INTRACELLULAR SARS-COV-2 FOUNDIN THE SPERMATOZOA OF CONVALESCENT MEN WITH NEGATIVE PCR SEMEN SAMPLES REVEALED BY ELECTRON MICROSCOPY

INTRODUCTION AND OBJECTIVE: SARS-CoV-2 can invade different testicular cell types, such as spermatogonia, spermatids, Sertoli, and Leydig cells. We investigated the viral presence inside the sperm of negative PCR infected men up to 3 months after discharge from the hospital. METHODS: This cross-sectional study included 13 of a 26 moderate-to-severe SARS-CoV-2 infected men cohort (mean 34.3 ± 6.5 years;range: 21-50 years old). Patients were enrolled 30 to 90 days after the diagnosis. Semen samples were obtained by masturbation and processed within one hour according to WHO guidelines. All patients were PCR negative for the virus in the ejaculate. Samples were liquefied for 30 min at room temperature in 0.1M phosphate buffer before centrifuging at 500 g for 10 min. The supernatant was removed, and pellets were fixed in 2,5% v/v glutaraldehyde in 0.1M phosphate buffer for 2h at 4°C, post-fixed in 1% OsO4 for 1h at 4°C, stained overnight in 1% aqueous uranyl acetate. Then, the pellets were dehydrated sequentially in 30%, 70%, and 100% ethanol and embedded in epoxy resin. Ultrathin sections (70nm) were obtained in an ultramicrotome, collected on nickel grids, and double-stained by uranyl acetate and lead citrate. Micrographs were obtained with a Jeol JEM 1010 electron microscope (Tokyo, Japan, 80 kV). RESULTS: We identified viruses inside spermatozoa in 9/13 patients up to 90 days after discharge from the hospital. Moreover, in all 13 men, a type of DNA-based extracellular traps, probably in a cfDNAdependent manner, like described in the COVID-19 systemic inflammatory response. FIGURE: High magnification electron micrograph of a spermatozoon with the nucleus (nu) displaying the typical condensed chromatin. The remained cytoplasm contains several viral particles (ranging in diameter from 90 to 110 nm). The inset corresponds to a higher magnification of the boxed area containing two virions, showing the SARS-CoV-2 characteristics: viral envelope (white arrowhead), nucleocapsids (black arrowhead), and spike-like projections (white arrow). CONCLUSIONS: Although SARS-CoV-2 is not found in the infected men's semen, it was intracellularly present in the spermatozoa. The potential implications for assisted conception should be addressed. (Figure Presented).

ELEVATED ROS LEVELS AND DNA FRAGMENTATION IN SPERM FROM CONVALESCENT MEN IN SARS-COV-2 INFECTION

INTRODUCTION AND OBJECTIVE: Current evidence has proven the systemic nature of COVID19, including its involvement in the male reproductive tract. We aimed to investigate seminal parameters of moderate-to-severe COVID-19 men during the convalescence phase. METHODS: This cross-sectional study included 18 to 50-yearold men with confirmed moderate-to-severe COVID-19. Patients were enrolled 15 to 45 days after the diagnosis. After a urologist's initial clinical evaluation, semen samples were obtained by masturbation and processed within one hour. Semen analysis was performed using the World Health Organization (WHO) manual (6th edition). Sperm function tests were conducted in an andrology laboratory, including Reactive oxygen species (ROS), DNA fragmentation, lipid peroxidation, and Creatine Kinase (CK) analysis. An essential endocrine evaluation was performed. Patients with a history of disorders that could impair testicular function were excluded. A group of pre-vasectomy baseline samples was used as a control group. Statistical analysis was performed using R version 4.0.5. One-tailed and paired T-tests were used for comparisons between groups. RESULTS: The sample size was 26 men (mean 34.3±6.5 years;range: 21-50 years). Sperm concentration (mean 38.74±32, P <0.01) and total motile count (mean 55.3±66.8, P <0.01) were significantly reduced in the COVID-19 group. The DNA fragmentation (mean 41.1±29.2) and ROS (mean 4.84±8.7) were significantly higher in post-infection patients. Other parameters such as WHO/ Kruger morphology and progressive motility were also reduced in the disease group, albeit not statistically significant. Total testosterone (mean 409.2±201.2) was lower in the convalescent men. All semen samples were negative for SARS-CoV-2 using the PCR analysis. CONCLUSIONS: Our findings indicate that male reproductive injury can be a relevant component of SARS-CoV-2 systemic infection. High DNA fragmentation and ROS, hallmarks of tissue injury, might signal a direct testicular involvement. The morphological and functional damage could represent significant impairment of the male reproductive health if persistent after convalescence.

