Emergence and evolution of SARS-CoV-2 genetic variants during the Cuban epidemic.

The SARS CoV-2 D614G variant circulated in Cuba in 2020. New viral variants were detected after the opening of the border in November 2020. We show the results of the genomic surveillance in Cuba from December 28, 2020, to September 28, 2021 and their relationship to the epidemiological situation in the country. A total of 1,406 nasopharyngeal exudates from COVID-19 patients were processed for RNA extraction and the 1836 bp fragment of the spike gene was amplified and sequenced. The mutations present were determined using the GISAID database. Prevalence ratios were estimated by fitting Poisson univariate and multivariate regression models to investigate associations between SARS-CoV-2 variant group (VOC, non-VOC) and disease outcome. Seventeen genetic variants were detected including VOC Alpha, Beta, Gamma and Delta, one variant of interest (VOI) (Lambda) and two previous VOI (A.2.5.1 and Zeta/P.2). Beta (34.77%), Delta (24.89%) and D614G (19%) variants were the most frequently detected. By June, Delta increased in frequency, displacing Beta. Disease severity increased significantly with age and VOC (PR =1.98, IC 95%: 1.33-3.05, p <0.05). Genomic surveillance allowed us to identify the upsurge of novel variants. Coinciding with the higher epidemic period, multiple variants were co-circulating. Although we cannot rule out that failure in the transmission containment measures occurred, the increase in the number of cases associated with the circulation of several variants, particularly the Beta and Delta variants is highly suggestive. A greater association of Beta variant with clinical severity and Delta variant with a greater transmissibility was observed.

Evaluación de ensayos ultramicroanalíticos para la detección de anticuerpos contra el SARS-CoV-2 / Evaluation of ultramicroanalytic assays to detect antibodies against SARS-CoV-2

Introducción: En el presente trabajo se muestran los resultados de la validación de los ensayos serológicos in vitro para la detección de anticuerpos IgM, IgG y anticuerpos totales contra el SARS-CoV-2 UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 y UMELISA SARS-CoV-2 IgG desarrollados por el Centro de Inmunoensayo (CIE). Métodos: Se utilizaron paneles de muestras de suero de individuos negativos y de casos confirmados de COVID-19 para determinar el desempeño analítico de cada ensayo. Resultados: La especificidad clínica de los ensayos UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 y UMELISA SARS-CoV-2 IgG fue del 100 por ciento en todos los ensayos y la especificidad analítica fue de 100 por ciento para los dos primeros ensayos y del 93,1 por cientopara el último. La sensibilidad clínica fue de 64,3, 80,8 y 97,5 por ciento, respectivamente. El valor predictivo positivo fue de 100 por ciento en todos los ensayos, en tanto que el negativo osciló entre 83,3 y 95,2 por ciento. La concordancia fluctuó entre 92,4 y 96,9 por ciento y el índice kappa de todos los ensayos fue muy bueno. La sensibilidad de los ensayos se incrementó a 82,76, 96,5 y 100 por ciento, respectivamente, en las muestras de suero colectadas con más de 14 días de iniciado el cuadro clínico. Conclusiones: Los ensayos demostraron una elevada sensibilidad y especificidad, lo que permite contar con herramientas basadas en una tecnología desarrollada en Cuba que posibilita la realización de estudios serológicos, vigilancia epidemiológica y de otro tipo, incluyendo los relacionados con vacunas en una plataforma con amplia distribución nacional(AU)

