Operational effectiveness of tafenoquine and primaquine for the prevention of Plasmodium vivax recurrence in Brazil: a retrospective observational study.

BACKGROUND: Prevention of Plasmodium vivax malaria recurrence is essential for malaria elimination in Brazil. We evaluated the real-world effectiveness of an updated treatment algorithm for P vivax radical cure in the Brazilian Amazon. METHODS: In this non-interventional observational study, we used retrospective data from the implementation of a P vivax treatment algorithm at 43 health facilities in Manaus and Porto Velho, Brazil. The treatment algorithm consisted of chloroquine (25 mg/kg over 3 days) and point-of-care quantitative glucose-6-phosphate dehydrogenase (G6PD) testing followed by single-dose tafenoquine 300 mg (G6PD normal, aged ≥16 years, not pregnant and not breastfeeding), 7-day primaquine 0·5 mg/kg per day (G6PD intermediate or normal, aged ≥6 months, not pregnant, and not breastfeeding or breastfeeding for >1 month), or primaquine 0·75 mg/kg per week for 8 weeks (G6PD deficient, aged ≥6 months, not pregnant, and not breastfeeding or breastfeeding for >1 month). P vivax recurrences were identified from probabilistic linkage of routine patient records from the Brazilian malaria epidemiological surveillance system. Recurrence-free effectiveness at day 90 and day 180 was estimated using Kaplan-Meier analysis and hazard ratios (HRs) by multivariate analysis. This clinical trial is registered with ClinicalTrials.gov, NCT05096702, and is completed. FINDINGS: Records from Sept 9, 2021, to Aug 31, 2022, included 5554 patients with P vivax malaria. In all treated patients of any age and any G6PD status, recurrence-free effectiveness at day 180 was 75·8% (95% CI 74·0-77·6) with tafenoquine, 73·4% (71·9-75·0) with 7-day primaquine, and 82·1% (77·7-86·8) with weekly primaquine. In patients aged at least 16 years who were G6PD normal, recurrence-free effectiveness until day 90 was 88·6% (95% CI 87·2-89·9) in those who were treated with tafenoquine (n=2134) and 83·5% (79·8-87·4) in those treated with 7-day primaquine (n=370); after adjustment for confounding factors, the HR for recurrence following tafenoquine versus 7-day primaquine was 0·65 (95% CI 0·49-0·86; p=0·0031), with similar outcomes between the two treatments at day 180 (log-rank p=0·82). Over 180 days, median time to recurrence in patients aged at least 16 years who were G6PD normal was 92 days (IQR 76-120) in those treated with tafenoquine and 68 days (52-94) in those treated with 7-day primaquine. INTERPRETATION: In this real-world setting, single-dose tafenoquine was more effective at preventing P vivax recurrence in patients aged at least 16 years who were G6PD normal compared with 7-day primaquine at day 90, while overall efficacy at 180 days was similar. The public health benefits of the P vivax radical cure treatment algorithm incorporating G6PD quantitative testing and tafenoquine support its implementation in Brazil and potentially across South America. FUNDING: Brazilian Ministry of Health, Municipal and State Health Secretariats; Fiocruz; Medicines for Malaria Venture; Bill & Melinda Gates Foundation; Newcrest Mining; and the UK Government. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Operational feasibility of Plasmodium vivax radical cure with tafenoquine or primaquine following point-of-care, quantitative glucose-6-phosphate dehydrogenase testing in the Brazilian Amazon: a real-life retrospective analysis.

BACKGROUND: To achieve malaria elimination, Brazil must implement Plasmodium vivax radical cure. We aimed to investigate the operational feasibility of point-of-care, quantitative, glucose-6-phosphate dehydrogenase (G6PD) testing followed by chloroquine plus tafenoquine or primaquine. METHODS: This non-interventional, observational study was done at 43 health facilities in Manaus (Amazonas State) and Porto Velho (Rondônia State), Brazil, implementing a new P vivax treatment algorithm incorporating point-of-care quantitative G6PD testing to identify G6PD status and single-dose tafenoquine (G6PD normal, aged ≥16 years, and not pregnant or breastfeeding) or primaquine (intermediate or normal G6PD, aged ≥6 months, not pregnant, or breastfeeding >1 month). Following training of health-care providers, we collated routine patient records from the malaria epidemiological surveillance system (SIVEP-Malaria) retrospectively for all consenting patients aged at least 6 months with parasitologically confirmed P vivax malaria mono-infection or P vivax plus P falciparum mixed infection, presenting between Sept 9, 2021, and Aug 31, 2022. The primary endpoint was the proportion of patients aged at least 16 years with P vivax mono-infection treated or not treated appropriately with tafenoquine in accordance with their G6PD status. The trial is registered with ClinicalTrials.gov, NCT05096702, and is completed. FINDINGS: Of 6075 patients enrolled, 6026 (99·2%) had P vivax mono-infection, 2685 (44·6%) of whom were administered tafenoquine. G6PD status was identified in 2685 (100%) of 2685 patients treated with tafenoquine. The proportion of patients aged at least 16 years with P vivax mono-infection who were treated or not treated appropriately with tafenoquine in accordance with their G6PD status was 99·7% (95% CI 99·4-99·8; 4664/4680). INTERPRETATION: Quantitative G6PD testing before tafenoquine administration was operationally feasible, with high adherence to the treatment algorithm, supporting deployment throughout the Brazilian health system. FUNDING: Brazilian Ministry of Health, Municipal and State Health Secretariats; Fiocruz; Medicines for Malaria Venture; Bill & Melinda Gates Foundation; Newcrest Mining; and the UK Government. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Obesity and its Relationship with Covid-19: A Review of the Main Pharmaceutical Aspects.

