COVID-19, a novel infectious disease, caused by SARS-CoV-2, affected millions of people around the world with a high mortality rate. Although SARS-CoV-2 mainly causes lung infection, gastrointestinal symptoms described in COVID-19 patients and detection of the viral RNA in feces of infected patients drove attentions to a possible fecal-oral transmission route of SARS-CoV-2. However, not only the viral RNA but also the infectious viral particles are required for the viral infection and no proof has been demonstrated the transmission of the infectious virus particles via the fecal-oral route yet. Growing evidence indicates the crosstalk between gut microbiota and lung, that maintains host homeostasis and disease development with the association of immune system. This gut-lung interaction may influence the COVID-19 severity in patients with extrapulmonary conditions. Severity of COVID-19 has mostly associated with old ages and underlying medical conditions. Since the diversity in the gut microbiota decreases during aging, dysbiosis could be the reason for older adults being at high risk for severe illness from COVID-19. We believe that gut microbiota contributes to the course of COVID-19 due to its bidirectional relationship with immune system and lung. Dysbiosis in gut microbiota results in gut permeability leading to secondary infection and multiple organ failure. Conversely, disruption of the gut barrier integrity due to dysbiosis may lead to translocation of SARS-CoV-2 from the lung into the intestinal lumen via circulatory and lymphatic system. This review points out the role of dysbiosis of the gut microbiota involving in sepsis, on the severity of SARS-CoV-2 infection. Additionally, this review aims to clarify the ambiguity in fecal-oral transmission of SARS- CoV-2. A novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was detected in China and affected millions of people around the world with a very high mortality rate , much more than the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) combined . The disease, with the symptoms of fever and dry cough, caused by the current coronavirus was named as COVID-19 by WHO and declared as pandemic due to the ongoing global health crisis which SARS- CoV-2 led to . Several coronavirus species have been identified that cause human diseases including SARS-CoV, MERS-CoV, and the current coronavirus, SARS- CoV-2. Human coronaviruses mostly have a long single-stranded RNA with positive polarity . SARS- CoV-2 virions have a double layer lipid envelope structure on the outer surface with various proteins such as spike (S) protein, envelope (E) protein, and membrane (M) protein embedded in . Phylogenetic analysis of complete genome sequences of SARS-CoV-2 revealed that the new virus shares 89.1% nucleotide sequence identity with SARS-like coronaviruses detected in bats. SARS-CoV-2 is closely related to SARS-CoV but more distant to MERS . SARS-CoV-2 RNA dependent RNA polymerase, which has been used as target for antiviral inhibitors, shares 90.1% sequence identity to SARS-CoV but 56.7% that of to MERS-CoV . While MERS-CoV uses dipeptidyl peptidase 4 (DPP4), SARS-CoV and SARS-CoV-2 use the angiotensin-converting enzyme 2 (ACE2) to enter human cells. It has been shown that receptor affinity of SARS-CoV-2 to ACE2 is higher than the strain of SARS-CoV in 2003 . Although the SARS-CoV-2 has spread more quickly compared to SARS-CoV and MERS-CoV due to possible increased globalization, they do not seem very different regarding their course of disease and there are several similarities in their clinical signs . The manner of development of this current infectious disease has not been uncovered yet and there is not a comprehensive explanation of the pathogenesis for COVID-19; however, the mechanism of SARS-CoV and MERS-CoV can give us quite valuable information on the pathogenesis of SARS-CoV-2 infection. Although the COVID-19 mortality has been seen in patients with varying age, older adults are still at the higher risk groups . Studies to date have mostly associate the disease severity with elders and patients who have underlying medical conditions. However, the contribution of leaky gut to the severity of COVID-19 due to dysbiosis in the gut microbiota has not been revealed clearly. This review points out the role of dysbiosis of the gut microbiota involving in sepsis, on the severity of SARS- CoV-2 infection. Additionally, this review aims to clarify the ambiguity in fecal-oral transmission of SARS- CoV-2.The main symptoms at onset of illness in COVID-19 patients from Wuhan were defined as fever, cough, and fatigue by the retrospective reports and the symptoms such as headache, diarrhea, vomiting, and abdominal pain were described as less common symptoms due to low incidence . However, as more information collected from different regions and large scale studies, the incidence of the patients with gastrointestinal (GI) symptoms has been increased . Due to the common symptoms, indicating respiratory tract disease, of patients infected with SARS-CoV-2, the main organ effected by the COVID-19 seems to be lung. However, during the course of the disease, development of dysfunction in organs such a liver and intestine or multiple organ failure has been reported . Later on, fecal samples collected from COVID-19 patients were found to be positive of SARS-CoV-2 nucleic acid . Despite clearance of viral RNA in the respiratory tract, the fecal samples of COVID-19 patients remained positive for about 11 days. The centers performing research on gut microbiota and fecal microbiota transplantation (FMT), therefore, suggested to screen FMT donors for SARS-CoV-2 and drew attention to possible fecal-oral transmission of the virus . This may suggest us a widespread immunopathology or presence of extrapulmonary SARS-CoV-2 infection. Previous studies reported that patients with SARS (2002) representing common symptoms similar to COVID-19 had diarrhea with 16–73% . This ratio ranged from 2% to 20% in the patients with COVID-19 . Although the ratio vary among reports, which is probably due to the diagnosing criteria used for diarrhea in different hospitals, we should not underestimate gastrointestinal signs along with the respiratory symptoms . It was suggested in some studies that SARS-CoV-2 associated diarrhea could appear even before the respiratory symptoms . Pan et al. investigating the clinical characteristics of COVID-19 with 204 patients documented digestive symptoms including diarrhea, vomiting, and abdominal pain in 50.5% of the patients . Some of the patients showed only the digestive symptoms. Moreover, they reported that gastrointestinal symptoms were declared more often among the patients, as the severity increased with a higher level of liver enzyme and lower monocyte count in patients with digestive symptoms.During SARS-CoV-2 infection process, spike protein of the virus recognizes ACE2 receptor to invade host cell via type II transmembrane serine protease (TMPRSS2) . These 2 proteins need to be coexpressed in the same cell for the virus to be able to enter the host cell. Zhang et al. examined different cell types for ACE2 and TMPRSS2 expression pattern with single-cell transcriptome analysis . They found that these 2 proteins are mainly coexpressed in lung tissue as well as epithelial and gland cells in esophagus and enterocytes in the ileum and colon. In addition to the data detecting viral RNA in feces previously, these results suggest that SARS-CoV-2 infection does not remain with the respiratory tract only and the gastrointestinal system contribute to the course of the disease as well. However, with the limited data until today, it is hard to propose a fecal-oral transmission route to explain the enteric symptoms in COVID-19 patients and claim that SARS-CoV-2 pass through stomach and reach intestine to infect the intestinal cells as enteric viruses accomplish. Xia et al. examined stool, urine, and serum of 73 patients infected with SARS-CoV-2 throughout their hospitalization for the viral RNA . Additionally, they investigated the viral nucleocapsid and ACE2 in gastrointestinal tissues from 1 patient by histologic staining. They detected ACE2, viral RNA and nucleocapsid in epithelium of esophagus, stomach, duedonum, and rectum and suggested fecal-oral transmission as an additional route for the virus to spread the body. These results are notable to understand the mechanism of action SARS-CoV-2 involved in; however, it would be too strong referring to a SARS-CoV-2 infection in gastrointestinal cells due to limitation on sample size, only 1 patient. Moreover, as mentioned above, for the virus to be able to enter the cell, both ACE2 and TMPRSS2 need to be expressed in the cell; it is, therefore, not quite possible to conclude viral entrance to the cell by evaluating only the ACE2 expression. Studies often infer SARS-CoV-2 infection in gastrointestinal system through fecal-oral transmission mostly based on tissue distribution of ACE2 and fecal shedding of SARS-CoV-2 RNA . However, viral RNA detection in some tissues and feces of COVID-19 patients may support the viral RNA load in the area but not the infectious particles. It could be possible but it requires further investigation and more data. Overall, it is not clear whether enteric symptoms or intestinal injury is the result of direct viral infection, inflammation associated or drug induced damage.The human gastrointestinal tract hosts over 1014 cells made up of 500 to 1000 bacterial species, which are referred to as the gut microbiota . There is a well-balanced bidirectional interaction between the microbiota and the immune system. While the microbiota plays a fundamental role in development and maturation of the immune system, the immune system shapes the microbiota composition and functions. Disruption of this balance can lead to human diseases . Studies on the composition of intestinal microbiota in animals and humans of varying different health conditions suggest that gut microbiota is a major determinant in health and disease, impacting immunity, digestion, and pathogenesis. It also serves as an organ participating in physiological and homeostatic functions . Intestinal microbiota, therefore, has found to be disturbed as the other functions in the elderly and diet that strengthen the gut epithelial integrity against pathogens and possible infections increasing immunosenescence has been suggested to elder adults . Diversity in the gut microbiota decreases during aging and microbiome composition altered to an imbalance state, so called dysbiosis, leads to immune dysfunction and generalized inflammation . Dysbiosis considered as an indicator of unhealthy microbiome has been linked to various chronic conditions such as asthma, arthritis, obesity, and type 2 diabetes . People ≥ 65 years old have found to be at higher risk of death from COVID-19 than those of <65 