Introduction: This paper shows the results obtained in the validation of in vitro serological assays to detect IgM, IgG antibodies, and total antibodies against SARS-CoV-2 UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 and UMELISA SARS-CoV-2 IgG developed by the Immunoassay Center. Methods: Panels of serum samples from negative and COVID-19 confirmed patients were used to determine the analytical performance of each assay. Results: UMELISA SARS-CoV-2 IgM, UMELISA ANTI-SARS-CoV-2 and UMELISA SARS-CoV-2 IgG assays demonstrated 100 percent clinical specificity for all assays; and 100 percent analytical specificity for the first two assays, and 93.1 percent for the last one. Clinical sensitivity was 64.3 percent, 80.8 percent and 97.5 percent, respectively. The positive predictive value was 100 percent in all assays, while the negative predictive value ranged from 83.3 percent to 95.2 percent. Concordance varied from 92.4 percent to 96.9 percent, and kappa index in every assay was very good. Assays sensitivity increased to 82.7 percent, 96.5 percent and 100 percent, respectively for serum samples collected more than 14 days after onset of the symptoms. Conclusions: The assays demonstrated high sensitivity and specificity, which allows us to have Cuban technology-based tools for serological, epidemiological surveillance, and other types of studies, including those related to vaccines on a platform with wide national distribution(AU)

SARS-CoV-2: theoretical analysis of the proposed algorithms to the enhancement and segmentation of high-resolution microscopy images-Part II.

Today is a reality that the novel coronavirus SARS-Cov-2 has become a global pandemic. For this reason, the study of real microscopic images of this coronavirus is of great importance, as it allows us to carry out a more precise research on it. However, as we pointed out in a former paper as reported by Roberto Rodríguez (SARS-CoV-2: Enhancement and Segmentation of High-Resolution Microscopy Images. Part I", Sent to Signal, Image and Video Processing Video Processing, Springer, New York, 2020), many times these microscopic images present some blurring problems, which are always susceptible to be improved. The aim of this work is to carry out a theoretical analysis of the proposed algorithms to enhancement and segmentation of these microscopic images, which is important for the design and development of future algorithms before new epidemics.

JM-20 treatment prevents neuronal damage and memory impairment induced by aluminum chloride in rats.

The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl3) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl3 -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration.

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

SARS-CoV-2: enhancement and segmentation of high-resolution microscopy images-Part I.

Possibly, and due to poor eating habits and unhealthy lifestyle, many viruses are transmitted to human people. Such is the case, of the novel coronavirus SARS-Cov-2, which has expanded of exponential way, practically, to whole world population. For this reason, the enhancement of real microscopic images of this coronavirus is of great importance. Of this way, one can highlight the S-spikes and visualizing those areas that show a high density, which are related to active zones of viral germination and major spread of the virus. The SARS-Cov-2 images were captured from nasopharyngeal samples of Cuban symptomatic individuals (RT-PCR positives for SARS-CoV-2) and processed via scanning electron microscopy. However, many times these microscopic images present some blurring problems, and the S-spikes do not look well defined. Therefore, the aim of this work is to propose new computational methods to carry out enhancement and segmentation of SARS-Cov-2 high-resolution microscopic images. The proposed strategy obtained very satisfactory results, and we validated its performance, together with specialist physicians, on a set of 1005 images. Due to the importance of the obtained results, this first work will be addressed to the application of the proposed algorithm. A second paper will deeply analyze the theory related to these algorithms.

Association of Early Nasopharyngeal Immune Markers With COVID-19 Clinical Outcome: Predictive Value of CCL2/MCP-1.

Early recognition of severe forms of coronavirus disease 2019 (COVID-19) is essential for an opportune and effective intervention, reducing life-risking complications. An altered inflammatory immune response seems to be associated with COVID-19's pathogenesis and progression to severity. Here we demonstrate the utility of early nasopharyngeal swab samples for detection of the early expression of immune markers and the potential value of CCL2/MCP-1 in predicting disease outcome.

Safety evaluation of the venom from scorpion Rhopalurus junceus: Assessment of oral short term, subchronic toxicity and teratogenic effect.

