Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays overall exhibit high sensitivity and specificity. This variant panel can be used as a Research Use Only screening tool for triaging SARS-CoV-2 positive samples prior to whole genome sequencing.

A Genotype-to-Phenotype Modeling Framework to Predict Human Pathogenicity of Novel Coronaviruses

Leveraging prior viral genome sequencing data to make predictions on whether an unknown, emergent virus harbors a phenotype-of-concern has been a long-sought goal of genomic epidemiology. A predictive phenotype model built from nucleotide-level information alone has previously been considered un-tenable with respect to RNA viruses due to the ultra-high intra-sequence variance of their genomes, even within closely related clades. Building from our prior work developing a degenerate k-mer method to accommodate this high intra-sequence variation of RNA virus genomes for modeling frameworks, and leveraging a taxonomic group-shuffle-split paradigm on complete coronavirus assemblies from prior to October 2018, we trained multiple regularized logistic regression classifiers at the nucleotide k-mer level capable of accurately predicting withheld SARS-CoV-2 genome sequences as human pathogens and accurately predicting withheld Swine Acute Diarrhea Syndrome coronavirus (SADS-CoV) genome sequences as non-human pathogens. LASSO feature selection identified several degenerate nucleotide predictor motifs with high model coefficients for the human pathogen class that were present across widely disparate classes of coronaviruses. However, these motifs differed in which genes they were present in, what specific codons were used to encode them, and what the translated amino acid motif was. This emphasizes the importance of a phenetic view of emerging pathogenic RNA viruses, as opposed to the canonical phylogenetic interpretations most-commonly used to track and manage viral zoonoses. Applying our model to more recent Orthocoronavirinae genomes deposited since October 2018 yields a novel contextual view of pathogen-potential across bat-related, canine-related, porcine-related, and rodent-related coronaviruses and critical adaptations which may have contributed to the emergence of the pandemic SARS-CoV-2 virus. Finally, we discuss the utility of these predictive models (and their associated predictor motifs) to novel biosurveillance protocols that substantially increase the pound-for-pound information content of field-collected sequencing data and make a strong argument for the necessity of routine collection and sequencing of zoonotic viruses.

Tratamento da forma mucosa de Leishmaniose sem resposta a glucantime, com anfotericina B liposomal / Treatment of mucosal leishmaniasis unresponsive to glucantime, with liposomal amphotericin B

Tratamos com ambisome (2 a 5g totais de dose) seis pacientes com leishmaniose mucosa sem resposta a tratametno com glucantime (20mg sbv/kg/dia). A dose diária usada foi 2 a 3mg/kg/dia, aplicada por um mínimo de 20 dias. Após 26 a 38 meses de acompanhamento, cinco pacientes estäo clinicamente curados. Um recidivou aos 6 meses. Näo foram observados efeitos colaterais além de cefaléia, após a injeçäo. O ambisome constitue uma opçäo terapêutica para os pacientes com leishmaniose mucosa sem resposta aos antimoniais.