Dynamic profiles of SARS-Cov-2 infection from five Chinese family clusters in the early stage of the COVID-19 pandemic

Although several cases of family clusters with SARS-Cov-2 infection have been reported, there are still limited data preventing conclusions from being drawn regarding the characteristics and laboratory findings in the COVID-19 population within family clusters. In the present study, we retrospectively collected five family clusters with COVID-19 and summarized the dynamic profiles of the clinical characteristics, laboratory findings, immune markers, treatment and prognosis of this population. Furthermore, we also compared clinical and laboratory data between the SARS-Cov-2 infection with family cluster (n = 21) and those without family cluster (n = 16). We demonstrated that the duration of SARS-Cov-2 replication might be varied based on the different family clusters due to their different genetic backgrounds. The onset improved lung radiology might start at the end of the SARS-Cov-2 positive period. Furthermore, the obtained results demonstrated that similar basic characteristics and clinical findings seem to exist between the cases with SARS-Cov-2 and without family clusters. The serum level of ferritin might have a different biological function and be a new biomarker for the family cluster. Further studies with larger numbers of patients are required.


Basic and dynamic information for the family clusters of COVID-19.
There were a total of five family clusters with 21 COVID-19 patients in this study, with an average age of 34.0 ± 21.49 years old and eight male patients (38.1%). Among all 21 patients, there were three asymptomatic individuals (14.3%) and 18 symptomatic individuals (85.7%), including 11 with cough (52.4%), eight with expectoration (38.1%), six with fever (28.6%) or fatigue (28.6%), four with pharyngeal pain (19.1%), three with chest pain (14.3%), and one with joint pain or headache (4.8%). In addition, the coexisting conditions included diabetes mellitus (n = 2, 9.5%), heart disease (n = 2, 9.5%), hypertension (n = 1, 4.8%), and chronic obstructive pulmonary disease (n = 1, 4.8%). The details for each of the family clusters with COVID-19 are shown with timelines in Fig. 1 and with mixed coloured panels in Fig. 2. Cluster 1. On Jan 14th 2020, the index case (Case 1) was identified as a 32-year-old man who travelled to Wuhan from Chengdu, Sichuan Province. After a 1 day stay in Wuhan, he returned to Jinan (Shandong Province) on Jan 15th. Unfortunately, he began to complain of pharyngeal pain and fever, with his highest temperature recorded as for 38.0 °C on Jan 23rd 2020, and his mother (Case 2), who was a 58-year-old woman, began to feel fatigue and muscle and joint pain one day later. On Jan 26, Case 1 was admitted to our hospital and was later confirmed as SARS-Cov-2 positive, with scattered ground glass lesions in both lungs on chest CT. On Jan 30, Case 2 was confirmed to be COVID-19 by RT-PCR and chest CT. Therefore, all the close contacts were tested for SARS-Cov-2, and the son of Case 1 (Case 3, 4-year-old), who was asymptomatic, was diagnosed and hospitalized in an isolation ward on Feb 1st. The symptoms of Case 1 lasted from 9 days and disappeared on Jan 31st, and the RT-PCR results for SARS-Cov-2 were positive for 13 days and were negative twice on Feb 7th. Additionally, for Case 1, there was improvement in the lung shadow on chest CT, which started on Feb 2nd. Finally, Case 1 was discharged from the hospital on Feb 16th and had no relapse of SARS-Cov-2 after one month of follow-up. Case 2 experienced 16 days of symptoms and 20 days of SARS-Cov-2 positive status, in addition to 14 days of unchanged status of the lung shadow on chest CT. Ultimately, she was discharged from the hospital on Feb 18th. Although Case 3 had no symptoms or signs, his SASR-Cov-2 positive status lasted for approximately 49 days. He was discharged from the hospital on Mar 22nd. Fortunately, we did not find a relapse of SARS-Cov-2 infection after one month of follow-up for either Case 2 or Case 3.