Rhopalurus junceus is the most common scorpion in Cuba and the venom is often used as a natural product for anti-cancer therapy. Despite this, no study has been published concerning its toxicological profile. The aim of the study was characterizing the short-term, subchronic toxicity and the teratogenic potential of Rhopalurus junceus scorpion venom by oral route in mice. Short-term oral toxicity was test in both sexes NMRI mice that received 100 mg/kg/day of scorpion venom extract for 28 days. For the subchronic study, mice were administered with three doses (0.1, 10, and 100 mg/kg) by oral route for 90 days. Teratogenic potential was tested in pregnant mice administered from day 6-15 post conception. Significant differences were observed in body weight and food intake of animal treated for short-term and subchronic assays. Variations in serum urea and cholesterol were observed after 90 days oral treatment. Spontaneous findings not related to the treatment were reveal in histology evaluation. Exposure in pregnant mice did not produce maternal toxicity. Signs of embryo-fetal toxicity were not observed. The current study provides evidence that exposure to low or moderate dose of Rhopalurus junceus scorpion venom by oral route did not affect health of animals and has low impact on reproductive physiology.

Vaccine-mismatched influenza B/Yamagata lineage viruses in Cuba, 2012-2013 season.

Annual trivalent influenza vaccines contain one of influenza B lineages; influenza B/Victoria-lineage or influenza B/Yamagata viruses. Theoretically, these vaccines should protect against viruses expected to circulate in the next influenza season. The National Influenza Centers, based on surveillance data from National Reference Laboratories, selects the strains composing each annual trivalent or tetravalent vaccine. Nevertheless, in some epidemics, vaccine strains do not match genetically with circulating strains. The aim of the present study is to compare the HA1-domain of 42 influenza B viruses circulating in Cuba during the 2012-2013 season with the vaccine strain B/Wisconsin/01/2010-like virus from the B/Yamagata lineage, included in the 2012-2013 Northern-Hemisphere Influenza vaccine. The efficacy of the influenza vaccine was also estimated. The analysis of the present study indicates that the B/Victoria and B/Yamagata lineages co-circulated in Cuba in the 2012-2013 season. In 2012-2013 season, according to the sequences analysis, trivalent vaccine did not match with the circulating strains. The present study also detected amino acid substitutions which could have altered the antigenic properties of HA gene. The results presented here suggest the need to consider a possible introduction of tetravalent influenza vaccine in Cuba, as has been recommended by the WHO to ensure higher levels of protection.

New genetic variants of influenza A(H1N1)pdm09 detected in Cuba during 2011-2013.

Influenza A(H1N1)pdm09 virus has evolved continually since its emergence in 2009. For influenza virus strains, genetic changes occurring in HA1 domain of the hemagglutinin cause the emergence of new variants. The aim of our study is to establish genetic associations between 35 A(H1N1)pdm09 viruses circulating in Cuba in 2011-2012 and 2012-2013 seasons, and A/California/07/2009 strain recommended by WHO as the H1N1 component of the influenza vaccine. The phylogenetic analysis revealed the circulation of clades 3, 6A, 6B, 6C and 7. Mutations were detected in the antigenic site or in the receptor-binding domains of HA1 segment, including S174P, S179N, K180Q, S202T, S220T and R222K. Substitutions S174P, S179N, K180Q and R222K were detected in Cuban strains for the first time.

Anti-hypernociceptive effect of mangiferin in persistent and neuropathic pain models in rats.

The present study examines the possible effect of the glucosylxanthone mangiferin (MG) on pain-related behaviors in a tonic acute pain model (formalin test at 5%) and in a chronic constriction injury (CCI) model to clarify the underlying transient and long-term mechanisms. Acute administration of MG (10-100mg/kg, i.p.) reduced licking/biting exclusivity in the tonic phase of formalin test in a naloxone and yohimbine-sensitive manner. This effect was enhanced by a nonselective nitric oxide synthase (NOS) inhibitor (NG-monomethyl-L-arginine) and by a non-competitive N-methyl-D-aspartate (NMDA) antagonist (ketamine), but it was reversed by the NOS substrate (L-arginine). Pre-treatment with intrathecal yohimbine prevented the anti-hypernociceptive effect of systemic MG. Pre-treatment during 4 days before surgical and 3 days after CCI with MG (50mg/kg, i.p.) reduced mechanical hypernociception and decreased the signs of Wallerian degeneration (WD) of the sciatic nerve. MG improved the PC-12 cellular viability exposure to glutamate-mediated neuronal death, also involved in neuropathic pain. The findings of this study suggest that MG shows ability to decrease tonic pain in the formalin test. A transient activity of this xanthone on nociceptive pathways mediated by α2 adrenergic receptors in cooperation with the opioid system could be involved, at least in part, in this effect. Its neuroprotective effect by preventing WD in mononeuropathic rats could be implicated in the mechano-antihypernociceptive long term mechanisms.

