Quality Control in RT-PCR Viral Load Assays: Evaluation of Analytical Performance for HIV, HBV, and HCV.

Introduction: Quality Control Management (QCM) in clinical laboratories is crucial for ensuring reliable results in analytical measurements, with biological variation being a key factor. The study focuses on assessing the analytical performance of the Reverse Transcription Polymerase Chain Reaction (RT-PCR) system for Human Immunodeficiency Virus (HIV), Hepatitis B (HBV), and Hepatitis C (HCV). Five models proposed between 1999 and 2014 offer different approaches to evaluating analytical quality, with Model 2 based on biological variation and Model 5 considering the current state of the art. The study evaluates the RT-PCR system's analytical performance through Internal Quality Control (IQC) and External Quality Control (EQC). Materials and Methods: The Laboratório Central de Saúde Pública do Estado do Ceará (LACEN-CE) conducted daily IQC using commercial kits, and EQC was performed through proficiency testing rounds. Random error, systematic error, and total error were determined for each analyte. Results: Analytical performance, assessed through CV and random error, met specifications, with HIV and HBV classified as "desirable" and "optimal." EQC results indicated low systematic error, contributing to total errors considered clinically insignificant. Conclusion: The study highlights the challenge of defining analytical specifications without sufficient biological variability data. Model 5 is deemed the most suitable. The analytical performance of the RT-PCR system for HIV, HBV, and HCV at LACEN-CE demonstrated satisfactory, emphasizing the importance of continuous quality control in molecular biology methodologies.

Development of novel species-specific and genus-specific primers for the detection of Babaco Mosaic Virus (BabMV).

Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.

Bixafen, Prothioconazole, and Trifloxystrobin Alone or in Combination Have a Greater Effect on Health Related Gene Expression in Honey Bees from Nutritionally Deprived than from Protein Supplemented Colonies.

The aim of this study was to evaluate whether alterations in food availability compromise the metabolic homeostasis of honey bees exposed to three fungicides alone or together. Ten honey bee colonies were used, with half receiving carbohydrate-protein supplementation for 15 weeks while another five colonies had their protein supply reduced with pollen traps. Subsequently, forager bees were collected and exposed by contact to 1 or 7 µg of bixafen, prothioconazole, or trifloxystrobin, either individually or in combination. After 48 h, bee abdomens without the intestine were used for the analysis of expression of antioxidant genes (SOD-1, CAT, and GPX-1), detoxification genes (GST-1 and CYP306A1), the storage protein gene vitellogenin, and immune system antimicrobial peptide genes (defensin-1, abaecin, hymenoptaecin, and apidaecin), through real-time PCR. All fungicide treatments induced changes in gene expression, with bixafen showing the most prominent upregulation. Exposure to 1 µg of each of the three pesticides resulted in upregulation of genes associated with detoxification and nutrition processes, and downregulation of immune system genes. When the three pesticides were combined at a dose of 7 µg each, there was a pronounced downregulation of all genes. Food availability in the colonies affected the impact of fungicides on the expression of the studied genes in forager bees.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/µL for DNA samples diluted in water, 10 fg/µL for Fasciola/snail DNA scramble, and 100 fg/µL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Original article: novelty of Canadian manufacture nasopharyngeal swabs for collection of samples being tested for SARS-CoV-2 in a pandemic setting.

Objectives: The COVID-19 pandemic caused a global shortage of nasopharyngeal (NP) swabs, required for RT-PCR testing. Canadian manufacturers were contacted to share NP swab innovations. The primary objective was to determine whether novel NP test swabs were comparable to commercially available swabs regarding user characteristics, ability to collect a specimen, and diagnostic performance using RT-PCR testing. Methods: Participants were randomized by swab (test/control) and nostril (left/right). A calculated positive percent agreement ≥90% was considered successful. Mean Ct values of viral genes and housekeeping gene (RNase P) were considered similar if a Ct difference ≤ 2 between control and test group was obtained. There also was a qualitative assessment of swabs usability. Results: 647 participants were enrolled from Huaycan Hospital in Lima, Peru, distributed over 8 NP swabs brands. Seven brands agreed to share their results. There were no statistically significant differences between the test swabs of these 7 brands and control swabs. Conclusion: All the seven brands are comparable to the commercially available flocked swabs used for SARS-CoV-2 regarding test results agreement, ability to collect a specimen, and user characteristics.