years old . The incidence of diarrhea in COVID-19 patients and the high mortality rate in elderly patients, taken together, point to a possible involvement of gut-lung axis in COVID-19 with association of dysbiosis. These may imply that directed alteration of gut microbiota could be utilized to prevent or treat unhealthy conditions in humans. Gut microbiota are also relevant to metabolic syndromes such as obesity and insulin resistance . Alterations in microbial composition may contribute to the development of metabolic diseases such as liver cirrhosis, nonalcoholic fatty liver disease, and diabetes . Changes in gut permeability impact the liver disease severity via intestinal and systemic inflammation and lead to bacterial infections . Metabolic diseases are associated with a decrease in gut epithelial integrity and an increased bacterial translocation from the lumen to the mucosa inducing systemic inflammation . Moreover, gut microbiota and intestinal barrier have found to be disrupted in pulmonary diseases . COVID-19 patients with digestive symptoms tend to experience liver injury more than those of without digestive symptoms . Dysbiosis, the perturbation of the healthy/normal gut microbiota, has been involved in a wide range of disorders or diseases as mentioned above. This explains why an antibiotic induced imbalance in the gut microbiota can cause diarrhea. Therefore, staying in balance is vital for health and this interaction between the gut microbiota and the immune system has a crucial importance in nonhealthy conditions that could increase COVID-19 severity with extrapulmonary damages.Clostridium difficile associated diarrhea (CDAD), and conversely, CDAD may cause AAD . Microbial translocation with the decrease in intestinal barrier integrity is followed by a secondary infection. The bacterial translocation from the gut to the lungs has been reported in sepsis and acute respiratory distress syndrome due to a possible barrier dysfunction . It is known that the gut and the respiratory tract have been linked to modulate immune responses and dysbiosis in gut microbiota contributes to disease pathogenesis in respiratory tract . It has been shown that chronic lung diseases, such as chronic inflammatory lung disease and asthma, are usually developed with GI tract diseases including inflammatory bowel syndrome (IBD) and patients of those have the structure and function of their intestinal mucosa and permeability altered . Furthermore, it has been reported that around 50% of IBD patients of adults have an impaired lung function with no respiratory disease history . Vice versa, infectious viruses could translocate from infected lung to distant organs via systemic circulation. Gu et al. studied the pathogenesis of SARS on patients infected with SARS-CoV as well as on autopsies of dead patients using in situ hybridization and electron microscopy . Monocytes and lymphocytes in blood vessels of lungs as well as circulating white blood cells, lymph nodes, and lymphoid tissues were found to be positive for the viral sequences. Moreover, they found pathological modifications in the digestive tract tissues and refer that immune cells infected by the viruses could circulate and invade the enteric cells resulting in gastrointestinal damage. These results suggest that the coronavirus could translocate to systemic circulation after damage in lung tissue and migrate to intestinal cells through circulatory and lymphatic system. In this manner, the virus can avoid the harsh environment of the gastrointestinal tract with gastric and intestinal fluid that can disrupt the lipid envelop of the virus and inhibit its infectivity. Taken together all this, we can think about other way of reaching to the intestine for SARS-CoV-2, rather than fecal-oral transmission implying ingesting the viral infectious agent . For a successful infection through fecal-oral transmission, virus must deal with biological barriers such as acid in the stomach and bile salts in the intestine after ingestion. Darnell et al. found SARS-CoV to be inactivated under acidic conditions, which is pH<3, suggesting that the virus can be deactivated by gastric secretion after ingestion unless consuming with a large meal that can buffer the acidic environment . Pratelli et al. working with canine coronavirus reported complete inactivation of the virus under pH 2.26 and pH 4.38 at 37 °C . There is almost no information about the survival of SARS-CoV-2 under acidic conditions. In one of the preprints, Sun et al. studied the survival of the SARS-CoV-2 under various environmental conditions such as wet, dry, and acidic Preprint. Although the virus survives under wet or dry conditions for up to 3 days, it can survive at pH 2.2 for up to 1 h only at high concentrations. Other than the gastric drawback, the virus needs to avoid bile salts to be able to infect the intestinal cells after ingestion. Bile salts are one of the various mechanisms used in host defense with its detergent action on lipid portion of infectious agent . Viruses are divided into subgroups based on presence or absence of a lipid envelope surrounding the nucleocapsid. Bile salts are very effective on viruses that have lipoprotein envelop while those without envelop are resistant to bile acid. SARS-CoV-2 contains outer lipid-containing membrane and is one of the enveloped viruses . Although viral RNA has been detected in fecal samples from COVID-19 patients , no evidence has been demonstrated the transmission of the infectious virus via the fecal-oral route yet. Recently, Zang et al. published a remarkable study on SARS-CoV-2 infection of enterocytes and impact of gastric and intestinal fluid on SARS-CoV-2 . The results showed that although the viruses are capable of entering the intestinal cells, they are not able to survive in the digestive tract due to gastric fluid with low pH and intestinal environment with bile and digestive enzymes. Moreover, no infectious virus was recovered from the fecal samples of COVID-19 patients. This supports that it is quite early to mention about fecal-oral route and enteric infection of SARS-CoV-2 with limited data we have to date and requires more experimental data about it. However, we should not underestimate the contribution of the gut microbiota to the severity of COVID-19 due to its bidirectional relationship with immune system and being the possible reason of gut permeability resulting in secondary infection and multiple organ failure. Coronaviruses are thought to cause damages in lung and lead pneumonia with imbalanced and hyper-immune responses . COVID-19 has been found to cause adverse outcomes related to immune response. Increased proinflammatory cytokines and lymphocytopenia are associated with severe SARS-CoV-2 infection . Enhanced cytokine and chemokine production tend to contribute to “cytokine storm” which lead to severe acute respiratory syndrome in lung and multiple organ failure . A retrospective study on mortality of COVID-19 with 150 patients from Wuhan, China suggest that COVID-19 mortality might be due to virally induced hyper-inflammation, so called cytokine storm . They reported that there were significant differences between death group and discharge group in their blood analysis such as white blood cell counts, lymphocyte counts, and IL-6 level, and moreover, 16% of death had secondary infections. Previously, SARS was characterized by excessive immune response with elevated Th2 cytokines in patients with fatal infections . SARS coronaviruses have found to infect immune cells in addition to lung epithelium as the primary injury site and hyper-reaction has an important role in immune damage and pathogenesis of the virus . Immune responses induced by other viral infections such as influenza drive changes in gut microbiota resulting in dysbiosis and increase gut permeability, which may cause secondary infection, bacterial pneumonia . The high levels of circulating proinflammatory cytokines by viral infections are capable of altering gut microbiota and disturbing intestinal integrity. A malfunction in small intestine leads to an altered gut microbiota and inflammation due to the well-balanced bidirectional interaction between the gut microbiota and the immune system. Increased inflammation in intestine leads to a leaky gut allowing bacterial antigens and toxins to translocate to the systemic circulation, further worsening the septic state of the patients with COVID-19. An inflammatory reaction, then, could be initiated leading to multiple organ failure . In a study with critically ill patients treated in an intensive care unit, multiple organ failure has found to be associated with increased intestinal permeability . Decrease in the gut barrier integrity involves in translocation of bacteria and inflammatory product to distant organs via intestinal lymphatics and leads to sepsis and multiple organ failure syndrome . Another reason for altered gut microbiota could be use of massive amount of antibiotics. A large number of patients in China received antibiotic therapy with 58–71% during COVID-19 treatment . Antibiotics result in dysbiosis and increase susceptibility to new infections and inflammatory disorders. Additionally, they can cause antibiotic associated diarrhea (AAD). AAD is a very common side effect of antibiotic usage and results from an imbalance in gut microbiota due to antibiotic use. Moreover, AAD in hospitalized patients can cause As a result, various hypothesis could be introduced to explain the gastrointestinal symptoms in COVID-19 patients; however, very little is known due to the sudden spike in COVID-19 cases and the further research studies are required to make the final conclusion. Detection of the viral RNA in feces but not the viral particles causing infection does not quite support the idea of fecal-oral transmission. We think that gut-lung axis involves in COVID-19 severity with the association of dysbiosis . Disruption of the gut barrier integrity due to dysbiosis may lead to translocation of SARS-CoV-2 from the lung into the intestinal lumen via circulatory and lymphatic system. This may explain the detection of the virus in feces rather than fecal-oral transmission route. In addition, since the diversity in the gut microbiota decreases during aging, dysbiosis could be the reason of older adults being at high risk for severe illness from COVID-19. Considering the studies on coronaviruses to date and the relationship between the gut microbiota and the immune system, we assume that altering the gut microbiota back to its normal state before the disturbance might be an alternative strategy to alleviate the symptoms of COVID-19 and to shorten the recovery period. In this review, role of the lung and the gut microbiota on COVID-19 was investigated and the importance of further research to fully understand the gut-lung axis in severity of COVID-19 was emphasized.