UTILIZAÇÃO DO PLASMA FRESCO CONVALESCENTE EM PACIENTES DE ALTO RISCO DURANTE FASES PRECOCES DE INFECÇÃO POR COVID-19

Objetivos: Dentre o arsenal terapêutico atual contra a COVID-19, a terapia com plasma convalescente proveniente de doadores recuperados da COVID-19 (CCP), pode ser benéfica em pacientes de alto risco que estejam em fase precoce desta infecção, especialmente até o quinto dia de sintomas. Relatamos abaixo dados iniciais de uma série de pacientes de alto risco com infecção por SARS-CoV-2 que receberam esta terapêutica. Material e métodos: As unidades de CCP foram coletadas por aférese e submetidas à titulação de anticorpos neutralizantes através da neutralização viral em placa. As unidades com títulos ≥ 160 foram liberadas para uso, constituídos pools de duas doações e submetidas ao tratamento de redução de patógenos pelo Intercept®. Os critérios de inclusão dos pacientes para o estudo foram: idade ≥ 60 anos, presença de comorbidades, diagnóstico confirmado de SARS-CoV-2 por RT-PCR e sintomas de COVID-19 com início ≤ 5 dias. Após solicitação médica e assinatura do termo de consentimento informado, os pacientes receberam pré-medicação (anti-histamínico) seguido de uma dose única de plasma convalescente (200 mL). O desfecho primário foi considerado óbito após 28 dias da transfusão e o secundário, internação após receber a transfusão ambulatorial. Resultados: Estudamos 120 pacientes entre 01/01/2021 e 31/07/2021, sendo 37 (30,8%) pacientes internados devido à condição clínica no momento da transfusão e 83 (69,2%) ambulatoriais;84 (70%) eram masculinos e 36 (30%) femininos. A média de idade foi de 66 anos. Quanto aos antecedentes vacinais: 40/120 (33,4%) haviam recebido pelo menos a primeira dose da vacina (período médio da vacina à transfusão de plasma de 46 dias) sendo 25/40 (62,5%) Coronavac, 14/40 (35%) Astrazeneca e 1/40 (0,5%) Pfizer. As comorbidades mais encontradas foram hipertensão arterial (57,5%), obesidade (36,7%), diabetes (28,4%), cardiopatia (25%), neoplasia (18,4%), imunodeficiência (13,4%) e pneumopatia (8,4%). Observamos 3 (2,5%) reações transfusionais leves (2 alérgicas, 1 RFNH), sem danos aos pacientes. Entre os pacientes internados, 5/37 (13,5%) foram a óbito, pela própria condição clínica de base, associada às comorbidades, principalmente imunossupressão. Tais óbitos ocorreram em média, 25 dias após a transfusão. Dos pacientes ambulatoriais, 14/83 (16,9%) internaram nos 28 dias seguintes à transfusão, permanecendo, em média, 15 dias no hospital. Neste grupo, houve apenas 1 (1,2%) óbito, por complicações hemorrágicas e cardiológicas, em paciente com importante imunossupressão por transplante hepático recente. Discussão: Nossos dados demonstram a viabilidade do tratamento precoce na infecção pelo SARS-CoV-2 utilizando-se plasma convalescente com altos títulos de anticorpos neutralizantes em pacientes de alto risco, internados ou não, prevenindo, inclusive, internações subsequentes.

Efficacy of ixekizumab vs. IL-2 vs. colchicine vs. Standard of care for the treatment of hospitalized patients with COVID-19: Preliminary results of a randomized clinical trial (struck: Survival trial using cytokine inhibitors)

Introduction: There are currently scarce approved treatments for Covid-19 and new options are needed. Objectives: To evaluate the efficacy of an IL-17 inhibitor vs. low-dose IL-2 vs. colchicine vs. standard of care for the treatment of hospitalized patients with Covid-19. Methods: This is an on-going multicenter, open-label, prospective, randomized, active-controlled, adaptive 4-arm study. Patients hospitalized with moderate to severe presentation of Covid-19 are being recruited according to predetermined eligibility criteria. Patients are randomized to one of the trial arms: ixekizumab (IL-17 inhibitor), low dose IL-2, colchicine (indirect IL-6 inhibitor), or standard treatment, each according to the pre-established periods and doses. The primary study outcome is the proportion of patients with clinical improvement, defined by an increase of two points on the WHO's ordinal scale at day 28. Secondary study outcome is in-hospital mortality. Results: Until May 27, 30 patients with similar baseline clinical and demographic features have been enrolled in the study. Table 1 describes the main efficacy outcomes for each of the study arms. Ixekizumab arm presented the greatest improvement in the WHO scale (71.4%), while colchicine arm presented the lowest mortality rate (0%), but no statistically significant differences have been observed, so far. Conclusion: These preliminary results suggest that Ixekizumab and colchicine should be further studied as potential therapeutic drugs for treating patients with moderate to severe Covid-19. (Figure Presented).