A novel multi-target ligand (JM-20) protects mitochondrial integrity, inhibits brain excitatory amino acid release and reduces cerebral ischemia injury in vitro and in vivo.

We previously showed that JM-20, a novel 1,5-benzodiazepine fused to a dihydropyridine moiety, possessed an anxiolytic profile similar to diazepam and strong neuroprotective activity in different cell models relevant to cerebral ischemia. Here, we investigated whether JM-20 protects against ischemic neuronal damage in vitro and in vivo. The effects of JM-20 were evaluated on hippocampal slices subjected to oxygen and glucose deprivation (OGD). For in vivo studies, Wistar rats were subjected 90 min of middle cerebral artery occlusion (MCAo) and oral administration of JM-20 at 2, 4 and 8 mg/kg 1 h following reperfusion. Twenty-four hours after cerebral blood flow restoration, neurological deficits were scored, and the infarct volume, histopathological changes in cortex, number of hippocampal and striatal neurons, and glutamate/aspartate concentrations in the cerebrospinal fluid were measured. Susceptibility to brain mitochondrial swelling, membrane potential dissipation, H2O2 generation, cytochrome c release, Ca2+ accumulation, and morphological changes in the organelles were assessed 24 h post-ischemia. In vitro, JM-20 (1 and 10 µM) administered during reperfusion significantly reduced cell death in hippocampal slices subjected to OGD. In vivo, JM-20 treatment (4 and 8 mg/kg) significantly decreased neurological deficit scores, edema formation, total infarct volumes and histological alterations in different brain regions. JM-20 treatment also protected brain mitochondria from ischemic damage, most likely by preventing Ca2+ accumulation in organelles. Moreover, an 8-mg/kg JM-20 dose reduced glutamate and aspartate concentrations in cerebrospinal fluid and the deleterious effects of MCAo even when delivered 8 h after blood flow restoration. These results suggest that in rats, JM-20 is a robust neuroprotective agent against ischemia/reperfusion injury with a wide therapeutic window. Our findings support the further examination of potential clinical JM-20 use to treat acute ischemic stroke.

Short-term intra-nasal erythropoietin administration with low sialic acid content is without toxicity or erythropoietic effects.

The objective of this investigation was to assess the toxicological potential of nasal formulation of erythropoietin with low sialic acid content (Neuro EPO) after 28 days of intra-nasal dosing in rats besides to evaluate the immunogenicity and erythropoietic effect of the test substance. Healthy Wistar rats of both sexes were used for 28 days subacute toxicity and immunogenicity assays. Doses evaluated were 3450, 4830 and 6900 UI/kg/day. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, antibodies determination, selected tissue weights and histopathological examination. Reversibility of toxic effects was evaluated at high dose 14 days after treatment period. Female B6D2F1 mice were used for evaluated erythropoietic effect of the nasal formulation. Hematological endpoints were examined every week during 28 days of intra-nasal dosing of 6900 UI/kg/day. Variations of hematological patterns were not observed after 28 days of intranasal dosing. A slight increase in glucose level of treated animals within the normal range was observed. This effect was not dose related and was reversible. Antibody formation was not observed in any of the test doses. Histopathological examination of organs and tissues did not reveal treatment induced changes. The administration of Neuro EPO in normocythaemic mice did not produce erythropoietic effect. These results suggest that Neuro EPO could be used as a neuroprotective agent, without significant systemic haematological side effects.

Absence of hematological side effects in acute and subacute nasal dosing of erythropoietin with a low content of sialic acid.

