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.

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress.

Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake.

BACKGROUND: In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES: The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS: The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS: Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS: There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Telomeric gene deletion and intrachromosomal amplification in antimony-resistant Leishmania.

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next-generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony-resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate-dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

Enhancing the epidemiological surveillance of SARS-CoV-2 using Sanger sequencing to identify circulating variants and recombinants.

Since the emergence of SARS-CoV-2 in December 2019, more than 12,000 mutations in the virus have been identified. These could cause changes in viral characteristics and directly impact global public health. The emergence of variants is a great concern due to the chance of increased transmissibility and infectivity. Sequencing for surveillance and monitoring circulating strains is extremely necessary as the early identification of new variants allows public health agencies to make faster and more effective decisions to contain the spread of the virus. In the present study, we identified circulating variants in samples collected in Belo Horizonte, Brazil, and detected a recombinant lineage using the Sanger method. The identification of lineages was done through gene amplification of SARS-CoV-2 by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). By using these specific fragments, we were able to differentiate one variant of interest and five circulating variants of concern. We were also able to detect recombinants. Randomly selected samples were sequenced by either Sanger or Next Generation Sequencing (NGS). Our findings validate the effectiveness of Sanger sequencing as a powerful tool for monitoring variants. It is easy to perform and allows the analysis of a larger number of samples in countries that cannot afford NGS.

Comparison of diagnostic performance of RT-qPCR, RT-LAMP and IgM/IgG rapid tests for detection of SARS-CoV-2 among healthcare workers in Brazil.

BACKGROUND: COVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential. METHODS: In this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests - RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP - and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies. RESULTS: Our results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2. CONCLUSION: Our data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress

Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.

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.

Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major.

The Leishmania aquaglyceroporin AQP1 plays an important physiological role in water and uncharged polar solutes transport, volume regulation, osmotaxis, and is a key determinant of antimony resistance. By targeted gene disruption, we generated a Leishmania major promastigote AQP1 null mutant. This required several attempts but a chromosomal null AQP1 mutant was obtained by loss of heterozygosity in the presence of a rescue plasmid encoding AQP1. Growth in the absence of selection led to the loss of the rescuing plasmid, indicating that AQP1 is not essential for Leishmania viability. The AQP1-null mutant was resistant to antimonyl tartrate (SbIII) and arsenite (AsIII) due to a decrease import of these metalloids. It also exhibited alterations in its osmoregulation abilities compared with wild-type cells. This is the first report of the generation of a genetic AQP1 null mutant in Leishmania parasite, confirming its physiological function and role in resistance to antimonials, the therapeutic mainstay against Leishmania.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.