A coordinated system of disease surveillance will be critical to effectively control the coronavirus disease 2019 (Covid-19) pandemic. Such systems enable rapid detection and mapping of epidemics and inform allocation of scarce prevention and intervention resources. Although many lower- and middle-income settings lack infrastructure for optimal disease surveillance, health and demographic surveillance systems (HDSS) provide a unique opportunity for epidemic monitoring. This protocol describes a surveillance program at the Africa Health Research Institute’s Population Intervention Platform site in northern KwaZulu-Natal. The program leverages a longstanding HDSS in a rural, resource-limited setting with very high prevalence of HIV and tuberculosis to perform Covid-19 surveillance. Our primary aims include: describing the epidemiology of the Covid-19 epidemic in rural KwaZulu-Natal; determining the impact of the Covid-19 outbreak and non-pharmaceutical control interventions (NPI) on behaviour and wellbeing; determining the impact of HIV and tuberculosis on Covid-19 susceptibility; and using collected data to support the local public-sector health response. The program involves telephone-based interviews with over 20,000 households every four months, plus a sub-study calling 750 households every two weeks. Each call asks a household representative how the epidemic and NPI are affecting the household and conducts a Covid-19 risk screen for all resident members. Any individuals screening positive are invited to a clinical screen, potential test and referral to necessary care – conducted in-person near their home following careful risk minimization procedures. In this protocol we report the details of our cohort design, questionnaires, data and reporting structures, and standard operating procedures in hopes that our project can inform similar efforts elsewhere. Models project continued risk until effective treatment or vaccines become available, or sufficient immunity is induced in the population to limit transmission3. The transmission and clinical patterns of the pandemic have been well documented in areas of the world where systematic data capture is in place7. However, data from low- and middle-income countries (LMIC) remain scant.The global emergence of the novel coronavirus, SARS-CoV-2, and the associated pandemic of coronavirus disease 2019 (Covid-19) in humans, has led to substantial morbidity and mortality12. There have long been calls for strengthening public health preparedness in such countries14, but despite some progress15, significant capacity shortfalls remain in many LMICs18. As a result, low case numbers in resource-limited settings should not necessarily be interpreted to reflect a lack of disease transmission. A coordinated system of disease surveillance will be critical to effective prevention, control, and response to the Covid-19 epidemic in LMICs to enable rapid detection, transmission mapping, and non-pharmaceutical interventions, and to inform allocation of scarce prevention and intervention resources.Multiple factors place resource-limited settings at considerable risk of epidemics of respiratory disease. Housing structures, large informal work sectors, high population density (particularly in urban slums and settlements), lack of running water and access to hygiene resources, and a health infrastructure unprepared for diseases requiring advanced medical equipment, all place LMICs at risk of worse outcomes than higher-income settings21. These routine data collection systems can serve as a vital tool in the public health response to Covid-19. HDSS data are typically collected through interviews that generate population-representative longitudinal cohorts, including information on demographics , health status and behaviours. Historically, they have been conducted in person, however, they have increasingly incorporated telephonic or online data collection23. Additionally, HDSS are well-positioned to evaluate the long-term impacts of the epidemic, by measuring: (i) disease penetration through serologic testing; (ii) causes of death during and after the epidemic through the use of verbal autopsies26; and epidemic impact on economic indicators, and non-Covid healthcare access and health outcomes through ongoing interviews.A unique opportunity for epidemic monitoring within the sub-Saharan Africa region is the presence of longstanding health and demographic surveillance systems (HDSS)This protocol paper describes the program developed by the Africa Health Research Institute (AHRI) to leverage a longstanding HDSS to perform Covid-19 surveillance in a rural, resource-limited setting with very high prevalence of HIV and tuberculosis. In this protocol we illustrate how we have leveraged existing expertise and population-based surveillance infrastructure to conduct comprehensive Covid-19 surveillance in a rural area without existing surveillance capacity for rapidly evolving epidemic disease. We also highlight measures taken to minimize Covid-19 transmission risk while conducting this research. We provide our methods and surveillance tools as potential sources of guidance for others working in similar settings.The aims of this study are to conduct active Covid-19 surveillance and to estimate the health and non-health impacts of the Covid-19 epidemic in rural South Africa. To achieve these aims we will implement longitudinal surveillance based on regular telephonic contact with households in a geographically defined population. This surveillance will allow us to meet the following programmatic and scientific objectives:a. Support the local public health response by the Department of Health (DoH) through screening, testing, case notification and linking Covid-19 cases to care;b. Describe the incidence and evolution of Covid-19 epidemic in a well-defined population in rural KwaZulu-Natal, including its distribution by age, gender and socio-demographic characteristics;c. Determine the impact of the Covid-19 outbreak and non-pharmaceutical control interventions on individual behaviour and wellbeing, household socio-economic status and health service delivery;d. Determine the impact of pre-existing health conditions, including HIV and tuberculosis on Covid-19 susceptibility, clinical presentation and outcomes.2; it is largely rural with one town of approximately 30,000 residents, and among the lowest-ranked areas in South Africa in terms of socioeconomic status27. A large proportion of the population live with HIV (~20% of men and ~40% of women aged 15–54 in 201728). Tuberculosis incidence in the district in 2015 was above the national average of 577 per 100,000 individuals29. Mobile phone ownership in the population is high, 90% of households report owning a phone and in 84% of households there is a valid phone number on record for at least one household member.The study will be conducted within the AHRI Population Intervention Platform surveillance area in the uMkhanyakude district of KwaZulu-Natal province. The study area covers approximately 850 km30. SAPRIN standardised the health and demographic surveillance protocols of the three established HDSS sites, in rural KwaZulu-Natal (AHRI), Limpopo and Mpumalanga32. Through SAPRIN, the three HDSS sites agreed on a common Covid-19 surveillance protocol, which each node then adapted to its specific local health priorities and linkage to care pathways.AHRI is a member of the South African Population Research Infrastructure Network (SAPRIN), an initiative funded by the National Department of Science and Innovation (DSI) and hosted by the South African Medical Research CouncilSince 2000 AHRI has conducted surveillance of approximately 20,000 households  to capture births, deaths, migrations and household socio-demographics. All households under surveillance are contacted once during each 15-week  surveillance cycle. All visits were initially face-to-face, but since 2017, two of the three data collection encounters each year have been collected using a telephonic interviewing platform, which allows for high-throughput surveillance. These interviews are conducted with one household senior representative, acting as proxy for all other members. In addition, residents aged 15 years and older are invited to participate individually in an annual biobehavioural survey. AHRI has also embedded data collection clerks within the eleven public health clinics and one district hospital serving the study population. These clerks electronically capture clinic visits and hospital admissions, and link these with the surveillance platform data for all consenting attendees. The population database is also routinely linked to the electronic tuberculosis and HIV treatment registers at these facilities. AHRI holds memoranda of understanding with the Provincial and District DoH that permit extraction of health record data from primary care and hospital sites for linkage to the household surveillance dataset.The overall flow of this study is described inThis protocol builds upon the underlying routine health and demographic surveillance. For the duration of the Covid-19 lockdown period (and until it is deemed safe by both the South African government and study investigators), physical household visits have been suspended and replaced by telephonic interviews with the household proxy respondent. In addition to the standard demographic surveillance questions, proxy respondents are asked to answer a Covid-19 questionnaire covering:a. A screen for symptoms of Covid-19 for each resident household member, completed either by the proxy informant or the household member, if available;b. Epidemiologic risk factors for Covid-19 including known contacts, healthcare contact, and travel;c. Household awareness of Covid-19;d. Behaviour changes in response to the epidemic;e. Recent visitors to the household and recent travel patterns;f. Economic and healthcare impacts of Covid-19 non-pharmaceutical interventions on the household;g. 34;Brief mental health assessments of the household respondent, using the Patient Health Questionnaire-2 and Generalized Anxiety Disorder-2 screening toolsThe Covid-19 questionnaire takes about 15-20 minutes to administer telephonically, whereas when it is integrated into the household demographic surveillance questionnaire, the complete questionnaire takes about 45 minutes in total.To improve the temporal resolution of Covid-19 surveillance, a cohort of 750 households  will also be invited to participate in more intensive surveillance, with calls every two weeks, during which all Covid-19 questions are repeated. The intensive household cohort were randomly selected from all households for which at least one member also participated in a recent population-wide HIV, tuberculosis, and non-communicable disease healthcare screening study.To ensure that detailed information on reaction to the Covid-19 pandemic is available on the full age-range of residents, we will sample 400 past participants in the healthcare screening study, stratifying by age and gender, and oversampling those with multiple non-communicable health conditions. These individuals will be invited to participate in two-weekly calls to report directly on how the epidemic has affected them, including detailed descriptions of their travel and direct personal contact with others.Additionally, planned qualitative work will gather in-depth information from a further sub-sample of the study population using telephone interviews to adhere with national social distancing regulations. This will allow