Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES: To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS: SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS: SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS: Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice.

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory-functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal.

Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.

Detection of Vaccinia Virus in Dairy Cattle Serum Samples from 2009, Uruguay.

We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out.

The role of Nav1.9 channel in the development of neuropathic orofacial pain associated with trigeminal neuralgia.

BACKGROUND: Trigeminal neuralgia is accompanied by severe mechanical, thermal and chemical hypersensitivity of the orofacial area innervated by neurons of trigeminal ganglion (TG). We examined the role of the voltage-gated sodium channel subtype Nav1.9 in the development of trigeminal neuralgia. RESULTS: We found that Nav1.9 is required for the development of both thermal and mechanical hypersensitivity induced by constriction of the infraorbital nerve (CION). The CION model does not induce change on Nav1.9 mRNA expression in the ipsilateral TG neurons when evaluated 9 days after surgery. CONCLUSIONS: These results demonstrate that Nav1.9 channels play a critical role in the development of orofacial neuropathic pain. New routes for the treatment of orofacial neuropathic pain focussing on regulation of the voltage-gated Nav1.9 sodium channel activity should be investigated.

Horizontal study of vaccinia virus infections in an endemic area: epidemiologic, phylogenetic and economic aspects.

Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7% of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.

Chemogenetic Silencing of NaV1.8-Positive Sensory Neurons Reverses Chronic Neuropathic and Bone Cancer Pain in FLEx PSAM4-GlyR Mice.

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging on activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.

Longitudinal study of humoral immunity against SARS-CoV-2 of health professionals in Brazil: the impact of booster dose and reinfection on antibody dynamics.

Introduction: The pandemic caused by SARS-CoV-2 has had a major impact on health systems. Vaccines have been shown to be effective in improving the clinical outcome of COVID-19, but they are not able to fully prevent infection and reinfection, especially that caused by new variants. Methods: Here, we tracked for 450 days the humoral immune response and reinfection in 52 healthcare workers from Brazil. Infection and reinfection were confirmed by RT-qPCR, while IgM and IgG antibody levels were monitored by rapid test. Results: Of the 52 participants, 19 (36%) got reinfected during the follow-up period, all presenting mild symptoms. For all participants, IgM levels dropped sharply, with over 47% of them becoming seronegative by the 60th day. For IgG, 90% of the participants became seropositive within the first 30 days of follow-up. IgG antibodies also dropped after this period reaching the lowest level on day 270 (68.5 ± 72.3, p<0.0001). Booster dose and reinfection increased the levels of both antibodies, with the interaction between them resulting in an increase in IgG levels of 130.3 arbitrary units. Conclusions: Overall, our data indicate that acquired humoral immunity declines over time and suggests that IgM and IgG antibody levels are not associated with the prevention of reinfection.

Equine Infectious Anemia Virus (EIAV): Evidence of Circulation in Donkeys from the Brazilian Northeast Region.

Equine infectious anemia (EIA) is listed by the World Organization for Animal Health (OIE) as one of the equine diseases that must be notified. No effective treatment or vaccine is available. EIA control is based on segregation and euthanasia of positive equids. The disease is caused by the equine infectious anemia virus (EIAV), a member of the genus Lentivirus of the Retroviridae family. Despite the importance of this disease in equids, EIA has been poorly studied in donkeys (Equus asinus). We evaluate the sanitary conditions related to EIAV in donkeys from a shelter of abandoned animals captured on the roads of the Ceará. A total of 124 donkeys were randomly selected, and three horses lived at the same shelter. The animals were clinically evaluated, and a group of the 20 animals was submitted to hematological tests. Three diagnostic tests for EIA were used, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) using EIAV recombinant protein gp90 (rgp90) and recombinant protein p26 (rp26) ELISA, and polymerase chain reaction (PCR) for detection of the EIAV tat-gag gene. From the donkeys, only 1 animal was positive using AGID 0.81% (1/124), compared to 21.8% (27/124) in the rgp90 and 10.5% (13/124) in the rp26 ELISA. Proviral DNA was detected by PCR tat-gag in 8.8% (11/124), and phylogenetic analysis confirms that the EIAV sequences of donkeys from the Brazilian Northeast grouped with Pantanal Brazilian sequences. Thus, in light of the results, we conclude that donkeys are carriers of EIAV and could be sources of infection.