Molecular characterization of Treponema pallidum isolates from Brazil.

Syphilis remains a public health concern in Brazil, and the data on the characterization and resistance of Treponema pallidum in Brazil is limited. The present study aimed to detect Treponema DNA in the lesions and blood samples obtained from individuals diagnosed with syphilis. The Brazilian isolates were submitted to the Enhanced Centers for Disease Control and Prevention (ECDC) scheme and also analyzed for resistance gene. Treponemal DNA from 18 lesions and 18 blood specimens were submitted for amplification using Polymerase Chain Reaction (PCR) and Polymerase Chain Reaction in Real Time (RT-PCR). Eight samples from lesions and eight from blood were positive in the RT-PCR analysis. Eight lesions and three blood samples were positive using PCR. Two samples exhibited azithromycin resistance. The Brazilian isolate types 14d/g, 14 d/c, 15d/c, and 15d/e were identified using the ECDC scheme. The three subtypes 14d/c, 15d/c, and 15d/e have been identified in Brazil for the first time.

Infrared spectroscopy combined with chemometrics in transflectance mode: An alternative approach in the photodiagnosis of COVID-19 using saliva.

The Coronavirus Disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has required the search for sensitive, rapid, specific, and lower-cost diagnostic methods to meet the high demand. The gold standard method of laboratory diagnosis is real-time reverse transcription polymerase chain reaction (RT-PCR). However, this method is costly and results can take time. In the literature, several studies have already described the potential of Fourier transform infrared spectroscopy (FTIR) as a tool in the biomedical field, including the diagnosis of viral infections, while being fast and inexpensive. In view of this, the objective of this study was to develop an FTIR model for the diagnosis of COVID-19. For this analysis, all private clients who had performed a face-to-face collection at the Univates Clinical Analysis Laboratory (LAC Univates) within a period of six months were invited to participate. Data from clients who agreed to participate in the study were collected, as well as nasopharyngeal secretions and a saliva sample. For the development of models, the RT-PCR result of nasopharyngeal secretions was used as a reference method. Absorptions with high discrimination (p < 0.001) between GI (28 patients, RT-PCR test positive to SARS-CoV-2 virus) and GII (173 patients who did not have the virus detected in the test) were most relevant at 3512 cm-1, 3385 cm-1 and 1321 cm-1 after 2nd derivative data transformation. To carry out the diagnostic modeling, chemometrics via FTIR and Discriminant Analysis of Orthogonal Partial Least Squares (OPLS-DA) by salivary transflectance mode with one latent variable and one orthogonal signal correction component were used. The model generated predictions with 100 % sensitivity, specificity and accuracy. With the proposed model, in a single application of an individual's saliva in the FTIR equipment, results related to the detection of SARS-CoV-2 can be obtained in a few minutes of spectral evaluation.

TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Evaluation of HNF1B, KLK3, ELAC2, TMPRSS2-ERG, and CTNNB1 polymorphisms associated with prostate cancer in samples of patients from HUPE-UERJ.