an in-depth understanding of perceptions of risks associated with Covid-19, and vulnerabilities experienced by the community. Data collection tools for this have been informed by risk perceptions contributory domains including knowledge, personal risk, severity and probability of possible outcomes, response efficacy as well as precautionary behaviour and use of information sources. Aspects of vulnerability within the tool focus on how likely the respondent thought he or she was to get the disease and aspects they thought made someone vulnerable to COVID-19. Working with local traditional leaders will help identify contextually appropriate solutions to deleterious effects of both Covid-19 epidemic and non-pharmaceutical interventions used to mitigate its effects.35), the individual is referred to an AHRI Covid-19 mobile testing centre for screening and a possible diagnostic test. These mobile testing centres will come to within walking distance of participant homes. The testing centres are staffed by AHRI research assistants and senior professional nurses and include strict infection control and personal protective equipment procedures35. A research assistant performs a symptom and epidemiologic screen to confirm PUI status. Those meeting PUI criteria are referred to a professional nurse who conducts a clinical examination for triage, including temperature, respiratory rate, and oxygen saturation monitoring. Nurses then complete a testing and referral questionnaire  on a tablet computer using web-based REDCap forms36. Responses trigger assignment to one of the following statuses:If a household member screens positive as a potential person under investigation (PUI) for Covid-19, based on the current South African Covid-19 screening guidelines  will replace the nasopharyngeal and oropharyngeal swabs. Specimens will be stored and shipped at 2–8°C to the AHRI Diagnostic Laboratory in Durban within 24 hours of sample collection, for processing and testing.Specimen collection consists of nasopharyngeal swab and oropharyngeal swabs placed together into a cryovial containing 2 ml viral transport medium labelled with a unique DataMatrix38 and undergo inactivation (at 60°C for 30 minutes). Following inactivation, extraction will be performed either manually using the QIAamp® Viral RNA kit  or on the ChemagicTM 360 Nucleic Acid Extractor  using ChemagicTM Viral DNA/RNA 300 Kit H96 . SARS-CoV-2 templates will be generated using the TaqManTM 2019-nCoV Assay Kit v1 or v2  and run on The Applied Biosystems QuantStudio 5 or 7 Real-Time PCR System by the KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP) or by AHRI on a CFXTM Touch Real-Time System  or Abbott m2000rt).On receipt in the AHRI Diagnostic Laboratory, the specimens will be registered and tracked on the Laboratory Information Management system (LIMS)39. WGS will be assembled using Genome Detective40 and analysed using phylogenetic software applications, such as BEAST41 and Nextstrain42. All genomes will be analysed with over 12,000 WGS publicly available in the GISAID database43.We will use comparative Ct to analyse 2019-nCoV assay data, with Ct <37 regarded as positive (CoV-2 RNA detected), Ct ≥37 but <40 considered indeterminate, and Ct ≥40 as negative. Indeterminate results will be repeated from the amplification process and if found to be indeterminate, the sample re-extracted and amplified; if the sample remains indeterminate it will be reported as suspected positive/positive. Positive cases with a Ct <20 will be genotyped using the ARTIC network SARS-CoV-2 whole genome sequencing (WGS) protocol at KRISPThe SARS-CoV-2 test results will be electronically transferred to the study database and presented for clinical review to the study physician.a. PUIs testing negative for SARS-CoV-2 will be informed of their results via short message service (SMS);b. PUIs testing positive for SARS-CoV-2 will be called by a study physician to be informed of their results. Covid-19 cases will be followed longitudinally with phone calls every three days for clinical monitoring until resolution of symptoms. Those with worsening symptoms will be referred to the AHRI mobile testing centre for further attention and, if warranted, referral to the regional Covid-19 treatment centre.national notifiable medical conditions web portal; a system-generated PUI (including a contacts list) form will also be submitted to the local public health service.All Covid-19 test results will be submitted to theAfter the Covid-19 outbreak and upon resumption of the parent AHRI surveillance protocol, which provides for an annual dried blood spot collection from resident individuals age 15 years and above, the protocol provides for measurement of SARS-CoV-2 seroprevalence, which will be conducted to determine contributions of asymptomatic disease to the epidemic.All participants with positive COVID-19 test results will be considered for enrolment in a related study titled, "Consequences of HIV and TB Co-Infection on COVID-19 Disease Dynamics, Severity and Immune Responses," which is a longitudinal disease characterization and sample collection protocol to establish mechanistic interactions between HIV, TB and COVID-19.We will collect and report Covid-19 surveillance data in real-time to local partners, including the DoH and the AHRI Community Advisory Board. At the conclusion of each week, we will report the following indicators to key stakeholders:1. Number of households screened by phone call;2. Number of individuals screened (through proxy);3. Proportion of households contacted and consented;4. Proportion of households and individuals with a positive case definition screen;5. Number of PUI screened at Covid-19 clinics;6. Number of Covid-19 tests conducted;7. Number and proportion of Covid-19 tests resulting positive;8. Number of individuals referred to clinic for moderate/severe Covid-19 disease;9. Population-representative prevalence of Covid-19 overall and by age and sex.At the conclusion of each 15-week surveillance block, we will additionally conduct a total population Covid-19 epidemiologic summary, including age, sex, and HIV-serostatus stratified Covid-19 prevalence and prevalence of moderate/severe disease. This project is designed to conduct surveillance of the entire HDSS population area and thus is not powered to test a specific hypothesis. However, with a total population sample size of 100,000, and assuming 60% participation in each 15-week block from at least one household member, we will have substantial power to estimate overall and stratified Covid-19 prevalence estimates within age and sex strata .The data collected will provide the opportunity to conduct a wide range of analyses. In particular, the 750-household cohort will allow local epidemic dynamics to be carefully mapped to changes in behaviour, individual wellbeing and household economic and healthcare access, using methods including time-to-event and interrupted time series analysis. Existing data, both collected by AHRI and routinely collected by others and linked to AHRI unique identifiers, will allow comparison of residents’ current situation with that in previous years.Data management. Data collected in this study will be centrally stored on AHRI’s secure data servers. Data will be collected through interviews at the call centre on central server using Survey Solutions44, and in the field on encrypted tablet computers using REDCap, and then securely uploaded to a central server. Collected specimens will be logged on CRF system and control lists generated to verify that all specimens have arrived at the laboratory. These lists will be electronically transferred to LIMS. An electronic study dashboard will assist operational teams in monitoring study progress. Database rules and constraints will be enforced on the master database to ensure that only valid data is stored. Personally identifiable data will be restricted to separate database tables accessible only to data management staff – these will never be shared with the research team.45, as well as research conduct guidance by national and international research oversight bodies. The main risk to study activities and participation is of SARS-CoV-2 transmission from in-person interactions between staff and study participants . Following a risk assessment35, we have implemented multiple control measures to minimize this risk, including: physical barriers; physical distancing; regular hand hygiene; and careful training on and use of standard operating procedures. Should it become impossible to comply with these provisions due to personal protective equipment supply shortages, case load, or other unforeseen challenges, surveillance will be paused, scaled back or terminated.The protocol adheres to the provisions of the Covid-19-specific National Infection Prevention and Control Strategic FrameworkThis protocol builds on the consent procedures in place for the household surveillance protocol it is embedded in. All households contacted for this study have previously provided written consent to: (i) be approached via a telephone call to be asked to participate in research studies; (ii) electronic linkage of surveillance study records with their medical records; and (iii) the sharing of anonymized data in data repositories accessible to the broader scientific community.35 prior to telephone interviews, which will be recorded in accordance with Covid-19 specific guidance from the University of KwaZulu-Natal Biomedical Research Ethics Committee46. Additionally, verbal consent for specimen collection and testing will be obtained (and recorded on the electronic case form) at the specimen collection point. Verbal recording of consent will remove the danger of transmitting SARS-COV-2 via the data collection tablet or a stylus. Any potential participants aged under 18 and not emancipated will be asked to provide recorded verbal assent after a responsible adult has provided informed consent.In this study, all participants are asked to provide verbal informed consentThe protocol was presented to both the DoH and the AHRI Community Advisory Board management committee and approved by both prior to submission for ethical approval. Bulk SMS messages are used to communicate Covid-19 information and study specific arrangements to the research population. Local radio stations will broadcast information on the study and feature interviews with study staff.The protocol received ethical approval from the University of KwaZulu-Natal Biomedical Research Ethics Committee on 2 April 2020 under reference BE290/16 and from the KwaZulu DoH Research Committee.The key findings of this study will be prepared for dissemination as rapidly as possible, given the urgency of the Covid-19 epidemic. These will be provided to the DSI and other government decision-makers at national and provincial levels, including the provincial DoH. They will also be submitted to open-access pre-print servers and as open-access articles to peer-reviewed journals.49. Proxy-based case identification will be augmented by documentation of cases from the study community presenting at the eleven primary health care clinics and one district hospital in the population study catchment area. Our ability to conduct a post-outbreak seroprevalence study will allow for detection of missed cases in this surveillance program, and ultimately serve a complementary role in epidemic description in this population.One limitation is the possibility of not re-interviewing the participant in the next round. On average, 12.0% (range 