RESUMO
Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT-PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~ 10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA extraction to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~ 50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.
Assuntos
Técnicas de Laboratório Clínico/métodos , Custos e Análise de Custo , Nanopartículas de Magnetita/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Teste para COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/diagnóstico , Países em Desenvolvimento , Humanos , Nanopartículas de Magnetita/economia , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economiaRESUMO
In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.
Assuntos
Vírus da Influenza B/classificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Brasil , Custos e Análise de Custo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/genética , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Fatores de TempoAssuntos
Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Técnicas de Química Analítica/economia , Técnicas de Química Analítica/métodos , Criança , Humanos , Fator de Transcrição Ikaros/análise , Pediatria , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Prognóstico , Isoformas de Proteínas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economiaRESUMO
OBJECTIVE: Evaluate the performance of clinical data and the rapid influenza diagnostic test (RIDT) in diagnosing influenza H1N1, and analyze the cost-benefit of using this diagnostic tool. METHODS: The RIDT was used for patients who came to four hospitals in Mexico City with an influenza-like illness (ILI) in October and November 2009. The diagnostic performance of the ILI clinical data and the RIDT was compared to that of the real-time reverse transcription polymerase chain reaction (rRT-PCR) test. The rRT-PCR test was conducted in a reference laboratory and blinded to the results of the RIDT. An economic evaluation also was conducted to estimate the budgetary impact of using the RIDT. RESULTS: The study included 78 patients, 39 of whom tested positive for influenza H1N1 and 6 tested positive for seasonal influenza A, according to the results of the rRT-PCR. The ILI clinical data yielded a sensitivity of 96% and specificity of 21%; the RIDT yielded a sensitivity of 76% and specificity of 82%; and the ILI clinical data and RIDT together yielded a sensitivity of 96% and specificity of 100%. The positive likelihood quotient for ILI-headaches was 31.5 and that of ILI-odynophagia, 330. The use of RIDT yielded savings of US$12.6 per each suspected case. CONCLUSIONS: Use of the RIDT to aid in the diagnosis of influenza H1N1 increases certainty and lowers the average cost per suspected and infected patient.
Assuntos
Imunoensaio/economia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Exame Físico/economia , Adolescente , Adulto , Idoso , Assistência Ambulatorial/economia , Antígenos Virais/análise , Antivirais/economia , Antivirais/uso terapêutico , Criança , Pré-Escolar , Sistemas Computacionais/economia , Análise Custo-Benefício , Erros de Diagnóstico , Diagnóstico Precoce , Feminino , Hospitalização/economia , Hospitais Urbanos , Humanos , Imunoensaio/métodos , Lactente , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , México , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Método Simples-Cego , Inquéritos e Questionários , Fatores de Tempo , Adulto JovemRESUMO
OBJETIVO: Evaluar el desempeño de los datos clínicos y la prueba rápida (PR) en el diagnóstico de influenza H1N1, y analizar el costo-beneficio que representa el uso de esta herramienta diagnóstica. MÉTODOS: Se aplicó la PR a pacientes que acudieron a cuatro hospitales en la ciudad de México con sintomatología similar a influenza (SSI) durante el período octubre y noviembre de 2009. Se comparó el desempeño diagnóstico de la SSI más la PR contra el de la reacción en cadena de la polimerasa en transcripción reversa en tiempo real (rRT-PCR). La rRT-PCR fue procesada en un laboratorio de referencia y cegado al resultado de la PR. Además, se llevó a cabo una evaluación económica a partir de la cual se estimó el impacto presupuestal relacionado con la utilización de la PR RESULTADOS: Se incluyó a 78 pacientes, de los cuales 39 fueron positivos para influenza H1N1 y 6 para influenza A estacional, de acuerdo al resultado de la rRT-PCR. La SSI mostró una sensibilidad de 96 por ciento y una especificidad de 21 por ciento, la PR de 76 por ciento y 82 por ciento y el conjunto de SSI más PR de 96 por ciento y 100 por ciento, respectivamente. El Cociente de Verosimilitud positivo de la SSI-cefalea fue de 31,5 y el de SSI-odinofagia fue de 330. El uso de PR mostró un ahorro de US$ 12,6 por cada caso sospechoso. CONCLUSIONES: El uso de la PR como auxiliar en el diagnóstico de influenza H1N1 incrementa la certeza y reduce el costo promedio por paciente sospechoso e infectado.
