Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples.

The Coronavirus Disease 19 (COVID-19) pandemic has caused an unexpected death toll worldwide. Even though several guidelines for the management of infectious corpses have been proposed, the limited number of post-mortem analyses during the pandemic has led to inaccuracies in the counting of COVID-19 deaths and contributed to a lack of important information about the pathophysiology of the SARS-CoV-2 infection. Due to the impossibility of carrying out autopsies on all corpses, the scientific community has raised the question of whether confirmatory analyses could be performed on exhumed bodies after a long period of burial to assess the presence of SARS-CoV-2 RNA. Post-mortem lung samples were collected from 16 patients who died from COVID-19 infection and were buried for a long period of time. A custom RNA extraction protocol was developed to enhance extraction of viral RNA from degraded samples and highly sensitive molecular methods, including RT-qPCR and droplet digital PCR (ddPCR), were used to detect the presence of SARS-CoV-2 RNA. The custom extraction protocol developed allowed us to extract total RNA effectively from all lung samples collected. SARS-CoV-2 viral RNA was effectively detected in all samples by both RT-qPCR and ddPCR, regardless of the length of burial. ddPCR results confirmed the persistence of the virus in this anatomical niche and revealed high viral loads in some lung samples, suggesting active infection at the time of death. To the best of our knowledge, this is the first study to demonstrate the persistence of SARS-CoV-2 viral RNA in the lung even after a long post-mortem interval (up to 78 days). The extraction protocol herein described, and the highly sensitive molecular analyses performed, could represent the standard procedures for SARS-CoV-2 detection in degraded lung specimens. Finally, the innovative results obtained encourage post-mortem confirmatory analyses even after a long post-mortem interval.

Current and innovative methods for the diagnosis of COVID­19 infection (Review).

The Coronavirus Disease 2019 (COVID­19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID­19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT­PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)­based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID­19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID­19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.

Association of Viral Infections With Oral Cavity Lesions: Role of SARS-CoV-2 Infection.

Different viral agents, such as herpesviruses, human papillomavirus, and Coxsackie virus, are responsible for primary oral lesions, while other viruses, such as human immunodeficiency virus, affect the oral cavity due to immune system weakness. Interestingly, it has been reported that coronavirus disease 2019 (COVID-19) patients can show cutaneous manifestations, including the oral cavity. However, the association between oral injuries and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still unclear. This narrative review aimed to summarize the available literature and provide an overview of oral lesions associated with COVID-19. An online literature search was conducted to select relevant studies published up to November 2020. The results of 17 studies showed variability in oral lesions associated with COVID-19, including ulcerations, aphthous-like lesions, and macules. The tongue, lips, and palate were the most frequent anatomical locations. According to current knowledge, the etiopathogenesis of multiple COVID-19-associated lesions seems to be multifactorial. The appearance of such lesions could be related to the direct or indirect action of SARS-CoV-2 over the oral mucosa cells, coinfections, immunity impairment, and adverse drug reactions. Nevertheless, COVID-19-associated oral lesions may be underreported, mainly due to lockdown periods and the lack of mandatory dispositive protection. Consequently, further research is necessary to determine the diagnostic and pathological significance of oral manifestations of COVID-19. All medical doctors, dentists, and dermatologists are encouraged to perform an accurate and thorough oral examination of all suspected and confirmed COVID-19 cases to recognize the disease's possible early manifestations.

Immune-checkpoint inhibitors from cancer to COVID­19: A promising avenue for the treatment of patients with COVID­19 (Review).

The severe acute respiratory syndrome associated coronavirus­2 (SARS­CoV­2) poses a threat to human life worldwide. Since early March, 2020, coronavirus disease 2019 (COVID­19), characterized by an acute and often severe form of pneumonia, has been declared a pandemic. This has led to a boom in biomedical research studies at all stages of the pipeline, from the in vitro to the clinical phase. In line with this global effort, known drugs, currently used for the treatment of other pathologies, including antivirals, immunomodulating compounds and antibodies, are currently used off­label for the treatment of COVID­19, in association with the supportive standard care. Yet, no effective treatments have been identified. A new hope stems from medical oncology and relies on the use of immune­checkpoint inhibitors (ICIs). In particular, amongst the ICIs, antibodies able to block the programmed death­1 (PD­1)/PD ligand-1 (PD­L1) pathway have revealed a hidden potential. In fact, patients with severe and critical COVID­19, even prior to the appearance of acute respiratory distress syndrome, exhibit lymphocytopenia and suffer from T­cell exhaustion, which may lead to viral sepsis and an increased mortality rate. It has been observed that cancer patients, who usually are immunocompromised, may restore their anti­tumoral immune response when treated with ICIs. Moreover, viral-infected mice and humans, exhibit a T­cell exhaustion, which is also observed following SARS­CoV­2 infection. Importantly, when treated with anti­PD­1 and anti­PD­L1 antibodies, they restore their T­cell competence and efficiently counteract the viral infection. Based on these observations, four clinical trials are currently open, to examine the efficacy of anti­PD­1 antibody administration to both cancer and non­cancer individuals affected by COVID­19. The results may prove the hypothesis that restoring exhausted T­cells may be a winning strategy to beat SARS­CoV­2 infection.