5.7%–16.9%) of HDSS participants are not re-interviewed the next year due to out migration, death, loss to follow-up or refusal. Over 80% of these losses are due to outmigration; however, 80% of these outmigrants remain household members, and are therefore accessible to collect outcome data. Additionally, for an acute infection with a relatively short symptom duration, the 15 weeks between surveillance contacts means that some proportion of cases will be missed. This limitation is mitigated to some extent by the two-weekly surveillance of 750 households, but the analytical power of this cohort will depend on the cumulative incidence of Covid-19 in the overall study population. Moreover, our study depends on symptomatic screening and proxy responses by heads of households, which will also be susceptible to under-reportingWidespread SARS-CoV-2 testing for surveillance and case identification has been established as a major priority in the epidemic response. Although testing capacity has been limited in many areas of the world, HDSS provide a pre-existing structure in resource-limited settings that is purpose-built for surveillance, with both expertise and infrastructure for conducting population-based surveys.We have leveraged a HDSS to develop and implement a Covid-19 surveillance program in rural South Africa. In doing so, we focused on the following major considerations to ensure both optimal scientific methods and the safety of our staff and study population:1. Total-population sampling, with enhanced surveillance of random sub-samples for early identification of high transmission areas and groups;2. Phone-based screening surveillance to minimize in-person contact;3. Limiting all in-person interactions to persons under investigation;4. Strict adherence to personal protective equipment use, well-trained staff using standardized operating procedures and risk mitigation plans, and hygiene measures in line with national and international guidelines;5. Laboratory procedure conducted in a clinical research laboratory within good laboratory clinical practice guidelines;6. Partnership with DoH and other government departments for case detection, reporting, contact tracing and to ensure all procedures fall within governmental guidance;7. Real-time reporting of surveillance results to inform epidemic response and resource allocation.8. Leveraging routinely collected data such as household socio-economic status, individual employment and education status and linkage to individual health service data to determine the impact of the Covid-19 pandemic on household socio-economic status and health care delivery.Herein we report the details of our cohort design, questionnaires, data and reporting structures, and standard operating procedures in hopes that our project can be used to help inform other similar programs elsewhere in the region.http://saprindata.samrc.ac.za/index.php/catalog). AHRI-specific data will be disseminated through the AHRI data repository (https://data.africacentre.ac.za/) within six months from the conclusion of the first 15-week surveillance cycle. Both datasets will be linkable to existing core demographic surveillance data.A core set of the Covid-19 surveillance indicators and data will made available for the three SAPRIN nodes in the publicly accessible SAPRIN data repository Suppl Mat 2.docx (Standard Operating Procedures)Suppl Mat 3.docx (Questionnaires)Suppl Mat 4.docx (Risk Assessment)Suppl Mat 5.docx (Informed consent wording)Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the Leveraging a demographic and health surveillance system for Covid-19 Surveillance in rural KwaZulu-Natal is an important implementation research programme in low-and-middle countries. This programme will allow monitoring and documenting COVID-19 in the context of data paucity. The project will also allow to evaluate the effect of public policies and other interventions in fighting the pandemic. The project is well designed and is build on an existing data platform and well known context. However, the protocol does not provide information on the phone coverage in the Demographic Surveillance Area. Do all households have a land line and/or mobile phone? What are the length of different questionnaires listed? What could be the influence of number of questionnaires (and questions) on the response rate, quality of answer and risk of abandon? I recommend indexing of this manuscript with minor comments described above.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Population and Health, evaluation, data sciences and Health SystemsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for the positive review. We have submitted a new version of the protocol description with details on the phone coverage in the surveillance area and also provided information of the average time it takes to complete the various questionnaires used. Overall an appropriate protocol whose study will provide meaningful insights on the extent of and the impact of Covid-19 on behaviour and wellbeing and susceptibility to Covid-19 due to HIV/TB in a rural population that could be generalised to similar settings in the region. The investigators leverage an existing health and demographic surveillance system built over several years in a rural setting in KZN with a high prevalence of HIV and TB to perform Covid-19 surveillance. The surveillance system is well developed and multiple surveys have been conducted within this community making the proposed work feasible. The protocol objectives are clear and how these objectives will be achieved is well described. The study population and how it will be accessed is documented and includes details on how risk to Covid-19 for study staff or the population will be mitigated. They further show how this work will supplement DOH activities and will ensure that any potential Covid-19 participants are linked to care. The protocol however needs additional information on how they will determine the impact of Covid-19 outbreak and non-pharmaceutical control interventions on individual behaviour and wellbeing, household socio-economic status and health service delivery. Additional information on the planned qualitative work, methodology, tools to be used would be helpful to enable others replicate this work.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:HIV patient outcomes implementation scienceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for the positive review. We have submitted a new version of the protocol description that clarifies the points raised by the reviewer regarding how we plan to determine impact and we also provide more details on the qualitative research component.


The world is in the middle of a historic public health crisis. As of March 30, 2020, over a third of the population in the United States were under “stay at home” orders given by state governors to protect the vulnerable and the unexposed. Unprecedented steps have been taken by governments globally to contain the novel coronavirus disease 2019 (COVID-19), a rapidly spreading pandemic. This has resulted in more than 690,000 cases and over 33,000 deaths worldwide . The indDoes COVID-19 Spread through Droplets Alone?While preventative measures have been imposed globally, the observed propagation of COVID-19 has noticeable differences among select nations. Epidemiologic data show that some countries have exponential increases in disease incidence, while others seem to have “flattened the curve.” This raises questions of whether a full scientific understanding of disease transmission modes has yet to be attained, and thus whether there are more effective ways to prevent its spread. This brings us to the fundamental question: To answer this question, we provide epidemiological observational data in conjunction with known molecular characteristics of SARS-CoV-2. We discuss the ability of this novel coronavirus to remain viable on environmental surfaces from hours to days and describe its increased virulence characteristics compared to the previous SARS-CoV-1. These biochemical and molecular properties likely allow this novel coronavirus to employ indirect methods of transmission, including fomites and aerosols, in addition to respiratory droplet transmission .Public health measures of this aggressive nature have the universal purpose of reducing the exponential rise in incidence rates of disease transmission. Observations made in health outcomes following the 1918 influenza pandemic have guided public health policy regarding these preventative measures. Importantly, during this pandemic, some U.S. cities chose more effective measures to address the spread of the disease, resulting in observable differences in mortality rates across the nation , 3. ThisThe current consensus regarding the transmission of SARS-CoV-2 is that it spreads person to person through respiratory droplets , 6. PrecSpecific epidemiological observations may provide evidence to suspect that the spread of SARS-CoV-2 may not be limited to respiratory droplets alone. For example, on February 4, 2020, the Diamond Princess Cruise ship carrying 3,711 passengers and crew members reported 10 cases testing positive for COVID-19 after their 14-day voyage. As a response to this, the ship was quarantined for 14 days while docked off the coast of Japan. Following this quarantine period, a total of 634 cases reportedly tested positive for COVID-19, despite droplet precautions and social distancing principles practiced on board . Public Tragically, another story that is unfolding in the COVID-19 pandemic is occurring within the country of Italy, which currently maintains a mortality rate of 9.3% . Once thfewer SARS-CoV-2 virions are necessary to establish an infection in humans. This, in part, could explain the rapid spread of the disease worldwide compared to the 2002–2003 SARS outbreak that infected approximately 8,100 individuals.Recent studies have indicated that SARS-CoV-2 demonstrated 10–20 times greater affinity to angiotensin-converting enzyme 2 (ACE2) receptors compared to SARS-CoV-1, making it a much more virulent virus , 10. Thiremain viable in the environment much longer than most other viruses transmitted through respiratory droplets.The primary mode of transmission of SARS-CoV-1 in the 2002–2003 outbreak was by respiratory droplets up to a distance of about 6 feet . Howeverindirect transmission routes. This can be either a certain level of airborne spread or vehicle-borne (fomites) transmission. Pathogens like influenza virus and rhinovirus that usually spread through respiratory droplets have some airborne transmission properties making it plausible that SARS-CoV-2 may have such characteristics as well , and aerosolized viral particles traveling beyond 6 feet could have exacerbated the volatile spread of COVID-19. In Italy, having houses or other domiciles close to one another may have transmitted the disease even with a limited level of aerosolization. This example may also better explain the current exponential spread of SARS-CoV-2 in many European nations and in the United States that are aggressively practicing social distancing.Today, the world is facing a particularly deadly disease to which there is no cure currently nor a vaccine. Based on the findings mentioned above, if SARS-CoV-2 is also transmitted through indirect contact, additional, yet practical methods of precaution may be indicated. There are ways to help prevent such spread. (1) First, it is essential to follow all droplet precautions including washing hands with soap