Cluster 2. This cluster included five patients with COVID-19, with four patients who had been living in
Wuhan and travelled to Jinan before the onset of illness on Jan 22nd. The index case (Case 4, a 34-year-old woman) first began to report fatigue and headache with pharyngeal pain on Jan 23rd. Subsequently, the husband of Case 4 (Case 5) began to suffer from cough, expectoration, fatigue and muscle pain on Jan 24th. Meanwhile, the mother-in-law of Case 4 (Case 6) showed symptoms of cough, expectoration and chest pain beginning on Jan 25th. Both of the sons of Case 4 (11-month-old twins, Case 7 and Case 8) showed no symptoms or signs. Unfortunately, the father-in-law of Case 4, who had not travelled to Wuhan, developed fever, with the highest reported temperature of 38.0 °C on Feb 1st. On Feb 4, all five family members were detected as being positive for SARS-Cov-2 and were transferred to our isolation ward. Chest CT showed shadows in both of the lungs for Cases 4, 5 and 9, as well as a shadow plus pleural effusion for Case 6. There were no positive findings on chest CT for Cases 7 and 8, showing that they might be asymptomatic individuals. Case 4 was symptomatic for 19 days, had a SARS-Cov-2 positive status for 14 days, and had unchanged status with regard to lung shadow for 10 days. The symptoms in Case 5 disappeared 10 days after the onset of the illness, and the detection of SARS-Cov-2 was negative twice after 10 days of hospitalization, and there was improvement of lungs after 3 days of hospitalization. Interestingly, negative results of SARS-Cov-2 were demonstrated for Cases 7 and 8 on the fourth day of hospitalization. All of the above 4 patients were discharged from the hospital on Feb 21st. Case 6 presented with a symptomatic period of 15 days, SARS-Cov-2 positive testing for 22 days and a solid period of chest shadow for 18 days. Case 9 recovered after 5 days of symptoms, 20 days of testing positive for SARS-Cov-2 and 14 days of    www.nature.com/scientificreports/ The characteristics of the family clusters compared with the non-family cluster. Table 1 demonstrates that age and sex were well matched between the family clusters and non-family cluster. There were no significant differences in disease severity (P > 0.05), coexisting conditions (P > 0.05), habits of smoking and alco- www.nature.com/scientificreports/ hol drinking (both P > 0.05) or exposure history in Wuhan between the family clusters and non-family cluster. Furthermore, we also present the symptom distribution, including the percentage of fever, expectoration, fatigue, diarrhoea, cough, headache, chest pain, muscle pain, joint pain and pharyngeal pain in Table 1. Although there seems to be little differences in expectoration (P = 0.082), fatigue (P = 0.086) and diarrhoea(P = 0.096) between the family clusters and non-family cluster, the derivations were not statistically significant (all P > 0.05, respectively).
The laboratory findings in the family clusters compared with the non-family cluster. Table 2 displays the laboratory findings, including routine blood tests, liver function, cardiac function and inflammatory factors, in the general population divided by the presence of family clusters. In detail, the values and the percentages of individual categories for each variable are included and compared. We did not find significant differences in routine blood tests, liver function or cardiac function (all P > 0.05, respectively). Regarding the inflammatory factors, the serum levels of interleukin (IL)-6, procalcitonin  Table 3 demonstrates that the treatments for all the patients were follows: lipinavir/ritonavir + interferon alpha inhalation (14/37, 38.9%), lipinavir/ritonavir + interferon alpha inhalation + Peginterferon (6/37, 16.7%), and lipinavir/ritonavir + interferon alpha inhalation + antibolics (6/37, 16.7%). Furthermore, we did not find any differences in the treatments between the family clusters and non-family cluster (P > 0.05).
The dynamic profiles of immune markers for the family clusters compared with the non-family cluster. The dynamic profiles of the percentages of immune cells has are showed as smooth curves in Fig. 3.
Here, we demonstrated the increase trend of the percentages of CD4 and CD8 immune cells as the length of hospital stay increase. On the contrary, the percentage of B cells seems to show a decreasing trend according to the days of illness. In detail, we did not find any significant discrepancy among T cells, B cells and natural killing (NK) cells between the family clusters and non-family cluster.

