Compared with Wuhan, the 29 cases of COVID-19 in Jinan exhibited mild or moderate symptoms, which are mainly imported cases from Wuhan's contact history (Tables 1, 2). Of the 29 cases, 44.8% had a history of contact with Wuhan person, while others were clustering disease. The average age of the patients was 38.2 years old (SD = 13), including male (11[37.9%]) and (18[62.1%]) female. SARS-CoV-2 nucleic acid was detected in all patients by real-time RT-PCR. But two of them were asymptomatic, and they were compulsively detected nucleic acid because of close contact with confirmed cases. Asymptomatic patients were also treated in hospital's isolation wards until their detection turned negative, with one patient of them showing stool positive before being discharged.
All patients were tested for the nucleic acid of Influenza A and B viruses (FluA, B) and respiratory syncytial virus (RSV). We did not find the co-infection of SARS-CoV-2 and Flu A, B, or RSV viruses in the patients because of the low sample size. Patients had clinical manifestations of fever (18 [62.1%] patients), cough and pharyngalgia (9 [31%] patients), pneumonia (25 [86.2%] patients), muscle aches (4 [13.8%] patients), fecal nucleic acid positive (4 [13.8%] patients). According to imaging examination, 25 [86.2%] patients showed pneumonia (Tables 1, 2). We sorted out 10 representative cases and combed the timeline of the positive throat and stool samples (Figure 1). Four cases are enrolled in Jinan, and the rest of the cases are reported by other agencies. In addition to patients whose initial symptoms of infection are diarrhea, there are some particular cases. After the throat swab turned to negative, their stool samples became positive again, and most of the clinical manifestations appeared 5-7 days after the pulmonary symptoms. Some patients with gastrointestinal infections were secondary to pulmonary infections, and during the patients' recovery period, the virus may still be released from the patient's gastrointestinal tract for 7 days, or even longer. We inferred virus being excreted through the intestine may be more beneficial to the recovery of patients. The replication and duration of SARS-CoV-2 detoxification is directly related to the prognosis of patients. Patients with different clinical symptoms may be closely related to the route of the virus during transmission. The infection sites of virus causing different clinical symptoms and directly affects the course of disease. We speculated several different clinical symptoms may reflect various transmission pathways of SARS-CoV-2 shown in Figure 2. The symptomless SARS-CoV-2 carriers were divided into two groups, i.e., the throat swab positive or the stool sample positive, which reflected the different target sites of a susceptible patient. Study on the first clinical symptoms and the progression of the disease, lungs-intestine transmission route showed that there are three kinds of routes: from the lungs to intestine, the self-infection from the intestine to lungs, co-infection of lungs, and intestine.
Combing the time points of patients' hospitalization may be helpful to provide a guideline for COVID-19 diagnosis and treatment. Whether the excretion of feces in vitro during the recovery period is infectious remains to be further studied. The criteria of patients discharged should be reassessed, and the nucleic acid testing of anal swabs or stool samples should be added.
Environmental samples detection of four patients with early diagnosis, including eye and conjunctival secretions, telephone surfaces, hands surface, masks, the surface of door handles and bed at home, and other samples in the isolation ward, were all negative except their masks. The virus nucleic acid testing of the blood and stool samples were all negative on admission. But the nucleic acid test of their stool samples became positive after the patients were hospitalized for 8-10 days, while nucleic acid test of throat swab turned to negative.
SARS-CoV-2 is mainly transmitted through the respiratory tract in the early stage and can be survival longer on the mask. The epidemiological survey showed the four patients had no recurrence of human transmission before isolated by the hospital.
Cases in Jinan are mainly mild symptoms, which indicated that timely diagnosis, early detection, early isolation are very effective approaches before the virus becomes highly contagious. There are no new cases reported for the 26 successive days in Jinan. The fundamental measure for controlling infectious diseases is to cut the source of infection. It is suggested that study on the transmission routes of the virus is a feasible pathway in controlling the disease effectively and we summarize the transmission routes of SARS-CoV-2 systematically.
Just like SARS-CoV, SARS-CoV-2 is a typical respiratory virus causing highly contagious potentially lethal disease. The mucous membranes infection is still the main transmission. Many clinical cases developed influenza-like symptoms, with a 2-4 days history of cough and subjective fever (Wang D. et al.,; Wu F. et al.,; Zhu et al.,). Coronavirus gains entry into host cells through recognizing and binding to the host receptor ACE2 distributed from the conjunctiva (Wan et al.,). Once exposed directly or indirectly to the virus (infectious droplets, body fluids, virus-carrying hands), the mucosal cells in the conjunctiva, mouth, nasal cavity, or throat were susceptibility infected by the virus for replicating (Gao et al.,; Zhou P. et al.,). A larger amount of virus was assembled and released into the human lungs through the respiratory tract, resulting in various types of fever, cough, or ground-glass opacity of lung on CT examination results and even respiratory failure.