or using an alcohol-based hand sanitizer for 20–40 s, (2) protecting viral entry points, (3) covering one's mouth when coughing or sneezing, and (4) appropriate social/physical distancing. In addition, (5) continually disinfecting contact surfaces can eliminate the risk of fomite-based transmission. (6) Furthermore, to prevent the possible spread of aerosolized SARS-CoV-2 infections, we will need to reevaluate the current recommendations of 6 feet of physical separation between individuals to possibly increasing it further. Also (7), infected hosts can help prevent the propagation of the virus by donning a face mask covering their mouth and nose to disrupt the airflow near the source. (8) CDC's latest recommendation that all individuals wear a cloth face mask addresses asymptomatic carriers, also known as silent spreaders, and will help protect susceptible hosts. Finally in areas at increased risk of COVID-19 transmission such as hospitals and patient care facilities, (9) appropriately fitted N95 respiratory (facemask), with other personal protective equipment (PPE) and (10) expanded use of special air handling and ventilation systems  need to be in place (TG compiled the epidemiological data, prepared the figures, and helped with the writing and editing of the manuscript. BP discussed the modes of disease spread and helped with the writing and editing of the manuscript. RG discussed the molecular basis of the study and helped with the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes of hsTnT, D-dimer and CRP were related; but only D-dimer was associated with mortality. Moreover, low molecular weight heparin showed attenuation of the mortality in the whole population, particularly in subjects with higher hsTnT. D-dimer possessed a strong relationship with hsTnT and mortality. Anticoagulation treatment showed greater benefits with regard to mortality. These findings suggest a major role of SARS-CoV-2 coagulopathy in hsTnT elevation and its related mortality in COVID-19. A better understanding of the mechanisms related to COVID-19 might pave the way to therapy tailoring in these high-risk individuals.High sensitivity troponin T (hsTnT) is a strong predictor of adverse outcome during SARS-CoV-2 infection. However, its determinants remain partially unknown. We aimed to assess the relationship between severity of inflammatory response/coagulation abnormalities and hsTnT in Coronavirus Disease 2019 (COVID-19). We then explored the relevance of these pathways in defining mortality and complications risk and the potential effects of the treatments to attenuate such risk. In this single-center, prospective, observational study we enrolled 266 consecutive patients hospitalized for SARS-CoV-2 pneumonia. Primary endpoint was in-hospital COVID-19 mortality. hsTnT, even after adjustment for confounders, was associated with mortality. D-dimer and CRP presented stronger associations with hsTnT than PaO The mechanisms underlying to troponin elevation in COVID-19 are thought to be multifactorial. Hypoxia related to interstitial pneumonia potentially leading to non-ischemic myocardial injury, as well as the exaggerated immune-inflammatory response, marked by an elevation of the C-reactive protein (CRP), are amongst the two most commonly cited mechanisms potentially leading to hsTnT elevation in these patients5.Elevated high sensitivity troponin T (hsTnT) at admission, a well-established marker of cardiac injury, has been shown to be common and highly predictive of adverse outcome in patients with Coronavirus Disease 2019 (COVID-19)6. An hyperactivation of the coagulation system, marked by an elevation of the D-dimer, has been associated to an increased risk of mortality in COVID-197. Post-mortem studies have confirmed the presence of a COVID-19-related coagulopathy characterized by the development of thrombotic microangiopathy in multiple organs, including the heart vessels8. Such anatomical alterations can contribute to the elevation of hsTnT independently from the severity of the inflammatory response, as they can further aggravate the delivery of an adequate oxygen supply to the myocardium. The importance of control the coagulatory homeostasis in COVID-19 is recently under attention as some studies have reported a beneficial effect of anticoagulation therapies in these patients10. However, beyond the large number of reports describing associations between hsTnT with mortality in COVID-19, no studies have comprehensively explored the relative contribution of coagulopathy, hypoxia and inflammation to the increase in hsTnT during SARS-CoV2 pneumonia, nor the importance of these pathways in defining the risk of mortality in COVID-19.More recently, activation of coagulation pathways has been described in a substantial proportion of patients with severe COVID-19 and their presence has been related to the development of multi-organ dysfunction, resulting in an increased risk of deathA better understanding of the plethora of ischemic and non-ischemic causes of myocardial injury related to COVID-19 might inform on the most appropriate treatment to be used in patients presenting with elevated levels of hsTnT.In this study, we evaluated the relative importance of the hypoxia, inflammatory response and activated coagulation in the elevation of the hsTnT levels in patients with a diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).We also assessed the complex interaction between hsTnT, D-dimer and C-reactive protein (CRP) in defining the risk of an adverse outcome or hospital complications in the same population. Finally, given the potential contribution of inflammation and coagulation abnormalities to the myocardial injury in COVID-19, we assessed the capacity of immunomodulating drugs or low molecular weight heparin to modify the relationship between high hsTnT levels and increased risk of mortality.11; hsTnT upper reference limit was 14 ng/L. Venous and arterial blood samples for standard biochemistry and arterial blood gas analysis were collected at the time of hospitalization and repeated according to clinical practice and depending on the patient clinical conditions. The study complied with the edicts of the 1975 Declaration of Helsinki and. All experimental protocols were approved by the institutional ethic committee Comitato Etico Area Vasta Nord Ovest (CEAVNO).This single-center, prospective, observational study included 266 consecutive patients hospitalized for SARS-CoV-2 pneumonia at the tertiary care University hospital of Pisa, Tuscany, Italy, between 4th March and 31st March 2020. All patients had a diagnosis of SARS-CoV-2 infection confirmed by positive results of PCR testing of a nasopharyngeal swab. People with high clinical and radiological suspicion of SARS-CoV-2 infection were tested at least twice (at admission and after 12 to 24 h) and infection was excluded only after at least two negative nasopharyngeal swab or two negative nasopharyngeal swab and a negative bronchoalveolar lavage testing, depending on the clinical suspicion. Pneumonia was established with a plain chest CT reviewed by trained radiologists. Demographic, clinical, laboratory, instrumental, treatment, and outcome data were collected as previously reported12 and the need for respiratory support by invasive or non-invasive mechanical ventilation.The primary endpoint of the study was considered the in-hospital mortality. Secondary endpoints were considered the admission to Intensive Care Unit (ICU), the development of acute respiratory distress syndrome i.e. according to sex, history of CVD, increased admission levels of hsTnT) were examined with the independent’s samples Student’s T Test or the non-parametric Mann–Whitney test and the chi-squared test for continuous and nominal variables, respectively. To identify independent determinants of admission hsTnT concentrations, we used multivariable linear regression models. Subsequently, we employed Cox proportional-hazards models to evaluate the association between admission variables and the occurrence of the primary endpoint . We used the Schoenfeld residuals to test the proportional hazards assumption. Kaplan Meier curves were plotted to illustrate the difference in survival probabilities (by implementing the Log rank test) according to admission hsTnT levels. Respectively, we used multivariable logistic regression analysis to appraise the association of hsTnT with secondary outcomes, including incidence of ARDS, invasive and/or non-invasive mechanical ventilation and transfer to ICU. Finally, we utilized two-level linear mixed models with random effects (random intercept and random coefficient) and unstructured variance–covariance matrix to assess whether longitudinal changes in hsTnT levels were associated with fluctuations in levels of CRP and D-dimer across the hospitalization. In all multivariable regression models , we controlled for a pre-specified set of biologically plausible covariates, including age, gender, history of CVD and creatinine levels. These were selected given their well-established association with the COVID-19 mortality risk reported in several previous reports14. In linear regression and linear mixed models, we ensured the normal distribution of the dependent outcome by transforming continuous hsTnT with the natural logarithm. Associations are presented as Hazard Ratio (HR) or Odds Ratio (OR) or coefficients with 95% confidence intervals (CI).Continuous variables are presented as mean ± standard deviation or median (interquartile range) for variables not following normal distribution. Nominal variables are summarized as absolute counts and percentage values (%). Normal distribution of all continuous variables was graphically assessed by histograms and quantile–quantile plots. Differences in variables of interest between pre-defined groups , we performed bootstrapping with 200 replicates and derived corrected confidence intervals.P < 0.05.Statistical analysis was conducted with STATA package, version 12.1 . All tests were 2-tailed. We deemed statistical significance at The study complied with the edicts of the 1975 Declaration of Helsinki. All experimental protocols were approved by Comitato Etico Area Vasta Nord Ovest (CEAVNO). Informed consent was obtained from all individual participants or their legal guardians included in the study.Informed consent for data publications was obtained from all individual participants or their legal guardiansincluded in the study.2 and PaO2/FiO2 on admission were also detected in patients who died during hospitalization. Remarkably, hsTnT was the parameter with the greatest difference between the group of patients who died during admission compared to subjects who survived (fivefold difference). In Cox regression models, hsTnT levels were strongly associated with the risk of in-hospital mortality, even after adjustment for age, sex, previous history of CVD, creatinine levels . Weaker o no associations were observed between hsTnT and risk of ARDS, ICU admission and ventilation. The association between hsTnT and mortality was significant in both sexes, as well as in people with and without previous history of CVD. Only age modified the relationship between hsTnT and risk of mortality, so that in subjects > 75 years old this relationship was stronger than in those < 75 years old .The baseline clinical characteristics of the population stratified by mortality status at follow up are reported in Table ls Table . AdditioP = 0.005). Conversely, CRP was not associated with an increased mortality risk, although it was related