Discussion
The transmission of family clusters with COVID-19 accounts for the majority of SARS-Cov-2 infections worldwide 10 . However, limited data have been reported to explore the dynamic progression of COVID-19 within family clusters. Here, we summarized the characteristics of the epidemiological characteristics and disease progression style from a series of five family clusters with COVID-19 in Jinan, China. Notably, we also tried to explore the potential difference of the clinical features and possible mechanisms of immune response in the family clusters compared with the non-family cluster. As reported for the five family clusters, we demonstrated that the duration of SARS-Cov-2 replication might be varied based on different family clusters due to different genetic background. The onset of improvement of lung radiology might start at the end of the SARS-Cov-2 positive period. Furthermore, the obtained results demonstrated that similar basic characteristics and clinical findings seem to exist between the family clusters and non-family cluster. The serum level of ferritin might have a different biological function and be a new biomarker for the family cluster. As a typical area of imported SARS-Cov-2 infection, Jinan is the local capital city of Shandong Province and has a resident population of more than 9 million people 16 . In the present study, a total of 21 COVID-19 patients from a series of five family clusters were collected, and the detailed information of disease progression were determined. The major features of these family clusters could be speculated on, and conclusion were drawn. First, without doubt, SARS-Cov-2 can be transmitted person to person. There is no specific population to be automatically immunized against the infection of SARS-Cov-2 17,18 . Our data showed that SARS-Cov-2 can infect one person regardless of the age and/or sex. Therefore, artificial isolation is a strong measure to protect from the transmission of SARS-Cov-2. Second, most of the COVID-19 cases might experience mild or common symptoms and signs and can completely recover from the SARS-Cov-2 infection [19][20][21] . Although there were three patients in the family clusters that showed relapse of SARS-Cov-2 replication, the duration of being positive SASR-Cov-2 usually lasts for no longer than ten days. Third, the duration of SARS-Cov-2 replication might be varied based on different family clusters due to different genetic backgrounds. In family cluster 5, all five family members showed a rather long duration of SARS-Cov-2 replication approximately 15 days. Meanwhile, in family cluster 3, two patients experienced only 4 days of a SARS-Cov-2 positive period. This discrepancy might be attributed to the varying strength of the immune response to SARS-Cov-2 on the basis of different genetic or epigenetic backgrounds [22][23][24] . HLA haplotypes have been reported to be associated with the disease susceptibility 25 . A recent meta-analysis revealed that 21 genes involved in toll like receptors and C-lectin pathways were associated with severe COVID-19 26 . Finally, we demonstrated that the onset of improvement of lung radiology might start at the end of the SARS-Cov-2 positive period in nearly all the family clusters. Although any increasing number of studies have revealed the mechanism of SARS-Cov-2 lung injury 27-29 , we still know little about the trend of disease progression under the same SARS-Cov-2 infection. Therefore, the interplay of virus and chest radiology should also be further studied.
The initial symptoms and signs of COVID-19 usually occur with the non-specific characteristics of the common cold or pneumonia. In the present study, there were 21.6% asymptomatic individuals and 78.4% Table 2. The laboratory findings for all the patients and the subgroup divided by family cluster.

Characteristics
All patient (N = 37)  www.nature.com/scientificreports/ www.nature.com/scientificreports/ symptomatic individuals, and the major symptoms were cough (46.0%), expectoration (27.0%), fever (24.3%) and fatigue (18.9%). Our data are in agreement with previous reports in China 21,30 . A current report from outside of China showed that impairment of taste and smell, extreme fatigue, cough, and loss of appetite were the best indicators of SARS-CoV-2 infection 31 . Impairment of taste and smell have also been reported in a small proportion of Chinese populations with SASR-Cov-2 infection 2,22,32,33 . Unfortunately, the medical records of the symptoms related to taste and smell from the COVID-19 patients in our ward were not collected. We also demonstrated the dynamic profiles of the basic characteristics, clinical findings, treatment outcomes and immune response of COVID-19 using the family clusters compared with the age-and sex-matched non-family cluster. We did not find any differences in the basic characteristics and laboratory findings between the two groups without the severe type of COVID-19. Of interest, we reported an increased level of ferritin in COVID-19 patients without family clusters compared with those with family clusters. However, we did not find a significant difference in the category of ferritin divided by 400 U/L between the two groups. Ferritin is a blood protein that contains iron, and a high level of ferritin might indicate that excessive storage of iron in the blood. Ferritin is not a specific marker for liver disease, rheumatoid arthritis, inflammatory conditions, cancers or hyperthyroidism 2,34 . Therefore, the significance of the P value might come from the relatively small number of samples or selection bias. However, it is still helpful to further determine the po0tential role of ferritin as a functional factor or biomarker of disease progression in COVID-19 patients.