PURPOSE: Prostate cancer (PCa) is the leading cause of death among men in 48 countries. Genetic alterations play a significant role in PCa carcinogenesis. For the hypothesis of this research, five unique polymorphisms (SNP) were investigated in different genes that showed to be associated in different ways with PCa: rs4430796, rs2735839, rs4792311, rs12329760, and rs28931588, respectively for the genes HNF1B, KLK3, ELAC2, TMPRSS2-ERG, and CTNNB1. PATIENTS AND METHODS: Blood samples from 426 subjects were evaluated: 290 controls (161 females and 129 males) and 136 PCa patients. SNP were determined by real-time polymerase chain reaction. TaqMan SNP genotyping assay. In the control samples, the SNPs were defined in association with the self-reported ethnicity, and in 218 control samples with markers with ancestry indicators. The genes were in Hardy-Weinberg equilibrium. One hundred and seventy control samples were matched by ethnicity for comparison with the PCa samples. RESULTS: The G allele at rs28931588 was monomorphic in both patients and controls studied. Significant differences were observed in allelic and genotypic frequencies between the control and Pca samples in rs2735839 (KLK3; p = 0.002 and χ2 = 8.73 and p = 0.01, respectively), by the global frequency and in the dominant model rs2735839_GG (odds ratio [OR] = 0.51, p = 0.02). AA and GA genotypes at rs4792311 (ELAC2) were more frequent in patients with Gleason 7(4 + 3), 8, and 9 (n = 37%-59.7%) compared to patients with Gleason 6 and 7(3 + 4) (n = 26%-40.0%) conferring a protective effect on the GG genotype (OR = 0.45, p = 0.02). The same genotype showed an OR = 2.71 (p = 0.01) for patients with low severity. The HNF1B-KLK3-ELAC2-TMPRSS2-ERG haplotypes: GAAT, AAAT, GAGT, and AAGT were more frequent in patients with Pca with OR ranging from 4.65 to 2.48. CONCLUSIONS: Higher frequencies of risk alleles were confirmed in the SNPs, KLK3 rs2735839_A, ELAC2 rs4792311_A, and TMPRSS2 rs12329760_T in patients with Pca. Rs2735839_A was associated with risk of Pca and rs4792311_A with severity and Gleason score of 7(4 + 3) or greater. There is a need for careful observation of rs2735839 and rs4792311 in association with the prostatic biopsy due to the increased risk of Pca.

Environmentally Relevant Concentrations of the Insecticide Fipronil Modulated Molecular Response in Chironomus riparius.

Pesticides employed worldwide for crop protection easily reach aquatic systems, which act as the main reservoirs, and become a risk factor for aquatic fauna. Fipronil is a broad-spectrum insecticide acting on the insect nervous system; however, other effects and systems unrelated to this mechanism could be affected in non-target organisms. Thus, the present study aimed to assess the impact of fipronil on the suborganismal response (gene expression and enzymatic activity) of Chironomus riparius larvae as a model organism in ecotoxicology. To this end, short-term toxicity tests were carried out with fourth-instar larvae exposed to 0.001, 0.01, and 0.1 µg L-1 of fipronil for 24 and 96 h. Messenger RNA levels of 42 genes related to diverse metabolic pathways were analyzed by real-time polymerase chain reaction, complemented with catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities. Few effects were observed at 24 h; however, after longer exposure (96 h), genes involved in the endocrine, detoxification, stress, and immune response pathways were altered. Moreover, fipronil at 96 h increased CAT and GST activity at 0.01 µg L-1 and AChE at the highest concentrations. The results demonstrate that even low environmentally relevant fipronil concentrations can modulate the molecular response of several cellular pathways in C. riparius after short-term exposure. These results bring new information about the underlying response of fipronil and its mode of action on a key aquatic invertebrate. Despite no effects on mortality, strong modulation at the suborganismal level emphasizes the advantage of biomarkers as early damage responses and the harmful impact of this pesticide on freshwater organisms. Environ Toxicol Chem 2024;43:405-417. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

D-dimer and HbA1c levels findings in COVID-19 Iraqi patients / Descobertas dos níveis de D-dímero e HbA1c em pacientes iraquianos com COVID-19