Identification of large genetic variations in the equine infectious anemia virus tat-gag genomic region.

The aetiological agent of equine infectious anaemia (EIA) is the retrovirus equine infectious anemia virus (EIAV) that infects all members of the Equidae family. The EIA is widely disseminated in the Brazilian territory with a high seroprevalence in the Brazilian Pantanal and is mainly diagnosed using agar gel immunodiffusion (AGID). There are few complete EIAV genome sequences available in GenBank, which had an impact on molecular detection studies. In this study, we conducted molecular detection and sequencing of EIAV proviral DNA from Brazilian horses. We analysed the genomic region from exon 1 of tat to gag (tat-gag). Comparative serological tests, comprising AGID and two enzyme-linked immunosorbent assays (ELISAs), were also conducted. Of the 133 samples, 58 were positive in the tat-gag PCR, and 49 nucleotide sequences of 272 bp were obtained. Using this developed tat-gag PCR EIAV proviral DNA was detected in 7% of the AGID-negative samples and 26% of the AGID-negative samples were positive in at least one of the ELISA tests used. Using phylogenetic analysis, the Brazilian Pantanal EIAV sequences grouped in a different clade of EIAV sequences from other countries. Thus, the EIAV sequences can contribute to the knowledge of the tat-gag genomic region in the circulating viruses in the Brazilian Pantanal, in addition to providing new information about the genetic diversity. In addition, the serological results demonstrate the greater sensitivity of the ELISAs used in this study compared to AGID for EIA diagnosis.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Synthetic derivatives of the alpha- and beta-amyrin triterpenes and their antinociceptive properties.

Fifteen different derivatives of an alpha- and beta-amyrin mixture were synthesized by acylation with appropriate anhydride or acid chlorides and oxidation in the presence of tert-butyl chromate or PCC. The molecular structures of the obtained compounds were confirmed by means of IR and (1)H NMR spectra. The compounds were screened for antinociceptive activity using the acetic acid pain model. The 3-O-acyl derivatives alpha- and beta-amyrin propionate 4, alpha- and beta-amyrin hexanoate 6, and alpha- and beta-amyrin octanoate 7 were found to be the most active compounds of the series. In addition, we also have found that alpha- and beta-amyrin octanoate 7 was able to reduce acetic acid-induced abdominal constriction when administered by oral route. Furthermore, this compound reduced the nociceptive response induced by intraplantar injection of formalin.

Adverse childhood experience and rheumatic diseases.

It has been suggested that the adaptive stress response may be disrupted by life adverse events such as childhood maltreatment. To investigate if the number of adverse childhood experiences (ACEs) increases susceptibility to systemic lupus erythematosus (SLE), spondyloarthritis (SpA), scleroderma (SSc), and rheumatoid arthritis (RA), we interviewed 315 patients with rheumatic disease (100 SLE; 40 SSc; 60 SpA; 115 RA) and 272 controls, using questions of the ACEs study questionnaire validated to ask about experiences of childhood abuse, negligence, domestic violence, and household dysfunctions. The questionnaire score ranges from zero (best result) to 8 (worst scenario). Patients and controls did not differ regarding the median number of ACEs (3 in both groups, patient IQR = 2.5-5 vs. control IQR = 2-5, p = 0.45). Among the patients, 63.8% (201/315) presented ACE score ≥ 3, compared with 59.9% (163/272) of the controls (p = 0.31). The proportion of patients with at least 3 ACEs did also not differ among those with different rheumatic diseases. Specifically, 64% (64/100) of those with SLE, 60% (24/40) of those with SSc, 60% (36/60) of those with SpA, and 66.9% (77/115) of those with RA reported at least 3 ACEs. There was also no difference between the distribution of ACE scores and number of individuals with ACEs ≥ 3 between patients with different rheumatic diseases and controls. Nevertheless, there was a trend for association between higher ACE score and susceptibility to RA (p = 0.06). In this setting, the occurrence of ACEs was not associated with susceptibility to rheumatic diseases in adulthood.

Antinociceptive action of ethanolic extract obtained from roots of Humirianthera ampla Miers.

