Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment.

BACKGROUND AIMS: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. METHODS: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. RESULTS: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. CONCLUSIONS: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation.

Chronic myeloid leukemia (CML) is a type of leukemia whose main genetic marker is the reciprocal translocation that leads to the production of the BCR::ABL1 oncoprotein. The expression of some genes may interfere with the progression and development of leukemias. MicroRNAs are small non-coding RNAs that have the potential to alter the expression of some genes and may be correlated with some types of leukemia and could be used as biomarkers in the diagnosis and prognosis of patients. Therefore, this project carried out an analysis of microRNA-type plasma biomarkers in patients with chronic myeloid leukemia at unique points, including follow-up analysis of patients from the Erasto Gaertner Hospital. 35 microRNAs were analyzed in different cohorts. Inside those groups, 70 samples were analyzed at unique points and 11 patients in a follow-up analysis. Statistically different results were found for microRNA-7-5p, which was found to be upregulated in patients with high expression of the BCR::ABL1 transcript when compared to healthy controls. This microRNA also had evidence of behavior related to BCR::ABL1 when analyzed in follow-up, but strong evidence was not found. In this way, this work obtained results that may lead to manifestations of a relationship between miR-7-5p and chronic myeloid leukemia, and evaluations of possible microRNAs that are not related to this pathology.

Germline variants in early and late-onset Brazilian prostate cancer patients.

BACKGROUND: The median age for Prostate Cancer (PCa) diagnosis is 66 years, but 10% are diagnosed before 55 years. Studies on early-onset PCa remain both limited and controversial. This investigation sought to identify and characterize germline variants within Brazilian PCa patients classified as either early or later onset disease. METHODS: Peripheral blood DNA from 71 PCa patients: 18 younger (≤ 55 years) and 53 older (≥ 60 years) was used for Targeted DNA sequencing of 20 genes linked to DNA damage response, transcriptional regulation, cell cycle, and epigenetic control. Subsequent genetic variant identification was performed and variant functional impacts were analyzed with in silico prediction. RESULTS: A higher frequency of variants in the BRCA2 and KMT2C genes across both age groups. KMT2C has been linked to the epigenetic dysregulation observed during disease progression in PCa. We present the first instance of KMT2C mutation within the blood of Brazilian PCa patients. Furthermore, out of the recognized variants within the KMT2C gene, 7 were designated as deleterious. Thirteen deleterious variants were exclusively detected in the younger group, while the older group exhibited 37 variants. Within these findings, 4 novel variants emerged, including 1 designated as pathogenic. CONCLUSIONS: Our findings contribute to a deeper understanding of the genetic factors associated with PCa susceptibility in different age groups, especially among the Brazilian population. This is the first investigation to explore germline variants specifically in younger Brazilian PCa patients, with high relevance given the genetic diversity of the population in Brazil. Additionally, our work presents evidence of functionally deleterious germline variants within the KMT2C gene among Brazilian PCa patients. The identification of novel and functionally significant variants in the KMT2C gene emphasizes its potential role in PCa development and warrants further investigation.

Screening of Exosome-Derived Proteins and Their Potential as Biomarkers in Diagnostic and Prognostic for Pancreatic Cancer.

In the oncological area, pancreatic cancer is one of the most lethal diseases, with 5-year survival rising just 10% in high-development countries. This disease is genetically characterized by KRAS as a driven mutation followed by SMAD4, CDKN2, and TP53-associated mutations. In clinical aspects, pancreatic cancer presents unspecific clinical symptoms with the absence of screening and early plasmatic biomarker, being that CA19-9 is the unique plasmatic biomarker having specificity and sensitivity limitations. We analyzed the plasmatic exosome proteomic profile of 23 patients with pancreatic cancer and 10 healthy controls by using Nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS). The pancreatic cancer patients were subdivided into IPMN and PDAC. Our findings show 33, 34, and 7 differentially expressed proteins when comparing the IPMN vs. control, PDAC-No treatment vs. control, and PDAC-No treatment vs. IPMN groups, highlighting proteins of the complement system and coagulation, such as C3, APOB, and SERPINA. Additionally, PDAC with no treatment showed 11 differentially expressed proteins when compared to Folfirinox neoadjuvant therapy or Gemcitabine adjuvant therapy. So here, we found plasmatic exosome-derived differentially expressed proteins among cancer patients (IPMN, PDAC) when comparing with healthy controls, which could represent alternative biomarkers for diagnostic and prognostic evaluation, supporting further scientific and clinical studies on pancreatic cancer.

