Long COVID: plasma levels of neurofilament light chain in mild COVID-19 patients with neurocognitive symptoms.

It is well known the potential of severe acute respiratory coronavirus type 2 (SARS-CoV-2) infection to induce post-acute sequelae, a condition called Long COVID. This syndrome includes several symptoms, but the central nervous system (CNS) main one is neurocognitive dysfunction. Recently it has been demonstrated the relevance of plasma levels of neurofilament light chain (pNfL), as a biomarker of early involvement of the CNS in COVID-19. The aim of this study was to investigate the relationship between pNfL in patients with post-acute neurocognitive symptoms and the potential of NfL as a prognostic biomarker in these cases. A group of 63 long COVID patients ranging from 18 to 59 years-old were evaluated, submitted to a neurocognitive battery assessment, and subdivided in different groups, according to results. Plasma samples were collected during the long COVID assessment and used for measurement of pNfL with the Single molecule array (SIMOA) assays. Levels of pNfL were significantly higher in long COVID patients with neurocognitive symptoms when compared to HC (p = 0.0031). Long COVID patients with cognitive impairment and fatigue symptoms presented higher pNfL levels when compared to long COVID patients without these symptoms, individually and combined (p = 0.0263, p = 0.0480, and 0.0142, respectively). Correlation analysis showed that levels of cognitive lost and exacerbation of fatigue in the neurocognitive evaluation had a significative correlation with higher pNfL levels (p = 0.0219 and 0.0255, respectively). Previous reports suggested that pNfL levels are related with higher risk of severity and predict lethality of COVID-19. Our findings demonstrate that SARS-CoV-2 infection seems to have a long-term impact on the brain, even in patients who presented mild acute disease. NfL measurements might be useful to identify CNS involvement in long COVID associated with neurocognitive symptoms and to identify who will need continuous monitoring and treatment support.

miRNAs: Targets to Investigate Herpesvirus Infection Associated with Neurological Disorders.

Herpesvirus is associated with various neurological disorders and a specific diagnosis is associated with a better prognosis. MicroRNAs (miRNAs) are potential diagnostic and prognostic biomarkers of neurological diseases triggered by herpetic infection. In this review, we discuss miRNAs that have been associated with neurological disorders related to the action of herpesviruses. Human miRNAs and herpesvirus-encoded miRNAs were listed and discussed. This review article will be valuable in stimulating the search for new diagnostic and prognosis alternatives and understanding the role of these miRNAs in neurological diseases triggered by herpesviruses.

Serological Evidence of Hepatitis E Virus Infection in Brazilian Equines.

Hepatitis E virus (HEV) infection has been demonstrated in various animal species; those recognized as potential zoonotic reservoirs pose a considerable risk to public health. In Brazil, HEV-3 is the only genotype identified in humans and swine nationwide, in a colony-breeding cynomolgus monkey and, recently, in bovines and capybara. There is no information regarding HEV exposure in the equine population in Brazil. This study aimed to investigate anti-HEV antibodies and viral RNA in serum samples from horses slaughtered for meat export and those bred for sport/reproduction purposes. We used a commercially available ELISA kit modified to detect species-specific anti-HEV, using an anti-horse IgG-peroxidase conjugate and evaluating different cutoff formulas and assay precision. Serum samples (n = 257) were tested for anti-HEV IgG and HEV RNA by nested RT-PCR and RT-qPCR. The overall anti-HEV seroprevalence was 26.5% (68/257) without the detection of HEV RNA. Most municipalities (53.3%) and farms (58.8%) had positive horses. Animals slaughtered for human consumption had higher risk of HEV exposure (45.5%) than those bred for sports or reproduction (6.4%) (p < 0.0001). The statistical analysis revealed sex and breeding system as possible risk-associated factors. The first serological evidence of HEV circulation in Brazilian equines reinforces the need for the surveillance of HEV host expansion in a one-health approach.

miRNAs in Neurological Manifestation in Patients Co-Infected with SARS-CoV-2 and Herpesvírus 6 (HHV-6).

Human herpesviruses (HHVs) can establish latency and be reactivated, also are neurotropic viruses that can trigger neurological disorders. HHV-6 is a herpesvirus that is associated with neurological disorders. Studies have reported the detection of HHV-6 in patients with COVID-19 and neurological manifestations. However, specific diagnoses of the neurological disorders caused by these viruses tend to be invasive or difficult to interpret. This study aimed to establish a relationship between miRNA and neurological manifestations in patients co-infected with COVID-19 and HHV-6 and evaluate miRNAs as potential biomarkers. Serum samples from COVID-19 patients in the three cohorts were analyzed. miRNA analysis by real-time polymerase chain reaction (qPCR) revealed miRNAs associated with neuroinflammation were highly expressed in patients with neurological disorders and HHV-6 detection. When compared with the group of patients without detection of HHVs DNA and without neurological alterations, the group with detection of HHV-6 DNA and neurological alteration, displayed significant differences in the expression of mir-21, mir-146a, miR-155 and miR-let-7b (p < 0.01). Our results reinforce the involvement of miRNAs in neurological disorders and provide insights into their use as biomarkers for neurological disorders triggered by HHV-6. Furthermore, understanding the expression of miRNAs may contribute to therapeutic strategies.

Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

The use of oral fluid (OF) samples for serological diagnosis of parvovirus B19 infection during outbreaks of erythema infectiosum had already been demonstrated, but the feasibility of using OF for the characterization of B19 genotypes circulating during outbreaks has not been described. The aim of this study was to assess the use of "in-house" PCR-based assays as a powerful tool for a rapid diagnosis and molecular characterization of B19 strains in OF samples during outbreaks. Paired serum and OF samples collected from anti-B19 IgM-positive patients, during two outbreaks of ertythema infectiosum (1999-2000 and 2004-2005), were tested by conventional (cPCR) and quantitative PCR (qPCR). qPCR was more sensitive than cPCR for detecting B19-DNA in both OF and serum. Overall, OF presented lower viral load (9.97 × 106 UI/mL) than serum (2.42 × 1010 UI/mL) and this difference was statistically significant. All OF samples obtained from patients in the age group < 14 years presented low viral load (< 104 IU/mL). No correlation was found between viral load and the number of days of onset of rash. Sequence analysis from PCR positive OF samples confirmed the circulation of subgenotype 1a (G1a) during these outbreaks. Our findings indicate that PCR-based assays may fail to detect B19-DNA in approximately 50% of OF compared to serum samples. Nevertheless, our study has shown for the first time that the genome sequence of the amplicon from non-invasive clinical sample is useful for molecular genotyping and may be a tool to clarify the genetic diversity of B19 strains circulating in distinct outbreaks.

Herpesvirus and neurological manifestations in patients with severe coronavirus disease.

BACKGROUND: Certain clinical manifestations of coronavirus disease (COVID-19) mimic those associated with human herpesvirus (HHV) infection. In this study, we estimated the prevalence of herpesvirus in patients with COVID-19 and determined if coinfection is associated with poorer outcomes and neurological symptoms. METHODS: We analyzed samples of 53 patients diagnosed with COVID-19. The samples were evaluated for the presence of alphaherpesviruses, betaherpesviruses, and gammaherpesviruses, and the viral loads were quantified using quantitative polymerase chain reaction (qPCR) method. RESULTS: Among the patients, in 79.2% had detection at least one type of herpesvirus. HHV-6 (47.2%), cytomegalovirus (43.3%), and HHV-7 (39.6%) showed the highest detection rates. Patients with a high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) load were more likely to show herpes simplex virus 1 detection (p = 0.037). Among patients coinfected with SARS-CoV-2 and HHVs, 26.4% showed central nervous system-associated neurological symptoms and herpetic manifestations. A statistically significant association was observed between neurological changes and HHV-6 detection (p = 0.034). CONCLUSIONS: The findings showed a high prevalence of herpesvirus in patients with COVID-19. Furthermore, even though SARS-CoV-2 and HHV coinfection was not associated with poorer outcomes, the findings demonstrated the association between neurological symptoms and HHV-6 detection.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Parvovirus B19 Infection in a Fatal Case of Acute Liver Failure.

B19V has been proposed as an etiologic agent for hepatitis, mainly in children, but this is a rare clinical occurrence. In this article, we report a case of non-A-E acute liver failure in an immunocompetent child with B19 infection. The clinical findings of severe anemia and pancytopenia combined with the detection of anti-B19 Immunoglobulin G (IgG), B19 DNA and B19 mRNA in liver indicate a persistent infection and suggest a diagnosis of parvovirus B19-associated acute liver failure.

A Rare Case of Transfusion Transmission of Hepatitis A Virus to Two Patients with Haematological Disease.

BACKGROUND: This paper describes the transmission of hepatitis A virus (HAV) to two blood recipients from a healthy donor that later presented to the blood bank with jaundice. METHODS: The RNA of HAV was detected by qualitative nested reverse transcription polymerase chain reaction (nested RT-PCR) and quantified by real-time RT-PCR. HAV RNA samples were genotyped by direct sequencing of PCR products. A sequence from a fragment of 168 bp from the VP1/2A HAV region was used to construct a phylogenetic tree. CASE REPORT: A 31-year-old male donor accepted for donation of a whole blood unit returned to the blood bank with clinical jaundice 20 days after donation. His serological and NAT tests were negative for HBV and HCV. Serological tests for HAV IgM and IgG were negative on donation sample but positive on follow-up sample, confirming donor's HAV acute infection. Both recipients of red blood cells (R1) and platelet concentrate (R2) from the same implicated donation were HAV IgM-negative and IgG-positive. Qualitative PCR was positive on samples from all three individuals and phylogenetic analysis of viruses proved HAV transmission to the two recipients of blood products. HAV viral load on donor follow-up sample and the platelet recipient was 1.3 and 1.5 × 10(3) IU/ml, respectively. The RBC recipient, also infected by HCV, was undergoing bone marrow transplantation and died from fulminant hepatitis, 26 days after the implicated HAV transfusion. CONCLUSION: The blood donor, a garbage collector, spontaneously returned to the blood bank when developing jaundice. This highlights the importance of donor education to immediately report to blood banks of any signs and symptoms related to infectious disease developed after blood donation. The fact that one immunocompromised patient with HCV infection died from fulminant hepatitis after receiving a HAV-contaminated platelet transfusion underpins the importance of a HAV vaccination program for these group of patients.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure.

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.