The use of human recombinant erythropoietin (EPO) as a neuroprotective agent is limited due to its hematological side effects. An erythropoietin along with a low content of sialic acid (rhEPOb), similar to that produced in the brain during hypoxia, may be used as a neuroprotective agent without risk of thrombotic events. The objective of this investigation was to assess the toxicological potential of a nasal formulation with rhEPOb in acute, subacute and nasal irritation assays in rats. Healthy Wistar rats (Cenp:Wistar) were used for the assays. In an irritation test, animals received 15 µl of rhEPOb into the right nostril. Rats were sacrificed after 24 h and slides of the nasal mucosa tissues were examined. Control and treated groups showed signs of a minimal irritation consisting of week edema and vascular congestion in all animals. In the acute toxicity test, the dose of 47,143 UI/kg was administered by nasal route. Hematological patterns, body weight, relative organ weight, and organ integrity were not affected by single dosing with rhEPOb. In the subacute toxicity test, Wistar rats of both sexes received 6,600 UI/kg/day for 14 days. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, selected tissue weights, and histopathological examinations. An increase of lymphocytes was observed in males that was considered to reflect an immune response to treatment. Histopathological examination of organs and tissues did not reveal treatment-induced changes. The administration of rhEPOb at daily doses of 6,600 UI/kg during 14 days did not produce hematological side effects. These results suggest that rhEPOb could offer the same neuroprotection as EPO, without hematological side effects.

Acute and subchronic oral toxicities of Calendula officinalis extract in Wistar rats.

We have studied the acute and subchronic oral toxicities of Calendula officinalis extract in male and female Wistar rats. A single acute C. officinalis extract dose of 2000 mg/kg dissolved in distilled water was administered by oral gavage for acute toxicity. Subchronic doses of 50, 250 and 1000 mg/kg/day were administered in drinking water. The major toxicological endpoints examined included animal body weight, water and food intake, selected tissue weights, and histopathological examinations. In addition, we examined blood elements: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count and blood clotting time and blood chemistry: glucose, total cholesterol, urea, total proteins, alkaline phosphatase, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In the acute study, there were no mortality and signs of toxicity. In the subchronic study, several of the blood elements were significantly affected in males and females after 90 days; hemoglobin, erythrocytes, leukocytes and blood clotting time. For blood chemistry parameters, ALT, AST and alkaline phosphatase were affected. Histopathological examination of tissues showed slight abnormalities in hepatic parenchyma that were consistent with biochemical variations observed. These studies indicate that the acute and subchronic toxicities of C. officinalis extract are low.

First report on fatal myocarditis associated with adenovirus infection in Cuba.

Myocarditis is caused frequently by viral infections of the myocardium. In the past, enteroviruses (EV) were considered the most common cause of myocarditis in all age groups. Other viruses that cause myocarditis are adenovirus and influenza viruses. Parvovirus B19 infection is associated sometimes with myocarditis. Members of the Herpesviridae family, cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) have been associated occasionally with myocarditis. During an atypical outbreak of acute febrile syndrome, eight children, with ages from 5 months to 15 years, died in cardiogenic shock due to myocarditis in July-August 2005, in the city of Havana, Cuba. Nested polymerase chain reaction (nPCR) and nested reverse transcription-PCR (nRT-PCR) were carried out on fresh heart muscle and lung tissue to analyze the genomic sequences of adenovirus, CMV, HHV-6, herpes simplex virus, Epstein-Barr virus (EBV), varizella zoster virus, influenza virus A, B, C, respiratory syncytial virus (RSV) A and B, parainfluenza viruses, rhinoviruses, coronavirus, flaviruses and enteroviruses. Evidence was for the presence of the adenovirus genome in 6 (75%) of the children. Phylogenetic analyses of a conserved hexon gene fragment in four cases showed serotype 5 as the causal agent. No others viruses were detected. Histological examination was undertaken to detect myocardial inflammation. After exclusion of other possible causes of death, the results indicated that viral myocarditis was the cause of death in patients with adenovirus infection.