with the risk of ARDS, ICU admission and requirement of respiratory ventilation , while no association was observed with the heart rate . The prognostic association between hsTnT or D-dimer and mortality remained highly significant also after adjustment for both these haemodynamic parameters, as shown in Table Both systolic and diastolic blood pressure were found significantly and directly associated to all-cause mortality  and CRP (P < 0.001) and lower PaO2 (P < 0.001) and PaO2/FiO2 ratio (P = 0.001).The Table 2, D-dimer and CRP as independent variables further adjusted for history of CVD, creatinine, sex and age, D-dimer and CRP presented a stronger association with the levels of hsTnT than PaO2 (Table P = 0.003). A weaker association was also detected between changes in CRP and changes of hsTnT concentrations after adjustment .In linear regression models, including PaOO2 Table . Linear P < 0.001). Thus, we tested whether the risk of mortality related to high hsTnT level was somewhat attenuated by the treatment with heparin. When added to the model testing the relationship between hsTnT levels and mortality, the interaction between hsTnT and use/non-use of heparin during hospitalization was highly significant , suggesting that heparin could modify the relationship between hsTnT levels and mortality risk. Indeed, in the group of subjects with levels of hsTnT above the median values the treatment with heparin significantly lowered the risk of mortality compared to untreated subjects , while the same difference was not detected in subjects with hsTnT lower than the median  , hydroxychloroquine, antibiotic, immunomodulating drugs (baricitinib and tocilizumab) and corticosteroids. In multivariable Cox regression models including the same drugs and controlling for age, sex, history of CVD, creatinine and hsTnT, only LMWH remained significantly associated with mortality after bootstrapping  elevation, is frequent in patients with COVID-19 and related with adverse outcomes18. In fact, while the most common clinical presentation of the SARS-CoV-2 infection is bilateral interstitial pneumonia often associated to respiratory failure, several reports have supported the capacity of cardiac injury, indirectly assessed by cardiac troponin due to the in-hospital real-life setting, to inform on the mortality risk in COVID-1917. Troponin elevation has been related with a large set of inflammatory serum markers, such as CRP, ferritin, IL-6, tumour necrosis factor-α and also thrombotic markers such as D-dimer. No previous studies17, however, have accurately explored the potential and complex interaction between these three biologic systems and thus defined the putative determinants of hsTnT elevation levels in COVID-19.In our observation we found hsTnT, among a large numbers of serum biomarkers, the parameter with the major difference at baseline between survived vs deceased patients hospitalized for COVID-19. Cardiac injury, marked by troponin  in patients with more severe interstitial pneumonia could partially explain the stronger relationship of D-dimer and hsTnT with the mortality risk compared to CRP. However, in ours and other studies, the pattern of coagulopathy observed during SARS-CoV-2 infection was substantially different to the DIC commonly seen in sepsis24. Indeed, our population did not show severe thrombocytopenia which usually accompanies the sepsis-related DIC, and the D-dimer concentrations often reached higher levels than those expected in DIC. It has been repeatedly emphasized that most patients with COVID-19 related coagulopathy would not be classified as having DIC according to the recommendations of the International Society on Thrombosis and Haemostasis24. Rather, the activation of the coagulation system observed during COVID-19 and its association with multiple organ dysfunction might reflect the generalised state of endothelitis which accompanies the SARS-CoV2 infection. Independently from its origins, the presence of a coagulopathy in COVID-19 is undebatable: in post-mortem studies, a microangiopathy characterized by typical microvascular platelet-rich thrombotic depositions has been described not only in small vessels of the lung parenchyma but also in other tissues, such as the myocardium8. Such microangiopathy could account more directly for the multi-organ failure and death in COVID-19 patients. Following this interpretation, the elevation of hsTnT might represent also an early marker of a systemic macroangiopathic involvement during COVID-19 infection, and its high prognostic capacity might depend on its higher sensitivity and specificity compared to other markers of tissue injury4.In our exploratory analysis we found that also CRP might explain hsTnT variability. This is consistent with several observations which described how both coagulation and inflammation are involved in hsTnT elevation25. Ayerbe et al. reported an association between heparin and lower mortality in 2075 patients admitted with COVID-19 in 17 hospitals in Spain9. In a retrospective study including 449 patients with severe COVID-19 in China, 99 of which received heparin, 28-day mortality of heparin users was lower than that of non-users in patients with D-dimer levels more than six-fold the upper limit of normal7. An observational study including 2,773 patients hospitalized with laboratory-confirmed COVID-19 within the Mount Sinai Health System in New York City showed that systemic anticoagulation was associated with improved outcomes6. We recently found that low molecular weight heparin was the only therapeutic factor independently associated with survival in a propensity-score adjusted analysis on 315 patients with COVID-1910. As for other systemic diseases with high prevalence in older population, the prescription of an anticoagulation treatment should be weighed against the risk of bleeding. Otherwise, this may lead to controversial results. Indeed, Pesavento et al. showed that, in a cohort of 324 patients showed that (sub)therapeutic doses of anticoagulants, when compared to standard prophylactic ones, are related to a major risk of bleeding  despite no beneficial effect on overall mortality26.In our study only the treatment with low molecular weight heparin showed convincing evidence for an attenuation of the mortality risk. This is in line with the current literature: observations about anticoagulation therapy show promising results and expert opinions have recommended the use of prophylactic low molecular weight heparin in patients with COVID-19 in absence of clinical contraindicationsIn our study, we report, for the first time, that the benefit from anticoagulation therapy was more evident in subjects with higher hsTnT elevation. The positive effects obtained from the prescription of low molecular weight heparin and particularly in patients with higher hsTnT levels confirm the contribution of systemic coagulopathy to the myocardial injury detected in COVID-19. Moreover, due to the bleeding risk implied with the LMWH treatment, the identification of parameters that enable selection of patients who could receive the greatest benefits from anticoagulation therapies might be crucial to optimize the risk–benefit balance related to this treatment. Our data suggest that a prophylactic dose of low molecular weight heparin during admission might be particularly indicated in patients presenting with mild to moderate elevation of hsTnT levels. If these patients might benefit from the use of higher dose thromboprophylaxis than is generally given remains unknown. However, ongoing multicentre, randomized, controlled trials will be able to address this question .27. Similarly, CRP is commonly used in clinical practice to monitor the severity of the inflammatory response resulting from acute infections and is also considered a good marker to assess the inflammatory-related risk of cardiovascular events28. The analysis on the beneficial effects of treatments should be interpreted cautiously, as it was not conducted on randomized groups and might be therefore affected by several measured and unmeasured confounding factors. Furthermore, we were not able to recover clear information on the indication for anticoagulation treatment.Our study has some strengths and limitations. The availability of makers informing on the severity of hypoxia, inflammatory response and coagulation abnormalities provided us with the opportunity to explore their relative importance in causing an increase in the hsTnT level and its association with mortality. In a subset of patients, serial measures of hsTnT, CRP and D-dimer were also available, making it possible, for the first time, to relate changes in hsTnT levels with the fluctuation of the inflammatory and coagulation responses to the disease. The presence of several outcome measurements enabled the assessment of the relative importance of the different pathways explored in the study with multiple complications of patients with COVID-19 infection. However, our study is monocentric, thus the results might be affected by local practice in the management of the COVID-19 infection and not be generalizable to other health care settings. Also, we used only D-dimer and CRP as markers of coagulation abnormalities and inflammation. Furthermore, given the pressure imposed by the elevated number of admissions in a short timeframe, we were unable to provide a better characterization of the myocardial injury with other, well-established circulating markers (such as cardiac natriuretic peptides) or with imaging studies (including echocardiography). This has also precluded the opportunity to explore which proportion of the described changes in the marker of cardiac injury could be attributed to pulmonary haemodynamic changes and their potential relationships with the inflammatory and coagulation abnormalities reported in our population. For the same reasons, we were not able to obtain a more accurate assessment of the COVID-19-related coagulopathy and inflammatory response. However, previous report have shown that the elevation in the circulating levels of D-dimer is amongst the most typical characteristics of the coagulopathy characterizing the SARS-CoV-2 infection and its assessment is commonly used in clinical practice as a screening tool for the presence of trombo-embolic events in other diseasesIn summary, our data support the hypothesis that the increase of hsTnT levels detected in COVID-19 infection is multifactorial, with inflammation and coagulation responses representing important factors contributing to this clinical manifestation. CRP can be used to monitor inflammation but might not represent the most accurate marker to discriminate between the severity of the respiratory and systemic inflammatory involvement during SARS-CoV-2-related pneumonia. Instead, an elevation of the D-dimer might sign the evolution towards a systemic involvement of the infection, with the development of a coagulopathy that could lead to multiorgan failure. Low molecular weight heparin is, indeed, a promising treatment to counteract the risk of mortality related to systemic COVID-19. Further studies are needed to clearly define the pathogenesis of COVID-19 and provide useful insight for tailored therapies. Troponin, in a context where risk stratification is of primary importance, due to its ability to predict mortality and the relevance of if prognostic capacity even in terms of identification of patients who would benefit more from specific treatments (such as anticoagulation), might play a fundamental role.