The treatment regimens and the outcome of the antiviral therapy seemed to exert similar results in the family clusters and non-family cluster, indicating that family clusters might not be a strong risk factor for the prognosis of COVID-19 patients. The assessments of peripheral immune cells might partly explain this issue. In the present study, we demonstrated the increasing trend of the percentages of CD4 and CD8 immune cells, as well as the decreasing trend accompanied by the recovery of the COVID-19 patients in the family cluster and nonfamily cluster. Immune function is a strong barrier against invasive pathogens. SARS-Cov-2 targets surface cells throughout the respiratory system including in the lungs; has an average incubation of six days; and has a slow disease progression 35 . The adaptive immune response may kick in before the target cells are depleted, slowing the infection and interfering with the innate immune response's ability to kill off most of the virus quickly [36][37][38] . Zhang et al. reported that patients with COVID-19 showed a strong interferon-α response and an overall acute inflammatory response 39 . They demonstrated that severe patients showed an altered interferon response, profound immune exhaustion with a skewed T cell receptor repertoire and broad T cell expansion 39,40 . However, the immune mechanism during the whole progression of this disease is still need.
There are some limitations of the present study. First, there is a relatively small number of samples, with a total of 21 patients from five family clusters. To date, there is not a large amount of family clusters available for exploring the specific progression style in such populations. Second, we summarized the details of the disease progression in COVID-19 patients with family clusters, as well as performed a comparative analysis with the nonfamily cluster to find possible discrepancies. We did not analyze genetic or epigenetic material using sequencing or molecular experiments. The molecular mechanism for the genetic or epigenetic material for the SARS-Cov-2 infection should be studied in the future.
In conclusion, we demonstrated that the duration of SARS-Cov-2 replication might be varied based in different family clusters due to their different genetic backgrounds. The onset of improvement onset in lung radiology might start at the end of the SARS-Cov-2 positive period. Furthermore, the obtained results demonstrated that similar basic characteristics and clinical findings seem to exist between the family clusters and non-family cluster. The serum level of ferritin might have a different biological function and be a new biomarker for the family clusters. Further study with a larger number of patients is needed.

Methods
Data sources. This retrospective study was performed in patients of COVID-19 using real-time polymerase chain reaction (RT-PCR) or gene sequencing from one isolation inpatient ward of Jinan Infectious Hospital from January 28, 2020, to April 10, 2020. The criteria for the diagnosis and treatment were based on the guidelines from the Chinese National Health Commission 15 . Epidemiological data, including a travel history to Wuhan within 14 days before the onset of illness, and a history of close contact with patients who were confirmed or suspected as having SARS-Cov-2 infection, were gathered. The exposure period was defined as the number of days from exposure to the onset of the presence of symptoms or signs for symptomatic individuals, or to the onset of SARS-Cov-2 positivity for asymptomatic individuals. Furthermore, a family cluster was defined as no less than three infections of SARS-Cov-2, and the index patient was speculated as the confirmed COVID-19 patient who had a history of travelling to Wuhan and/or who had the first symptoms and signs of pneumonia of unknown cause in the family. Data including basic characteristics, clinical symptoms and signs, laboratory results, chest radiological information, frequency for immune cell subgroups and details of treatment and prognosis, were extracted from medical records. This study conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Commission of Jinan Infectious Diseases Hospital, Cheeloo College of Medicine, Shandong University with a waiver of informed consent (2020-JC-07).
Laboratory findings for patients with SARS-Cov-2 Infection. Specimens including sputum, stool, and nasopharyngeal swabs were obtained and the presence of SARS-Cov-2, were confirmed using a RT-PCR assay (Shanghai BioGerm Medical Biotechnology Co., Ltd, Shanghai, China) according to the published protocol 5 . The SARS-Cov-2 specific antibodies from serum were determined using the New Coronavirus Antibody Detection Kit" (Innovita Biological Technology Co., Ltd, Beijing, China) in accordance with the guideline 5 . The serum biochemical markers (COBAS integra 800, Roche Diagnostics, Germany) including aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, creatine kinase isoenzymes, and myoglobin Scientific Reports | (2020) 10:22048 | https://doi.org/10.1038/s41598-020-79035-1 www.nature.com/scientificreports/ were collected. Haematological markers (Sysmex XE-2100, Sysmex Corporation, Kobe, Japan) included white blood cells, lymphocyte, neutrophils and platelet counts. The serum concentrations of type B natriuretic peptide, IL-6, PCT and CRP were determined using the relevant enzyme-linked immunosorbent assays according to the standard protocol. The serum ferritin concentration was detected by an immunoradiometric assay. All the methods were carried out in accordance with standard regulations in the Clinical Laboratory of Jinan Infectious Diseases Hospital, Cheeloo College of Medicine, Shandong University.