The SARS-CoV-2 mainly spread from person to another through small respiratory droplets from the nose or mouth when a person confirmed COVID-19 coughs or exhales (Figures 3, 4). These droplets land on objects and surfaces around the person. Other people may catch the virus by breathing in droplets or touching these objects or surfaces, then touching their eyes, nose, or mouth. The risk of catching SARS-CoV-2 from someone with no symptoms is very low. However, many people with SARS-CoV-2 experience only mild symptoms, particularly true in the early stages of the disease. Some cases reported that conjunctivitis was the first symptom and they were infected while had a history of close contact with a patient with COVID-19 (Chen L. et al.,; Lu C. W. et al.,). It speculates that there may be a risk of tears and conjunctival transmission. Growing evidence shows that the virus attacks multiple organs in the body.
During the outbreak of SARS-CoV-2, some patients developed symptoms of conjunctivitis. Some patients even suffered the ocular diseases in clinical diagnosis before fever and cough (Chen L. et al.,). There have been case reports in which many ophthalmologists were found to be infected through routine diagnosis and treatment with only his eyes unprotected (Chan et al.,; Xia et al.,). Therefore, if conjunctivitis as the initial symptom of confirmed COVID-19 patients was neglected and contacted without comprehensive measures, the infectious tears and body fluids containing the virus could infect other persons (Belser et al.,; Lu C. W. et al.,). Those results suggested that the eyes route of transmission existed (Chan et al.,; Huang et al.,). Deng et al. demonstrated that macaques can be infected with SARS-CoV-2 via the conjunctival route.
We proposed that the SARS-CoV-2 transmitted and infected through eyes including two routes (Figure 3). One is direct contact and the other is indirect contact transmission. The direct route is that droplets with virus enter through the eyes. For example, in a fever clinic, a relatively closed environment, there is a high risk of eye infections. When medical personnel performs close-up operations, virus droplets (aerosols pollution) will spray out which may splash into the eyes and cause infections, so medical staff are strongly recommended wearing goggles or a mask. A virus presented in these body fluids may affect our precautionary practices and sites of sampling for diagnostic tests. Recently, Jiang et al. found that the virus was present both on surfaces and in the air (Jiang et al.,). The two positive areas were the surfaces of the nurse station in the isolation area with suspected patients and the air of the isolation ward with an intensive care patient. So high contraction of nucleic acid may exist in the aerosol and influence operator, even the test result. Another route is through indirect contact infection, that is, accidentally touching the virus droplets with your hands and rubbing your eyes or noses may cause conjunctival infection.
Therefore, once SARS-CoV-2 infected and replicated in eyes, it will be transmitted through two ways (Figure 3), one is outward transmission, eye secretions, or tears with virus contaminating the hands, and then there is a risk of viral transmission through hands. Another route is an inward transmission. If the virus infects person through eyes, conjunctival secretions, and tears can flow into the mouth through the nasopharyngeal tube which will ultimately reach the lungs or gastrointestinal tract and more infections may occur.
Routes of transmission of SARS-CoV-2 other than respiratory droplets and stool are still enigmatic. The proportion of patients confirmed SARS-CoV-2 with conjunctivitis is much smaller than respiratory symptoms, which reflect that the eyes are not the most important organ for propagating the virus. For instance, the eyes cannot generate infectious aerosol unless an eye ezamination has performed. But we still insist that the eyes are important portals of entry for virus.
Moreover, increasing reports each day suggesting that SARS-CoV-2 cases began with eye redness and tingling as the leading symptoms, and the literature suggests that viruses can infect the human body through conjunctiva (Lu C. W. et al.,; Wang W. et al.,). These results showed that a few new cases of COVID-19 began with conjunctivitis as the first symptom, and the SARS-CoV-2 containing in the eye surface may enter into the nasal cavity and throat through drain tears.
SARS-CoV-2 can be transmitted through the nose-eye, possibly through the way of increased oral pressure caused by coughing or sneezing, and reverse transmission of the virus through the nasolacrimal duct to the dacryocyst and then infect the conjunctival cornea (Sun et al.,; Zhou Y. et al.,). Thus, this route is a two-way transmission route (Figure 4). The lacrimal route, via drainage of tear fluid including virus from punctum in the upper and lower eyelid through canaliculi to the lacrimal sac, and further through the nasolacrimal duct to the nasal cavity, would be another pathway available for SARS-CoV-2 infection. During replication in the ocular tract there will be a continuous influx of virions to the nasal cavity, and respiratory infection may be established. The possibility of subclinical and/or prolonged virus replication in the eye, followed by continuous transfer to the respiratory tract cannot be excluded.