On March 11, 2020, the World Health Organization (WHO) declared a new coronavirus infection caused by the SARS-CoV-2 virus as a pandemic, making it the 11th pandemic of the 20th and 21st centuries. This study investigated the clinical and laboratory results (D-dimer, conventional coagulation, and HbA1c biomarker concentrations) of 150 patients (75 male and 75 female) with confirmed COVID-19 pneumonia and 50 controls (25 male and 25 female). For disease diagnosis, all COVID-19 patients were given a Real-Time Reverse Transcription Polymerase Chain Reaction Assay (RT-PCR). The findings revealed that D-dimer and HbA1c levels in COVID-19 patients were significantly higher (P 0.001) at the time of admission; In COVID-19 patients, there was also a strong correlation between D-dimer levels and HbA1c levels (P 0.001). In conclusion, COVID-19 patients are more likely to have a poor prognosis if their D-dimer and HbA1c levels remain uncontrolled over a lengthy period. To lower the likelihood of a bad prognosis in COVID-19, patients with higher levels of D-dimer and HbA1c should be continuously monitored.

Em 11 de março de 2020, a Organização Mundial da Saúde (OMS) declarou uma nova infecção por coronavírus causada pelo vírus SARS-CoV-2 como uma pandemia, tornando-a a 11ª pandemia dos séculos XX e XXI. Este estudo investigou os resultados clínicos e laboratoriais (D-dímero, coagulação convencional e concentrações de biomarcadores HbA1c) de 150 pacientes (75 homens e 75 mulheres) com pneumonia por COVID-19 confirmada e 50 controles (25 homens e 25 mulheres). Para o diagnóstico da doença, todos os pacientes com COVID-19 receberam um Ensaio de Reação em Cadeia da Polimerase com Transcrição Reversa em Tempo Real (RT-PCR). Os achados revelaram que os níveis de D-dímero e HbA1c em pacientes com COVID-19 foram significativamente maiores (P 0,001) no momento da admissão. Em pacientes com COVID-19, também houve uma forte correlação entre os níveis de D-dímero e os níveis de HbA1c (P 0,001). Em conclusão, os pacientes com COVID-19 têm maior probabilidade de ter um prognóstico ruim se seus níveis de D-dímero e HbA1c permanecerem descontrolados por um longo período. Para diminuir a probabilidade de um mau prognóstico na COVID-19, os pacientes com níveis mais altos de D-dímero e HbA1c devem ser monitorados continuamente.

Detection of NTRK gene fusions in solid tumors: recommendations from a Latin American group of oncologists and pathologists.

NTRK gene fusions have been detected in more than 25 types of tumors and their prevalence is approximately 0.3% in solid tumors. This low prevalence makes identifying patients who could benefit from TRK inhibitors a considerable challenge. Furthermore, while numerous papers on the evaluation of NTRK fusion genes are available, not all countries have guidelines that are suitable for their setting, as is the case with Latin America. Therefore, a group of oncologists and pathologists from several countries in Latin America (Argentina, Chile, Ecuador, Mexico, Peru and Uruguay) met to discuss and reach consensus on how to identify patients with NTRK gene fusions in solid tumors. To do so, they developed a practical algorithm, considering their specific situation and limitations.

Multiplex Reverse Transcription Polymerase Chain Reaction Combined with a Microwell Hybridization Assay Screening for Arbovirus and Parasitic Infections in Febrile Patients Living in Endemic Regions of Colombia.

Acute febrile syndrome is a frequent reason for medical consultations in tropical and subtropical countries where the cause could have an infectious origin. Malaria and dengue are the primary etiologies in Colombia. As such, constant epidemiological surveillance and new diagnostic tools are required to identify the causative agents. A descriptive cross-sectional study was conducted to evaluate the circulation and differential diagnosis of six pathogens in two regions of Colombia. The results obtained via multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) were comparable to those obtained using rapid tests conducted at the time of patient enrollment. Of 155 patients evaluated, 25 (16.1%) and 16 (10.3%) were positive for malaria and dengue, respectively; no samples were positive for any of the other infectious agents tested. In most cases, m-RT-PCR-ELISA confirmed the results previously obtained through rapid testing.

Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis

Abstract The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min, a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery > GeneFinderTM> WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct> 30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be aceptable.