SARS-CoV-2 pathophysiology and its clinical implications: An integrative overview of the pharmacotherapeutic management of COVID-19.

Common manifestations of COVID-19 are respiratory and can extend from mild symptoms to severe acute respiratory distress. The severity of the illness can also extend from mild disease to life-threatening acute respiratory distress syndrome (ARDS). SARS-CoV-2 infection can also affect the gastrointestinal tract, liver and pancreatic functions, leading to gastrointestinal symptoms. Moreover, SARS-CoV-2 can cause central and peripheral neurological manifestations, affect the cardiovascular system and promote renal dysfunction. Epidemiological data have indicated that cancer patients are at a higher risk of contracting the SARS-CoV-2 virus. Considering the multitude of clinical symptoms of COVID-19, the objective of the present review was to summarize their pathophysiology in previously healthy patients, as well as in those with comorbidities. The present review summarizes the current, though admittedly fluid knowledge on the pathophysiology and symptoms of COVID-19 infection. Although unclear issues still remain, the present study contributes to a more complete understanding of the disease, and may drive the direction of new research. The recognition of the severity of the clinical symptoms of COVID-19 is crucial for the specific therapeutic management of affected patients.

Cancer Management during COVID-19 Pandemic: Is Immune Checkpoint Inhibitors-Based Immunotherapy Harmful or Beneficial?

The coronavirus disease 2019 (COVID-19) is currently representing a global health threat especially for fragile individuals, such as cancer patients. It was demonstrated that cancer patients have an increased risk of developing a worse symptomatology upon severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) infection, often leading to hospitalization and intensive care. The consequences of this pandemic for oncology are really heavy, as the entire healthcare system got reorganized. Both oncologists and cancer patients are experiencing rescheduling of treatments and disruptions of appointments with a concurrent surge of fear and stress. In this review all the up-to-date findings, concerning the association between COVID-19 and cancer, are reported. A remaining very debated question regards the use of an innovative class of anti-cancer molecules, the immune checkpoint inhibitors (ICIs), given their modulating effects on the immune system. For that reason, administration of ICIs to cancer patients represents a question mark during this pandemic, as its correlation with COVID-19-associated risks is still under investigation. Based on the mechanisms of action of ICIs and the current evidence, we suggest that ICIs not only can be safely administered to cancer patients, but they might even be beneficial in COVID-19-positive cancer patients, by exerting an immune-stimulating action.

Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection.

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) is the gold standard method for the diagnosis of COVID­19 infection. Due to pre­analytical and technical limitations, samples with low viral load are often misdiagnosed as false­negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT­qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID­19 were analyzed by droplet digital PCR (ddPCR) and RT­qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)­approved probe for the SARS­CoV­2 N gene were used. SYBR­Green RT­qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT­qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT­qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10­fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT­qPCR for the diagnosis of COVID­19 infection in false­negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID­19 and the follow­up of positive patients until complete remission.

Cancer therapy and treatments during COVID-19 era.

The COVID-19 pandemic has put a serious strain on health treatments as well at the economies of many nations. Unfortunately, there is not currently available vaccine for SARS-Cov-2/COVID-19. Various types of patients have delayed treatment or even routine check-ups and we are adapting to a virtual world. In many cases, surgeries are delayed unless they are essential. This is also true with regards to cancer treatments and screening. Interestingly, some existing drugs and nutraceuticals have been screened for their effects on COVID-19. Certain FDA approved drugs, vitamin, natural products and trace minerals may be repurposed to treat or improve the prevention of COVID-19 infections and disease progression. This review article will summarize how the treatments of various cancer patients has changed during the COVID-19 era as well as discuss the promise of some existing drugs and other agents to be repurposed to treat this disease.