Generally, many respiratory pathogens, such as influenza, SARS-CoV and MERS-CoV, cause enteric symptoms, so is SARS-CoV-2 (Holshue et al.,). Diarrhea was observed in a considerable number of patients. In early reports from Wuhan, 2-10% of patients with COVID-19 had gastrointestinal symptoms such as diarrhea or vomiting (Chen N. et al.,; Wang D. et al.,). Abdominal pain was reported more frequently in patients admitted to the intensive care unit than in individuals who did not require intensive care unit care, and 10% of patients presented with diarrhea and nausea 1-2 days before the development of fever and respiratory symptoms (Yeo et al.,).
The study found that the detection of SARS-CoV-2 nucleic acid positive in a few feces of patients with confirmed COVID-19 cases indicated the presence of a live virus. Nanshan Zhong and Lanjuan Li teams have isolated SARS-CoV-2 from the fecal swab specimens of the pneumonia patient with COVID-19 separately. These findings demonstrated the presence of live viruses in the feces of patients. The recent occurrence of two COVID-19 patients in the same building in HongKong also provide the evidence of fecal transmission. Indeed, the SARS-CoV-2 receptor ACE2 is highly expressed on differentiated enterocytes. SARS-CoV-2 can infect enterocyte lineage cells in a human intestinal organoid model (Lamers et al.,).
Fecal transmission mode accounts for a small proportion of respiratory virus transmission. Most of the virus transmitted through the feces are enteroviruses, and respiratory viruses are mainly transmitted through droplets and contact. However, more than 15% of cases showed that the anal test of several patients had become positive at the later stage, while the chest radiographic evidence and viral clearance in respiratory samples from the upper respiratory tract showed significant improvement, so fecal formation route cannot be ignored. Pan et al. reported that the viral loads of stool samples were less than those of respiratory samples, so whether the excretion of feces in vitro during the recovery period is infectious remains to be further studied.
Considering the evidence of fecal excretion for SARS-CoV-2, the virus can also be transmitted via the fecal-oral transmission route or re-transmitted through the formation of aerosols in virus-containing feces. The transmission route of the tract may not be a single transmission. It may be a medium channel for other routes (Figure 4). Therefore, the standard procedure of stool sample collection and examination in patients with SARS-CoV-2 is important to protect medical staff and reduce the risk of infection.
Cytokines (CK) are key factors regulating the immune response caused by many infectious pathogens that can significantly damage host organs and tissues. In the early stages of SARS-CoV infection, cytokine levels in the blood, such as Il-6, Il-8, and TNF-alpha, are rapidly elevated, the elevation of which is associated with the progression of lung invasion and injury (Wong et al.,). MERS-CoV infection was also reported inducing increased concentrations of proinflammatory cytokines (IFNgamma, TNFalpha, IL15, and IL17) (Mahallawi et al.,).
Huang et al. reported 41 patients with laboratory confirmed SARS-CoV-2 infection and those high concentrations of cytokines recorded in plasma of critically ill patients (Huang et al.,). The concentrations of various cytokines (Il-6) in the blood of severe patients with SARS-CoV-2 infection were significantly higher than those of non-serious patients. The concentration of cytokines can indicate the severity of the disease. Liu et al. analyzed the blood immunological indexes of 33 patients with new coronary pneumonia and found that after SARS-CoV-2 infection, the pathogenic T cells were quickly activated to produce granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-6 (Liu J. et al.,). GM-CSF further activates CD14, CD16 inflammatory monocytes, producing more IL-6 and other cytokines, resulting in a cytokine storm that leads to severe immune damage to the lungs and other organs. More and more cases reported that the brain, kidney, and heart impairment induced by the virus infection, may contribute to multi-organ failure and death eventually (Li et al.,; Wu C. et al.,).
Those cases with SARS-CoV-2 indicate that there is a dissemination way (lymphatic, hematogenous, direct invasion of adjacent tissues, and pathogenic implantation) in blood vessels of viral infection, which usually occurs in critical patients. This potential way for the viral spread is that SARS-CoV-2 enters into the lung from mouth and throat and then infects cells. The virus replicates in the cell and releases more new viruses. Massive accumulation of SARS-CoV-2 leads to a surge of immune cells and more and more pro-inflammatory cytokines, resulting in a rapid increase in CK levels in the blood, or releasing more virus particles into the blood circulation. The virus and cytokines positively induce high expression of ACE2 in the intestinal epithelium and other organs which accelerated over-expression of ACE2 and viral binding, causing systemic infections with the virus (Figure 5). The model might explain why the SARS-CoV-2 nucleic acid testing in the stool of some patients turns to positive occurs in the later days of treatment.