Molecular BCR::ABL1 Quantification and ABL1 Mutation Detection as Essential Tools for the Clinical Management of Chronic Myeloid Leukemia Patients: Results from a Brazilian Single-Center Study.

Chronic myeloid leukemia (CML) is a well-characterized oncological disease in which virtually all patients possess a translocation (9;22) that generates the tyrosine kinase BCR::ABL1 protein. This translocation represents one of the milestones in molecular oncology in terms of both diagnostic and prognostic evaluations. The molecular detection of the BCR::ABL1 transcription is a required factor for CML diagnosis, and its molecular quantification is essential for assessing treatment options and clinical approaches. In the CML molecular context, point mutations on the ABL1 gene are also a challenge for clinical guidelines because several mutations are responsible for tyrosine kinase inhibitor resistance, indicating that a change may be necessary in the treatment protocol. So far, the European LeukemiaNet and the National Comprehensive Cancer Network (NCCN) have presented international guidelines on CML molecular approaches, especially those related to BCR::ABL1 expression. In this study, we show almost three years' worth of data regarding the clinical treatment of CML patients at the Erasto Gaertner Hospital, Curitiba, Brazil. These data primarily comprise 155 patients and 532 clinical samples. BCR::ABL1 quantification by a duplex-one-step RT-qPCR and ABL1 mutations detection were conducted. Furthermore, digital PCR for both BCR::ABL1 expression and ABL1 mutations were conducted in a sub-cohort. This manuscript describes and discusses the clinical importance and relevance of molecular biology testing in Brazilian CML patients, demonstrating its cost-effectiveness.

Circulating Cell-Free Nucleic Acids as Biomarkers for Diagnosis and Prognosis of Pancreatic Cancer.

A lack of reliable early diagnostic tools represents a major challenge in the management of pancreatic cancer (PCa), as the disease is often only identified after it reaches an advanced stage. This highlights the urgent need to identify biomarkers that can be used for the early detection, staging, treatment monitoring, and prognosis of PCa. A novel approach called liquid biopsy has emerged in recent years, which is a less- or non-invasive procedure since it focuses on plasmatic biomarkers such as DNA and RNA. In the blood of patients with cancer, circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) have been identified such as DNA, mRNA, and non-coding RNA (miRNA and lncRNA). The presence of these molecules encouraged researchers to investigate their potential as biomarkers. In this article, we focused on circulating cfNAs as plasmatic biomarkers of PCa and analyzed their advantages compared to traditional biopsy methods.

microRNA-based Genetic Therapy in Leukemia: Properties, Delivery, and Experimental Models.

Leukemia is a type of cancer that affects white blood cells. In this disease, immature blood cells undergo genetic mutations, leading to excessive replication and reduced cell death compared to healthy cells. In cancer, there may be the activation of oncogenes and the deactivation of tumor suppressor genes that control certain cellular functions. Despite the undeniable contribution to the patient's recovery, conventional cancer treatments may have some not-so-beneficial effects. In this case, gene therapy appears as an alternative to classical treatments. Gene therapy delivers genetic material to cells to replace or modify dysfunctional genes, a safe method for neoplasms. One of the types of nucleic acids explored in gene therapy is microRNA (miRNA), a group of endogenous, non-proteincoding, small single-stranded RNA molecules involved in the regulation of gene expression, cell division, differentiation, angiogenesis, migration, apoptosis, and carcinogenesis. This review aims to bring together the most recent advances found in the literature on cancer gene therapy based on microRNAs in the oncological context, focusing on leukemia.