OBJECTIVE: Evaluate the performance of clinical data and the rapid influenza diagnostic test (RIDT) in diagnosing influenza H1N1, and analyze the cost-benefit of using this diagnostic tool. METHODS: The RIDT was used for patients who came to four hospitals in Mexico City with an influenza-like illness (ILI) in October and November 2009. The diagnostic performance of the ILI clinical data and the RIDT was compared to that of the real-time reverse transcription polymerase chain reaction (rRT-PCR) test. The rRT-PCR test was conducted in a reference laboratory and blinded to the results of the RIDT. An economic evaluation also was conducted to estimate the budgetary impact of using the RIDT. RESULTS: The study included 78 patients, 39 of whom tested positive for influenza H1N1 and 6 tested positive for seasonal influenza A, according to the results of the rRT-PCR. The ILI clinical data yielded a sensitivity of 96 percent and specificity of 21 percent; the RIDT yielded a sensitivity of 76 percent and specificity of 82 percent; and the ILI clinical data and RIDT together yielded a sensitivity of 96 percent and specificity of 100 percent. The positive likelihood quotient for ILI-headaches was 31.5 and that of ILI-odynophagia, 330. The use of RIDT yielded savings of US$12.6 per each suspected case. CONCLUSIONS: Use of the RIDT to aid in the diagnosis of influenza H1N1 increases certainty and lowers the average cost per suspected and infected patient.
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Imunoensaio/economia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Exame Físico/economia , Assistência Ambulatorial/economia , Antígenos Virais/análise , Antivirais/economia , Antivirais/uso terapêutico , Sistemas Computacionais/economia , Análise Custo-Benefício , Erros de Diagnóstico , Diagnóstico Precoce , Hospitalização/economia , Hospitais Urbanos , Imunoensaio/métodos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , México , Inquéritos e Questionários , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Método Simples-Cego , Fatores de TempoRESUMO
DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.
Assuntos
Leishmania/isolamento & purificação , Leishmaniose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Replicação de Sequência Autossustentável/métodos , Animais , Biópsia , Sangue/parasitologia , Brasil , Humanos , Leishmania/genética , Leishmaniose/parasitologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/economia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Replicação de Sequência Autossustentável/economia , Sensibilidade e Especificidade , Pele/parasitologia , Fatores de TempoRESUMO
Short nucleotide repetitions (STRs) are commonly used as genetic markers; thus their detection and analysis constitutes a very important tool for the mapping of genetic diseases, as well as for gathering information about genetic polymorphisms at the population level. STRs can be detected with agarose- or acrylamide-based electrophoretic techniques, followed by visualization of the DNA sample with ethidium bromide, silver nitrate, or fluorophore labeling. In this work, we analyzed genomic DNA from five individuals affected with type II diabetes mellitus (T2DM) and five controls (unaffected individuals) in order to know the most precise and reproducible technique for the analysis of the existing polymorphism in the STR DG10S478 of the TCF7L2 gene. The combination of PCR with labeling of the products with the CY5 fluorophore, followed by detection on an ALFexpress sequencer, offered the required resolution to detect the variability in this STR, based solely on size analysis. Our methodology offers similar accuracy and reproducibility at lower costs than existing methods based on the sequencing of PCR products, and is a faster alternative when applied to genotyping studies.
Assuntos
Eletroforese em Gel de Poliacrilamida , Repetições de Microssatélites/genética , Fatores de Transcrição TCF/genética , Alelos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Eletroforese em Gel de Poliacrilamida/economia , Estudos de Viabilidade , Marcadores Genéticos , Genoma Humano , Genótipo , Humanos , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Fatores de Transcrição TCF/química , Proteína 2 Semelhante ao Fator 7 de TranscriçãoRESUMO
BACKGROUND: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. METHODS: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127). RESULTS: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (> 95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%-51%. The quantification range was 50-10,000,000 IU/mL. Viral loads for subtypes A-D, F-J, AE, and AG yielded mean differences of 0.31 log(10) compared with Amplicor in the 10(3)-10(4) IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) (P < 0.001 for all). CONCLUSIONS: This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.