Resumen La explosión de casos de COVID-19 resaltó el papel fundamental que desempeñan las pruebas de diagnóstico en la toma de decisiones médicas y de salud pública para contener y mitigar la pandemia de SARS-CoV-2. Este estudio reporta la evaluación y la implementación de diferentes test para la detección molecular de SARS-CoV-2 en la región central de Argentina. Evaluamos tres kits de RT-PCR en tiempo real (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit y WGene SARS-CoV-2 RT Detection), dos métodos de extracción de ácidos nucleicos (MagaBio plus Virus DNA/RNA Purification Kit II [BioFlux, 35-min vs. 9-min), un reactivo pre-analítico (FlashPrep®) y dos test de amplificación isotérmica (Neokit Plus and ELA CHEMSTRIP®). El orden de rendimiento de los tres kits de RT-PCR en tiempo real evaluados fue el siguiente: DisCoVery GeneFinder™ WGene. Los dos métodos de extracción de RNA mostraron buenos y similares resultados; se seleccionó MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min debido a su rápido tiempo de procesamiento. El reactivo FlashPrep® mostró excelentes resultados para realizar detección directa de RNA. Los ensayos de amplificación isotérmica mostraron valores de sensibilidad y de especificidad aceptables (80%), excepto en muestras con Ct 30. Nuestros resultados muestran kits de RT-PCR en tiempo real óptimos, como así también métodos moleculares alternativos para el diagnóstico de SARS-CoV-2 que resultan aceptables para su uso en contextos adversos, de descentralización y en diferentes escenarios epidemiológicos, para la detección rápida y precisa del SARS-CoV-2.

Estudio comparativo entre un test de antígeno y la prueba de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) para el diagnóstico de infección por COVID-19 en pediatría / Comparative study between an antigen test and the reverse transcription-polymerase chain reaction (RT-PCR) test for the diagnosis of COVID-19 in pediatrics

Introducción. La pandemia por COVID-19 ha puesto de manifiesto la necesidad de pruebas diagnósticas rápidas. La prueba de referencia es la reacción en cadena de la polimerasa en tiempo real (RT-PCR). Requiere un equipo y personal capacitado, y su resultado puede llevar un tiempo de espera prolongado. El sistema BD Veritor® es el método rápido cromatográfico utilizado para la detección del antígeno del coronavirus de tipo 2 del síndrome respiratorio agudo grave, en individuos sintomáticos. El objetivo primario del siguiente trabajo es evaluar sensibilidad y especificidad del test de antígeno (TA) comparadas con la RT-PCR en población pediátrica. Población y métodos. Estudio prospectivo, de prueba diagnóstica. Se incluyó a todo menor de 17 años en los primeros 5 días de inicio de síntomas, que consultó desde julio de 2021 hasta febrero de 2022. Se calculó un mínimo de 300 muestras para lograr una precisión de ± 8,76 % y de ± 3,68 % para sensibilidad y especificidad respectivamente. Se analizaron en paralelo las muestras por ambas metodologías. Resultados. De 316 muestras pareadas, 33 fueron positivas por ambos métodos; 6 fueron positivas solo por RT-PCR. La especificidad del TA fue del 100 %; la sensibilidad, del 84,6 %, con un valor predictivo positivo y negativo del 100 % y del 98 % respectivamente. Conclusiones. El TA demostró ser útil en el diagnóstico de pacientes pediátricos con COVID-19 en los primeros 5 días de inicio de síntomas, aunque aquellos con TA negativo y alta sospecha clínica deberían confirmar su resultado con la RT-PCR.

Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR requires equipment and trained personnel, and results may take a long waiting time. The BD Veritor® System is a rapid chromatographic method used for the detection of severe acute respiratory syndrome coronavirus 2 antigen in symptomatic individuals. The primary objective of this study is to assess the sensitivity and specificity of the antigen test (AT) compared to the RT-PCR in the pediatric population. Population and methods. Prospective study with a diagnostic test. All children younger than 17 years in the first 5 days of symptom onset, who consulted between July 2021 and February 2022, were included. A minimum of 300 specimens was estimated to achieve an accuracy of ±8.76% and ±3.68% for sensitivity and specificity, respectively. Specimens were analyzed in parallel using both methodologies. Results. Of 316 paired samples, 33 were positive by both methods; 6 were positive only by RT-PCR. The specificity of the AT was 100%; sensitivity was 84.6%, with a positive and negative predictive value of 100% and 98%, respectively. Conclusions. The AT proved to be useful in the diagnosis of pediatric patients with COVID-19 in the first 5 days of symptom onset, although those with a negative AT and high clinical suspicion should confirm their result with a RT-PCR.

Material genético do SARS-CoV-2 em superfícies de equipamentos utilizados na prática de atividades físicas / Genetic material of SARS-CoV-2 on the surface of equipment used in the practice of physical activities

A humanidade foi impactada por uma Pandemia que expôs a população ao contato com um vírus de elevado contágio e com o índice de letalidade alarmante. Este estudo objetivou avaliar a possibilidade da persistência de material genético do SARS-CoV-2 na superfície dos equipamentos de estabelecimentos de prática de atividades físicas indoor e outdoor. Foram coleta das amostras de equipamentos utilizados para a prática de exercícios físicos em cinco academias, cinco praças e entre os frequentadores desses ambientes. Aplicou-se a técnica RT-PCR para a detecção doRNA do SARS-CoV-2 e posterior análise dos resultadose foi detectada a existência de partículas de RNA viral do SARS-CoV-2 em 48,57% das amostras coletadas dos equipamentos das academias e 12,85% das amostras coletadas nas praças, evidenciando uma incidência menor em equipamentos utilizados em locais abertos em todas as áreas comparadas.Além disso, constatou-se que 35,7% dos participantes do estudo testaram positivo para COVID-19.Os casos positivos para COVID-19 detectados apresentaram sintomas classificados como levesa moderados e uma recuperação rápida.A presença de material genético nos equipamentos,por sua vez, leva-nos a perceber a importância da higienização adequada das superfícies, como forma de prevenção.

Humanity was impacted by a Pandemic that exposed the population to contact with a highly contagious virus with an alarming lethality rate. The present study aimed to evaluate the possibility of the persistence of genetic material from SARS-CoV-2 on the surface of equipment used to practice indoor and outdoor physical activities. A sample of equipment used for the practice of physical activity was collected in five gyms and five squares and among the regulars of these environments. The RT-PCR technique was applied to detect the RNA of SARS-CoV-2 and subsequent analysis of the results. The existence of SARS-CoV-2 viral RNA particles was detected in 48.57% of the samples collected from gym equipment and 12.85% of the samples collected in squares, evidencing a lower incidence in equipment used in open spaces in all areas compared and it was found that 35.7% of the study participants tested positive for COVID-19. The positive cases for COVID-19 detected, had symptoms classified as mild to moderate and a quick recovery. The presence of genetic material in the equipment, in turn, leads us to realize the importance of proper cleaning of surfaces, as a form of prevention.

Diagnostic Efficacy of Chest Computed Tomography with a Dual-Reviewer Approach in Patients Diagnosed with Pneumonia Secondary to Severe Acute Respiratory Syndrome Coronavirus 2.

To compare the diagnostic effectiveness of chest computed tomography (CT) utilizing a single- versus a dual-reviewer approach in patients with pneumonia secondary to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we conducted a retrospective observational study of data from a cross-section of 4809 patients with probable SARS-CoV-2 from March to November 2020. All patients had a CT radiological report and reverse-transcription polymerase chain reaction (PCR) results. A dual-reviewer approach was applied to two groups while conducting a comparative examination of the data. Reviewer 1 reported 108 patients negative and 374 patients positive for coronavirus disease 2019 (COVID-19) in group A, and 266 negative and 142 positive in group B. Reviewer 2 reported 150 patients negative and 332 patients positive for COVID-19 in group A, and 277 negative and 131 positive in group B. The consensus result reported 87 patients negative and 395 positive for COVID-19 in group A and 274 negative and 134 positive in group B. These findings suggest that a dual-reviewer approach improves chest CT diagnosis compared to a conventional single-reviewer approach.

