler ultrasonography of the whole abdomen revealed double renal cyst without other abnormalities.Color doppler ultrasonography of the neck suggested the possibility of nodular goiter without lymph node enlargement.
The patient was mainly diagnosed with confirmed moderate COVID-19 according to the diagnostic criteria 1 and suspected early lung tumor with hypoxemia.She received antivirals with abidor and recombinant human interferon α-2b spray and other therapeutic drugs including traditional Chinese medicine, ulinastatin, thymalfasin, human granulocyte colony stimulating factor, amlodipine, etc.She was also administering oxygen inhalation by nasal cannula at 2L/minute.After 2 days of treatment, the patient developed cough and expectoration although fatigue disappeared and appetite improved.On illness day 10 (9 days after initial positive virus test), the patient's condition became better with the disappearance of symptoms and the improvement of blood indexes (Table 1, 2).Repeated chest CT revealed progress of partial lesions with absorption of partial lesions in both lungs (Fig. 2B).On illness day 13, the detection of serum antibodies against SARS-CoV-2 based on gold immnnochromatography (GICA) which was performed using a COVID-19 antibody detection kit [Inot (Tangshan) Biotechnology Co.,Ltd., China)] was positive for IgG and negative for IgM.On illness day 32, repeated chest CT showed significant absorption of infected lesions in both lungs (Fig. 2C).The throat swab samples were continuously negative for SARS-CoV-2 RNA on illness days 34 and 37 and the patient was discharged on illness day 38.According to the post-discharge isolation management 1 , the patient was transferred to the fixed isolation point for continuous solitary isolation and observation for 2 weeks.
On illness day 51 (the 14 th day of the isolation observation after discharge), the patient's throat swab was re-detectable positive (RP) for SARS-CoV-2 RNA, although she had no special discomfort.She was transferred to our hospital again for solitary isolation on the same day.Retest of IgG against SARS-CoV-2 was positive and the other laboratory results were normal.Chest CT showed continuously absorption of residual infection lesions in both lungs and the suspected cancerous nodule in the LLL-S 6 was slightly larger than that before (1.2 cm×0.9 cm in size).The patient was given antiviral drug with recombinant human interferon α-2b spray, immunopotentiator with thymalfasin and hypotensive drugs with amlodipine and benazepril.The throat swab test was still positive for SARS-CoV-2 RNA on illness day 72.On illness days 76 and 80, the throat swabs were continuously negative for SARS-CoV-2 RNA.The dectection of IgG against SARS-CoV-2 was reduced to a weak positive and the antibody titer was only 1:10 on illness day 81.The patient kept well and she was discharged again to be continuous solitary isolation and observation at the fixed isolation point for 2 weeks on illness day 81.
The sample of throat swab was rechecked with a negative result of SARS-CoV-2 RNA 14 days after the second discharge.Then the patient went home for continuous solidary isolation and observation.Surprisingly, on illness day 110 (the 16 th of home quarantine), the patient's throat swab was RP for SARS-CoV-2 RNA again and she was transferred to our hospital without any symptoms for the third times.Chest CT revealed that a small number of residual infection lesions were similar to those before, and the initial solid nodule in the LLL-S 6 was slightly larger (1.4cm×1.0cm in size).The re-detection of SARS-CoV-2-IgG was weakly positive and the antibody titer was reduced to be 1:1 on illness day 110 (Tabel 2).Elevated of CK, CK-MB and LDH levels and reduced T lymphocyte subgroups were noted again (Tabel S1, 2 and Fig. 1).Tumor markers were normal.The serum 1,3-β-D-glucan (G) test, galactomannan (GM) test and cryptococcus capsular antigen test were all negative.The patient was treated with thymalfasin for immuno-enhancement and hypotensive drugs with amlodipine and benazepril.After treatment, LDH, CK, CKMB and ESR all returned to normal.T lymphocyte subgroups initially increased to normal, whereas the indexes declined again after a while.On illness day 121, the titer of SARS-CoV-2-IgG remained 1:1.
However, the antibodies of IgG and IgM against SARS-CoV-2 were both negative on illness day 139 (Table S2).An additional GICA test was performed using a kit from a different manufacturer (Guangzhou Wondfo Biotech Co., Ltd., China) and the result was also negative for IgG and IgM.Negative RT-PCR results of SARS-CoV-2 nucleic acid of throat swabs were found on illness days 125 and 129, whereas the samples were RP on illness days 132 and 146.On illness days 151, 153, 157 and 163, the throat swabs were continuously negative for SARS-CoV-2 RNA.The re-detections of IgG and IgM against SARS-CoV-2 remained negative on illness days 149 and 164.Meanwhile, repeated chest CT revealed that a few residual infection lesions were similar to those before, and the initial solid nodule in the LLL-S 6 continuously enlarged (1.5cm×1.3cm in size).On illness day 164, the patient was discharged to be continue solitary isolation and observation for the third times at the fixed isolation point for 2 weeks and subsequent home quarantine for 2 weeks.The latest outpatient follow-up was September 30 th , 2020 (the 190 th day of illness onset), the patient remained well without reccurence, except for a slightly reduced T lymphocyte subgroups (Fig. 1 and Tabel 2).