Genetic and non-genetic risk factors for early-onset pancreatic cancer.

BACKGROUND: Early-onset pancreatic cancer (EOPC) represents 5-10% of all pancreatic ductal adenocarcinoma (PDAC) cases, and the etiology of this form is poorly understood. It is not clear if established PDAC risk factors have the same relevance for younger patients. This study aims to identify genetic and non-genetic risk factors specific to EOPC. METHODS: A genome-wide association study was performed, analysing 912 EOPC cases and 10 222 controls, divided into discovery and replication phases. Furthermore, the associations between a polygenic risk score (PRS), smoking, alcohol consumption, type 2 diabetes and PDAC risk were also assessed. RESULTS: Six novel SNPs were associated with EOPC risk in the discovery phase, but not in the replication phase. The PRS, smoking, and diabetes affected EOPC risk. The OR comparing current smokers to never-smokers was 2.92 (95% CI 1.69-5.04, P = 1.44 × 10-4). For diabetes, the corresponding OR was 14.95 (95% CI 3.41-65.50, P = 3.58 × 10-4). CONCLUSION: In conclusion, we did not identify novel genetic variants associated specifically with EOPC, and we found that established PDAC risk variants do not have a strong age-dependent effect. Furthermore, we add to the evidence pointing to the role of smoking and diabetes in EOPC.

Lyophilization of Molecular Biology Reactions: A Review.

Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.

Metagenomic insights into the plasma virome of Brazilian patients with prostate cancer

Growing evidence suggests that metavirome changes could be associated increased risk for malignant cell transformation. Considering Viruses have been proposed as factors for prostate cancer induction. The objective of this study was to examine the composition of the plasma metavirome of patients with prostate cancer. Blood samples were obtained from 49 male patients with primary prostate adenocarcinoma. Thirty blood donors were included as a control group. The obtained next-generation sequencing data were analyzed using a bioinformatic pipeline for virus metagenomics. Viral reads with higher abundance were assembled in contigs and analyzed taxonomically. Viral agents of interest were also confirmed by qPCR. Anelloviruses and the Human Pegivirus-1 (HPgV-1) were the most abundant component of plasma metavirome. Clinically important viruses like hepatitis C virus (HCV), cytomegalovirus and human adenovirus type C were also identified. In comparison, the blood donor virome was exclusively composed of torque teno virus types (TTV) types. The performed HPgV-1 and HCV phylogeny revealed that these viruses belong to commonly detected in Brazil genotypes. Our study sheds light on the plasma viral abundance in patients with prostatic cancer. The obtained viral diversity allowed us to separate the patients and controls, probably suggesting that malignant processes may influence virome composition. More complex and multiple approach investigations are necessary to examine the likely causal relationship between metavirome and its nvolvement in prostate cancer.

Association of laboratory markers and cerebral blood flow among sickle cell anemia children.

Background: Stroke is one of the highest complications of sickle-cell anemia (SCA). The Transcranial Doppler (TCD) has been adopted worldwide as a gold standard method for detecting alterations in the blood velocity in cerebral arteries. In this study, we investigated the association between laboratory parameters and increased cerebral blood flow velocity in Brazilian SCA pediatric patients. Methods: The study included 159 pediatric patients with SCA, submitted to TCD velocity screening, and the time-averaged maximum mean velocity (TAMMV) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). We compared cerebral blood flow in patients stratified by the following: TCD1-defined as normal, with TAMMV inferior to 170 cm/s; TCD2-conditional, with TAMMV above 170 cm/s, but less than 199 cm/s; TCD3-altered, with TAMMV greater than or equal to 200 cm/s. Results: TAMMV was negatively correlated with age and weight (p < 0.05). Moreover, TAMMV was associated or correlated with reductions in HbF, RBC, hemoglobin, hematocrit, HDL, and haptoglobin and, increases in MCV, MCH, RDW, reticulocytes, WBC, lymphocytes, monocytes, eosinophils, total and indirect bilirubin, LDH, AST, ALT, glucose, ferritin, and AAT (p < 0.05). Conclusion: The current study highlights the importance of the investigation of hemolytic and inflammatory biomarkers for monitoring the clinical outcome of SCA pediatric patients, to avoid acute or chronic stroke. Moreover, glucose and HDL-C appear useful for predicting higher TAMMV.