Co-circulation of Chikungunya virus, Zika virus, and serotype 1 of Dengue virus in Western Bahia, Brazil.

Chikungunya, mayaro, dengue, zika, and yellow fever are mosquito-borne viral diseases caused, respectively, by Chikungunya virus, Mayaro virus (CHIKV and MAYV, respectively: Togaviridae: Alphavirus), Dengue virus, Zika virus, and Yellow fever virus (DENV, ZIKV, and YFV, respectively: Flaviviridae: Flavivirus). These viruses have an important epidemiological impact worldwide, especially in Brazil. Western Bahia is one of the less studied regions in that country regarding the circulation of these pathogens. In this study, we aimed to apply molecular biology assays to better know the mosquito-borne viruses circulating in Barreiras and Luís Eduardo Magalhães, two main cities of Western Bahia. From March to June 2021, we enrolled 98 patients with the clinical diagnosis of dengue. Personal information (gender and age) were retrieved at the moment of enrollment. Serum samples were obtained from volunteers and used in molecular detection of CHIKV, MAYV, DENV, ZIKV, and YFV by reverse transcription followed by real-time polymerase chain reaction as well as in genome sequencing aiming phylogenetic analysis. As the main result, we found that from the 98 patients 45 were infected by CHIKV, 32 were infected by serotype 1 of DENV (DENV-1) and six were infected by ZIKV, while 15 were negative for all arboviruses tested. In addition, phylogenetic analysis revealed that all CHIKV-positive samples were of the East/Central/South African (ECSA) genotype, while all DENV-1-positive samples were of the V genotype. These results clearly show that epidemiological surveillance cannot be based only on clinical evaluations. Laboratory diagnosis is important in arbovirus infection that are prevalent in a particular area. These findings also demonstrate the co-circulation of many arboviruses in Western Bahia in 2021.

Mapping of SARS-CoV-2 in Waldeyer's lymphatic ring and visceral biopsies: the age and the illness duration's impact.

OBJECTIVE: To study the impact of age and the interval between disease diagnosis and death on the organotropism of SARS-CoV-2. METHOD: Patients underwent post-mortem biopsies from lungs, Waldeyer ring, heart, liver, kidneys and bone marrow between 2020‒2021. SARS-CoV-2 organotropism was mapped by using molecular RT-PCR analyses for SARS-CoV2 targeting the Envelope gene (E), the RNA Polymerase Gene (RdRp), and the Nucleocapsid gene (N). Statistical and linear regression analysis was performed to study the impact of age and illness duration in SARS-CoV-2 organotropism. RESULTS: We performed 158 postmortem biopsies in 21 patients, with a mean age of 76 years old. The mean interval between the diagnosis of the infection to the death was 23 days. The RNA of the SARS-CoV-2 was detected in 100% of lung biopsies, 76%‒82% of Waldeyer's ring biopsies, 55% of heart biopsies, 40% of kidney biopsies, 33% of liver and 25% of bone marrow biopsies. Patients who died before the day 9, presented extensive visceral dissemination of SARS-CoV-2 RNA. Most of the patients older than 80 years (90%) presented visceral dissemination of SARS-CoV-2 RNA, while among younger patients, only 3/11 patients presented visceral dissemination of the virus. The relationship between "age" and "illness duration" and multitropism of the virus was statistically significant (p<0.001). CONCLUSION: Disease interval and age were factors that were significantly associated with RT-PCR positive results in multiple organs. Critical COVID-19 patients have multiorganic viral dissemination in early stages. In the critical older patients, multiorganic viral dissemination is the rule.