Discussion
As seen in this case, the phenomenon of RP for SARS-CoV-2 RNA lasted for about 5 months after the onset of the disease.Although quantitative viral nucleic acid test, isolation of live virus and viral genome sequencing could not be performed due to the laboratory limitations in our hospital, the case was considered to be prolonged viral shedding but not a COVID-9 re-infection for several reasons as follows: First, the patient was quarantined alone after the first discharge, including the period of isolation and observation outside our hospital and the two re-hospitalizations, and she did not contact with any new source of infection.Second, except for the slight increase of LDH, CK, CKMB and ESR within a short time during the third hospitalization, the important inflammatory markers such as CRP and IL-6 kept normal after the first hospital discharge.Third, there were no new clinical symptoms or exacerbation of viral infection on dynamic chest CT after the first hospital discharge.
Prolong viral shedding in this case was considered to be related to the aging and the accompanying underlying diseases which might result in immune dysfunction [2,7], and the insufficient antiviral effect of IgG antibody.First, the solid nodule with lobulated sign and spiculation in the LLL-S 6 gradually increased in size within 5 months while viral pneumonia had been obviously absorpted, indicating a high probability of tumor.That was to say, the elderly COVID-19 patient had three underlying diseases with hypertension, total hysterectomy for hysteromyoma and suspected lung tumor.Moreover, the total lymphocyte count and T lymphocyte subsets count were repeatedly decreased in dynamic monitoring.
All the above factors suggested cellular immune deficiency in this patient which was the important reason of the difficulty in completely eliminating the virus [7,10].Second, IgG against SARS-CoV-2 in this patient was initially tested with positive result by qualitative detection method on illness day 13, whereas the antibody concentration was only 1:10 based on semi-quantitative detection method on illness day 81 during the second hospitalization, indicating IgG antibody titer in vivo was already extremely low at that time.The titer of IgG antibody continued to decline progressively to 1:1 on illness day 110 during the third hospitalization, and it was undetectable by two different test methods on illness day 139 when the throat swab was still positive at that time.Although the peak of IgG antibody titer in our patient was unknown at the begining of the disease due to absence of quantitative detection, it was certain that the IgG concentration had been at a very low level about 2.5 months after onset, which was similar to previous reports [11,12].It was suggested the ability of the IgG antibodies against SARS-CoV-2 decreased rapidly at the early course of the disease and nearly disappeared at the middle and later course of the disease in this case.Therefore, cellular immune deficiency and insufficient humoral immune response in the older patient as seen in our case resulted in a prolonged virus removal.Additionally, as seen in this patient, the immune status obviously fluctuated with frequent decrease of lymphocyte during the whole course of the disease.Reduced immunity could cause repeat increase of the number of viruses in the body, which appeared as the phenomenon of intermittent virus replication [13].This may explain alternately positive and negative results of SARS-CoV-2 detection in this patient.

Conclusions
This case suggests prolonged virus shedding is prone to occur in an immunocompromised host [5,6].With the change of host immune status, SARS-CoV-2 detection can be repeatedly positive.A recent case report has confirmed that SARS-CoV-2 can persistently survive with repeat replication more than 5 months after initial infection [6], suggesting that prolonged virus shedding could be associated with prolonged infectivity.Therefore, for such patients, it is necessary to increase the frequency of SARS-CoV-2 nucleic acid testing and enhance the post-discharge isolation management and health monitoring [1].Notably, the titer of IgG antibody in this case decayed rapidly at the early couse of disease onset and the antibody completely disappeared less than 5 months after disease onset before the virus removal, indicating that there may be certain limitations on the protective effect of antibody against SARS-CoV-2, especially in immuno-compromised hosts.As is known to all, most of the current vaccine studies take antibody response as the main evaluation index of vaccine efficacy.However, except for a strong anti-SARS-CoV antibody response, inducing a strong virus-specific memory T cell response should be the future direction of vaccine research [14,15].

Fig 1 Fig 2
Fig 1 Dynamics of T-lymphocyte subgroups and total lymphocyte after illness onset.After

Figure 1
Figures

Table 1 .
Dynamics of laboratory indexes