Humirianthera ampla Miers is a member of the Icacinaceae family and presents great amounts of di and triterpenoids. These chemical constituents in roots of Humirianthera ampla sustain not only the ethnopharmacological use against snake venom, but also some anti-inflammatory and analgesic properties of the plant. In this study we investigated the antinociceptive action of the ethanolic extract (EE) from roots of the Humirianthera ampla in chemical and thermal models of pain in mice. The oral treatment with ethanolic extract dose-dependently inhibited glutamate-, capsaicin- and formalin-induced licking. However, it did not prevent the nociception caused by radiant heat on the tail-flick test. The ethanolic extract (30 mg/kg) caused marked inhibition of the nociceptive biting response induced by glutamate, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), N-methyl-d-aspartate (NMDA) and substance P. The antinociception caused by ethanolic extract was significantly attenuated by naloxone, l-arginine, WAY100635, ondansetron or ketanserin, but not by caffeine or naloxone methiodide. In conclusion, the ethanolic extract from roots of Humirianthera ampla produces antinociception against neurogenic and inflammatory models of nociception. The mechanisms of antinociception involve nitric oxide, opioid, serotonin and glutamate pathways. Therefore, our results support the ethnopharmacological use of the Humirianthera ampla against inflammatory and painful process caused by snake venom.

Cross-sectional study involving healthcare professionals in a Vaccinia virus endemic area.

Orthopoxviruses (OPV) are emerging viruses with great importance in human and veterinary medicine, such as Vaccinia virus (VACV), which causes outbreaks of bovine vaccinia (BV) in South America. The clinical aspects of BV are similar to other vesicular infections, complicating the clinical diagnosis. This cross-sectional study evaluated the knowledge of Healthcare Professionals about BV and revealed their unpreparedness about BV in a VACV hyper-endemic area in Brazil, highlighting the public health issues associated with VACV infections. This study presents an opportunity to discuss the importance of vaccination for healthcare professionals who work in areas of VACV circulation and brings an educational measure on VACV infections for health professionals around the world.

Etiological agents of viral meningitis in children from a dengue-endemic area, Southeast region of Brazil.

Meningitis is a disease with a global distribution that constitutes a worldwide burden, with viruses as the primary etiologic agents. The range of viral meningitis severity depends mainly on age, immune status and etiological agent. The aim of this work was to investigate the suspected cases of viral meningitis using molecular techniques to confirm the viral infection. The diagnosed virus was correlated with clinical findings and cytochemical parameters in cerebrospinal liquid (CSF) of patients. CSF of 70 children with the presumptive diagnosis of viral meningitis was analyzed by real time PCR (qPCR). Viruses were identified by qPCR in 44 CSF samples (62.9%). Among them, 31 were identified as Enterovirus (ENTV) (70.4%), six as Human herpes virus 3 (HHV-3) (13.6%), five as Dengue virus (DENV) (11.7%), one as Human herpes virus 1-2 (2.3%) and one as Human herpes virus 5 (2.3%). Patients in the HHV-positive groups had increased percentage of polymorphonuclear neutrophils (PMN) (mean of 81%) while the groups of patients with DENV and ENTV had a mean of 30.9%. This study contributes to the knowledge of the epidemiological distribution of viral agents in CNS infections in children. In addition, it raises the relevance of DENV as an agent of CNS infection, and reinforces the importance for molecular in the cases of CNV infection.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Sodium Channels in Pain and Cancer: New Therapeutic Opportunities.

Voltage-gated sodium channels (VGSCs) underpin electrical activity in the nervous system through action potential propagation. First predicted by the modeling studies of Hodgkin and Huxley, they were subsequently identified at the molecular level by groups led by Catterall and Numa. VGSC dysfunction has long been linked to neuronal and cardiac disorders with some nonselective sodium channel blockers in current use in the clinic. The lack of selectivity means that side effect issues are a major impediment to the use of broad spectrum sodium channel blockers. Nine different sodium channels are known to exist, and selective blockers are now being developed. The potential utility of these drugs to target diseases ranging from migraine, multiple sclerosis, muscle, and immune system disorders, to cancer and pain is being explored. Four channels are potential targets for pain disorders. This conclusion comes from mouse knockout studies and human mutations that prove the involvement of Nav1.3, Nav1.7, Nav1.8, and Nav1.9 in the development and maintenance of acute and chronic pain. In this chapter, we present a short overview of the possible role of Nav1.3, Nav1.7, Nav1.8, and Nav1.9 in human pain and the emerging and unexpected role of sodium channels in cancer pathogenesis.