Protein palmitoylation, the reversible addition of palmitate to proteins, is a dynamic post-translational modification. Both membrane  and cytoplasmic proteins  are substrates. In mammals, palmitoylation is mediated by 23-24 palmitoyl acyltransferases (PATs), also called ZDHHCs for their catalytic aspartate-histidine-histidine-cysteine (DHCC) domain. PATs are integral membrane proteins found in cellular membranes. In the palmitoylation cycle, palmitate is removed by the depalmitoylation enzymes, acyl palmitoyl transferases (APT1 and 2), and α/β Hydrolase domain-containing protein 17 (ABHD17A-C). These are cytoplasmic proteins that are targeted to membranes where they are substrates for PATs. The second class of depalmitoylating enzymes are palmitoyl thioesterases, PPT1 and 2, discovered through their association with infantile neuronal ceroid lipofuscinosis. These are secreted proteins found in the lumen of intracellular organelles, primarily lysosomes, where their function as depalmitoylating enzymes is unclear.Our understanding of protein palmitoylation is still in its infancy, with the most basic questions still to be addressed. Have all of the proteins involved in the process of palmitoylation been identified? What are the molecular details for how palmitoylation and depalmitoylation occurs and how substrates are targeted to and bind to PATs and deplamitoylating enzymes? What roles do the different PATs and deplamitoylating enzymes play? How is palmitoylation tied to and regulated by lipid metabolism and other metabolic pathways? How do other post-translational modifications at overlapping sites on proteins, such as phosphorylation and nitrosylation, regulate palmitoylation and vice versa?One area where progress is being made is characterizing the different functions of palmitoylation in neurons. Here several groups have investigated the role of palmitoylation in different cell biological processes involved in axon growth, synapse formation, synaptic plasticity, and lysosomal function at synapses. Of special interest are the links these processes have to neurodegenerative diseases, in particular infantile neuronal ceroid lipofuscinosis and Huntington's Disease.Matt et al. review the dynamic cycling of protein palmitoylation and its role in activity-dependent synapse formation. Palmitoylation of postsynaptic proteins ranges from neurotransmitter receptors such as the N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) to scaffolding proteins such as postsynaptic density protein 95 (PSD-95). Especially, PSD-95 palmitoylation is a critical regulator of many aspects of synaptic plasticity. For example, another study from the same group demonstrates a mechanism by which a reduction in PSD-95 palmitoylation mediates chemically induced long-term depression (Chowdhury and Hell). Following calcium influx, binding of Ca2+/calmodulin to PSD-95 triggers a reduction in PSD-95 palmitoylation, and disperses the scaffolding protein and associated AMPARs out of the postsynaptic membrane. Remarkably, palmitoylation regulates PSD-95 conformation and orientation, thereby controlling the retention of AMPARs in PSDs  complex 1 (mTORC1) signaling complex on lysosomal membranes. Interestingly, lysosomes translocate to dendritic spines and regulate local protein degradation, thereby facilitating synaptic remodeling  and HIP14-like (HIP14L), directly interact with the huntingtin protein (Htt), and mutant Htt disrupts this interaction. Koster and Yoshii). Especially, Sapir et al. identified the lack of PPT1 function results in immature dendritic spines and a reduction in LTP expression. Another recent study also demonstrates the loss of PPT1 function results in the stagnation of the developmental subunit switch of the NMDAR subunit that correlates with immature spine formation. Consequently, the NMDAR dysregulation underlies the susceptibility to excitotoxicity (Koster et al., The lysosomal depalmitoylation enzyme PPT1 is associated with infantile neuronal ceroid lipofuscinosis. PPT1 is a crucial regulator of the autophagy-lysosome pathway and controls multiple steps of neuronal differentiation ranging from axonal guidance as well as pre- and post-synaptic functions (Progress in this field has been driven by new technical advantages that have allowed palmitoylation to be quantitatively studied, large numbers of substrates to be assayed, and the enzymes involved in the palmitoylation cycle identified. New tools for real-time analysis are finally becoming available. For example, it is now feasible to visualize the palmitoylated form of a particular protein (Gao and Hannoush, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The latest outbreak of pneumonia caused by SARS-CoV-2 presents a significant challenge to global public health and has a major impact on clinical microbiology laboratories. In some situations, such as patients in coma condition, the oropharyngeal or nasopharyngeal sampling is seldom feasible, and blood sampling could be an alternative. In the current article, a comprehensive literature search has been conducted for detecting coronavirus disease 2019 (COVID-19) using plasma or serum samples. To date, twenty-six studies have used SARS-CoV-2 nucleic acid in plasma or serum (RNAaemia) to diagnose COVID-19. The pros and cons are discussed in this article. While the detection of SARS-CoV-2 viral load in respiratory specimens is commonly used to diagnose COVID-19, detecting SARS-CoV-2 RNA in plasma or serum should not lose sight and it could be considered as an alternative diagnostic approach. The latest pneumonia epidemic caused by a novel coronavirus  termed coronavirus disease 2019 (COVID-19) has raised a major risk for global public health. It has had a significant impact on clinical microbiology laboratories over the last few months. Compared to the tests developed for other SARS-CoV diagnosis, the optimal test method to detect COVID-19 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) in respiratory samples , as for The review by Wang, distinctly showed that the nucleic acids were presented in the serum or plasma samples for all novel coronaviruses, but the timeout of viremia remained unclear . In anotOn February 2, 2020, the Hubei Province Center for Disease Control and Prevention conducted follow-up tests on plasma samples by reporting poor positive results close to the limit of detection. After a week, donor-collected throat swab specimens became rather positive, suggesting the presence of an extremely low viral load in plasma samples . Peng etViral RNA in blood has been detected in COVID-19 patients on the first 2 to 3 days after the onset of symptoms; however, there is no evidence on the plasma and serum viral load during the incubation period , 11. ZheChen et al. reported that the combination of the substantial level of IL-6 with a meaningful Ct (cycle threshold) value of viral RNA in serum samples can be considered as an effective and accurate biomarker unveiling adverse outcomes . Since tIn a recent study, Zhang et al.  reportedIn conclusion, while the detection of SARS-CoV-2 viral load in respiratory specimens is commonly used to diagnose COVID-19, detecting SARS-CoV-2 nucleic acid in plasma or serum should not lose sight. In some situations, such as patients in coma condition, the oropharyngeal or nasopharyngeal sampling is seldom feasible, and thus blood sampling could be an alternative. Moreover, the blood test could have less harm for the hospital staffs who conduct the sampling with the respiratory swaps. It should be noted that the detection of viral RNAaemia might not reveal active infection, but rather at times of post-viral infection. Furthermore, RNA detection by itself does not necessarily indicate the presence of live virus and only patients having positive blood tests with clinical symptoms should receive medical treatment.However, it remains unclear that whether the RNAaemia is associated with the frequency of cytokine storms or it is related to a particular type of patients. Another challenge for the identification of viral RNA in blood samples may be due to the small amounts of RNA in plasma or serum samples. In addition, large amounts of background RNA can mask the low-abundance of viral RNA fragments leading false negative outcomes. Further investigations in future studies are needed to lower the false negative results. We should keep in mind that although the sampling of oropharyngeal/nasopharyngeal compartments may be challenging, it may not be impossible. This may be justified further as blood specimens may not help in better yields. Once more, further studies are needed.


This cross-sectional study reviews the medical records of 1524 patients with cancer treated at a single tertiary care hospital in Wuhan, China, to evaluate the characteristics associated with transmission of the SARS-CoV-2 virus. Moreover, cancer treatments such as chemotherapy and radiotherapy are immunosuppressive. Here, we report the incidence and outcomes of SARS-CoV-2 infection in cancer patients who were treated at a tertiary cancer institution in Wuhan.In December 2019, an outbreak of 2019 novel coronavirus disease (COVID-19) occurred in Wuhan, Hubei, which has been linked to the severe adult respiratory syndrome coronavirus 2 (SARS-CoV-2). It is characterized by rapid human-to-human transmission from droplet contamination.We reviewed the medical records, including demographic, clinical, and treatment data of 1524 patients with cancer who were admitted to the Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, from December 30, 2019, to February 17, 2020 (data cutoff date). COVID-19 pneumonia was diagnosed based on the updated COVID-19 Diagnostic Criteria, 5th Edition . OutcomeThis retrospective study was approved by the Zhongnan Hospital of Wuhan University ethics committee (2020039). Waiver of informed consent was approved for the aggregated data; verbal informed consent was obtained from the living patients with COVID-19.We estimated the infection rate of SARS-CoV-2 in patients with cancer from our single institution at 0.79% . This was higher than the cumulative incidence of all diagnosed COVID-19 cases that was reported in the city of Wuhan over the same time period . Clinical details on the cancer diagnoses and treatment history are summarized in We also interrogated the association of SARS-Cov-2 infection with age and concurrent NSCLC diagnosis. Of the 1524 patients with cancer who were screened, 228 had NSCLC. We found that patients with NSCLC older than 60 years had a higher incidence of COVID-19 than those aged 60 years or younger (4.3% vs 1.8%) .4 This study highlights the following observations: patients with cancer from the epicenter of a viral epidemic harbored a higher risk of SARS-CoV-2 infection  compared with the community. However, fewer than half of these infected patients were undergoing active treatment for their cancers. Next, we observed that older patients (>60 years) and patients with NSCLC may be at risk of COVID-19. Nonetheless, a population study of 1099 patients with COVID-19 did not indicate that age was associated with susceptibility to infection.5 A larger sample size in patients with cancer will resolve these potential associations. Finally, our findings imply that hospital admission and recurrent hospital visits are potential risk factors for SARS-CoV-2 infection.It is hypothesized that patients with cancer may be susceptible to an infection during a viral epidemic owing to their immunocompromised status.We propose that aggressive measures be undertaken to reduce frequency of hospital visits of patients with cancer during a viral epidemic going forward. For patients who require treatment, proper isolation protocols must be in place to mitigate the risk of SARS-CoV-2 infection.