Dynamics of the Immune Response in Hospitalized SARS-CoV-2-infected Cancer Patients.

Seroconversion rates were compared between oncological and nononcological patients infected with SARS-CoV-2 during a 14-day hospitalization time. All COVID-19 non-oncological and solid malignancies patients reached 100% seroconversion at day 14, while less than half of the hematological patients were seroconverted at the same time point. Despite the limited number and variability of the patient's cohort, we conclude that there is a delayed seroconversion in the hematological malignancies group, which may be linked to changes in the hematological parameters, immune suppression and/or oncological treatments that are typically associated with these patients.

Plasma Exosome-Derived microRNAs as Potential Diagnostic and Prognostic Biomarkers in Brazilian Pancreatic Cancer Patients.

Pancreatic cancer represents one of the leading causes of oncological death worldwide. A combination of pancreatic cancer aggressiveness and late diagnosis are key factors leading to a low survival rate and treatment inefficiency, and early diagnosis is pursued as a critical factor for pancreatic cancer. In this context, plasma microRNAs are emerging as promising players due to their non-invasive and practical usage in oncological diagnosis and prognosis. Recent studies have showed some miRNAs associated with pancreatic cancer subtypes, or with stages of the disease. Here we demonstrate plasma exosome-derived microRNA expression in pancreatic cancer patients and healthy individuals from Brazilian patients. Using plasma of 65 pancreatic cancer patients and 78 healthy controls, plasma exosomes were isolated and miRNAs miR-27b, miR-125b-3p, miR-122-5p, miR-21-5p, miR-221-3p, miR-19b, and miR-205-5p were quantified by RT-qPCR. We found that miR-125b-3p, miR-122-5p, and miR-205-5p were statistically overexpressed in the plasma exosomes of pancreatic cancer patients compared to healthy controls. Moreover, miR-205-5p was significantly overexpressed in European descendants, in patients with tumor progression and in those who died from the disease, and diagnostic ability by ROC curve was 0.86. Therefore, we demonstrate that these three microRNAs are potential plasma exosome-derived non-invasive biomarkers for the diagnosis and prognosis of Brazilian pancreatic cancer, demonstrating the importance of different populations and epidemiological bias.

Sample Preparation for Lab-on-a-Chip Systems in Molecular Diagnosis: A Review.

Rapid and low-cost molecular analysis is especially required for early and specific diagnostics, quick decision-making, and sparing patients from unnecessary tests and hospitals from extra costs. One way to achieve this objective is through automated molecular diagnostic devices. Thus, sample-to-answer microfluidic devices are emerging with the promise of delivering a complete molecular diagnosis system that includes nucleic acid extraction, amplification, and detection steps in a single device. The biggest issue in such equipment is the extraction process, which is normally laborious and time-consuming but extremely important for sensitive and specific detection. Therefore, this Review focuses on automated or semiautomated extraction methodologies used in lab-on-a-chip devices. More than 15 different extraction methods developed over the past 10 years have been analyzed in terms of their advantages and disadvantages to improve extraction procedures in future studies. Herein, we are able to explain the high applicability of the extraction methodologies due to the large variety of samples in which different techniques were employed, showing that their applications are not limited to medical diagnosis. Moreover, we are able to conclude that further research in the field would be beneficial because the methodologies presented can be affordable, portable, time efficient, and easily manipulated, all of which are strong qualities for point-of-care technologies.

Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis.

The detection of BCR-ABL1 mRNA transcripts is essential to molecular chronic myeloid leukemia (CML) diagnosis. In most cases, the RT-qPCR technique is performed as the gold standard diagnosis tool for clinical cases. However, this method requires expensive reagents and equipment, such as a real-time thermal cycler, probes and master mix. Consequently, the development and validation of simple and low-cost methods are essential for a rapid CML diagnosis in less specialized and equipped centers. In this study, we develop and demonstrate an accessible, rapid, and low-cost method using RT-LAMP for BCR-ABL1 detection in both cell lines and CML clinical samples, using fluorescent and colorimetric assays. Both methods demonstrated diagnostic specificity of 100% and while diagnostic sensitivity reaches more than 90% in samples with RT-qPCR cycle threshold above 31. The obtained data indicates that the proposed method here described is robust, specific and rapid approach for CML diagnosis with outstanding performance, especially for CML diagnostic procedure where present high BCR-ABL1 expression.

Mesenchymal stromal cells-based therapy in a murine model of elastase-induced emphysema: Simvastatin as a potential adjuvant in cellular homing.

Chronic Obstructive Pulmonary Disease - COPD is characterized by the destruction of alveolar walls associated to a chronic inflammatory response of the airways. There is no clinical therapy for COPD. In this context, cell-based therapies represent a promising therapeutic approach for chronic lung disease. The goal of this work was to evaluate the effect of simvastatin on cell-based therapy in a mice emphysema model. Female FVB mice received intranasal instillation of elastase (three consecutive doses of 50 µL) in order to promote pulmonary emphysema. After 21 days of the first instillation, the animals were treated with Adipose-Derived Mesenchymal Stromal Cells (AD-MSC, 2.6 × 106) via retro-orbital infusion associated or not with simvastatin administrated daily via oral gavage (15 mg/kg/15d). Before and after these treatments, the histological and morphometrical analyses of the lung tissue, as so as lung function (whole body plethysmography) were evaluated. PAI-1 gene expression, an upregulated factor by ischemia that indicate a low survival of transplanted MSC, was also evaluated. The result regarding morphological and functional aspects of both lungs, presented no significant difference among the groups (AD-MSC or AD-MSC + Simvastatin). However, significant anatomical difference was observed in the right lung of the both groups of mice. The results shown a higher deposition of cells in the right lung, with might to be explained by anatomical differences (slightly higher right bronchi). Decreased levels of PAI-1 were observed in the simvastatin treated groups. The pulmonary ventilation was similar between the groups with only a tendency to a lower in the elastase treated animals due to a low respiratory frequency. In conclusion, the results suggest that both AD-MSC and simvastatin treatments could promote an improvement of morphological recovery of pulmonary emphysema, that it was more pronounced in the right lung.

ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.

Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis.

BACKGROUND: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. METHODS: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. RESULTS: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. CONCLUSION: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

Obesity as a Possible Risk Factor for Progression from Monoclonal Gammopathy of Undetermined Significance Progression into Multiple Myeloma: Could Myeloma Be Prevented with Metformin Treatment?

Obesity is increasingly associated with the transformation of monoclonal gammopathy of undetermined significance (MGUS) into multiple myeloma (MM). Obesity, MGUS, and MM share common etiopathogenesis mechanisms including altered insulin axis and the action of inflammatory cytokines. Consistent with this interconnection, metformin could predominantly exert inhibition of these pathophysiological factors and thus be an attractive therapeutic option for MGUS. Despite the possible clinical significance, only a limited number of epidemiological studies have focused on obesity as a risk factor for MGUS and MM. This review describes multiple biological pathways modulated by metformin at the cellular level and their possible impacts on the biology of MGUS and its progression into MM.