Important physiological changes are observed in patients with obesity, such as intestinal permeability, gastric emptying, cardiac output, and hepatic and renal function. These differences can determine variations in the pharmacokinetics of different drugs and can generate different concentrations at the site of action, which can lead to sub therapeutic or toxic concentrations. Understanding the physiological and immunological processes that lead to the clinical manifestations of COVID-19 is essential to correlate obesity as a risk factor for increasing the prevalence, severity, and lethality of the disease. Several drugs have been suggested to control COVID- 19 like Lopinavir, Ritonavir, Ribavirin, Sofosbuvir, Remdesivir, Oseltamivir, Oseltamivir phosphate, Oseltamivir carboxylate, Hydroxychloroquine, Chloroquine, Azithromycin, Teicoplanin, Tocilizumab, Anakinra, Methylprednisolone, Prednisolone, Ciclesonide and Ivermectin. Similarly, these differences between healthy people and obese people can be correlated to mechanical factors, such as insufficient doses of the vaccine for high body mass, impairing the absorption and distribution of the vaccine that will be lower than desired or can be linked to the inflammatory state in obese patients, which can influence the humoral immune response. Additionally, different aspects make the obese population more prone to persistent symptoms of the disease (long COVID), which makes understanding these mechanisms fundamental to addressing the implications of the disease. Thus, this review provides an overview of the relationship between COVID-19 and obesity, considering aspects related to pharmacokinetics, immunosuppression, immunization, and possible implications of long COVID in these individuals.

Effectiveness of Hospital-directed Wellness Interventions in COVID-19: A Cross-sectional Survey.

INTRODUCTION: Hospitals have implemented various wellness interventions to offset the negative effects of coronavirus disease 2019 (COVID-19) on emergency physician morale and burnout. There is limited high quality evidence regarding effectiveness of hospital-directed wellness interventions, leaving hospitals without guidance on best practices. We sought to determine intervention effectiveness and frequency of use in the spring/summer 2020. The goal was to facilitate evidence-based guidance for hospital wellness program planning. METHODS: This cross-sectional observational study we used a novel survey tool piloted at a single hospital and then distributed throughout the United States via major emergency medicine (EM) society listservs and closed social media groups. Subjects reported their morale levels using a slider scale from 1 (lowest) to 10 (highest) at the time of the survey and, retrospectively, at their respective COVID-19 peak in 2020. Subjects also rated effectiveness of wellness interventions using a Likert scale from 1 (not at all effective) to 5 (very effective). Subjects indicated their hospital's usage frequency of common wellness interventions. We analyzed results using descriptive statistics and t-tests. RESULTS: Of 76,100 EM society and closed social media group members, 522 (0.69%) subjects were enrolled. Study population demographics were similar to the national emergency physician population. Morale at the time of the survey was worse (mean [M] 4.36, SD 2.29) than the spring/summer 2020 peak (M 4.57, SD 2.13) [t(458)=-2.27, P=0.024]. The most effective interventions were hazard pay (M 3.59, SD 1.12), staff debriefing groups (M 3.51, SD 1.16), and free food (M 3.34, SD 1.14). The most frequently used interventions were free food (350/522, 67.1%), support sign display (300/522, 57.5%), and daily email updates (266/522, 51.0%). Infrequently used were hazard pay (53/522, 10.2%) and staff debriefing groups (127/522, 24.3%). CONCLUSION: There is discordance between the most effective and most frequently used hospital-directed wellness interventions. Only free food was both highly effective and frequently used. Hazard pay and staff debriefing groups were the two most effective interventions but were infrequently used. Daily email updates and support sign display were the most frequently used interventions but were not as effective. Hospitals should focus effort and resources on the most effective wellness interventions.

Antibiotics prescribing habits of Brazilian general dental practitioners during periodontal treatments / Hábitos de prescrição de antibióticos de dentistas brasileiros durante tratamentos periodontais

Introdução: O tratamento periodontal envolve procedimentos que visam reduzir a carga bacteriana, envolvendo ou não o uso de antibióticos locais ou sistêmicos. Objetivo: O objetivo deste estudo foi avaliar o conhecimento dos cirurgiões-dentistas brasileiros sobre a prescrição de antibióticos durante o tratamento periodontal. Material e método: Foi disponibilizado um questionário online solicitando aplicação sistêmica de prescrição de antibióticos locais ou sistêmicos (frequência e sequência com tratamento periodontal mecânico) e em relação a diferentes doenças periodontais. A utilização de serviços de diagnóstico microbiano e dados demográficos dos voluntários também foram elucidados. Os dados foram analisados estatisticamente (Teste Binomial, p<0,05). Resultado: Trezentos e noventa e três voluntários responderam ao questionário. Vinte e três por cento do sexo masculino e 76,2% do sexo feminino. A idade média foi de 27,7 anos. A minoria (19,2%) dos voluntários relatou indicar antibióticos sistêmicos para tratamento de gengivite ou periodontite estágios I e II (antiga periodontite crônica leve e moderada). A maioria dos profissionais indica para tratar abscesso periodontal, gengivite ou periodontite ulcerativa necrosante, periodontite em imunodeficiências graves, periodontite agressiva antiga e periodontite estágios III e IV (periodontite crônica grave antiga). Raramente (2,5%) a prescrição foi sem a associação com terapia mecânica. Os exames microbiológicos como método auxiliar de diagnóstico raramente (3,1%) são solicitados por falta de informação ou custo. As respostas mais frequentes sobre o número de vezes que os profissionais relataram prescrever antibióticos sistêmicos como parte do tratamento periodontal por trimestre foram "pelo menos uma vez", seguido de "2 vezes". Conclusão: Pode-se concluir que os cirurgiões-dentistas brasileiros possuem conhecimento sobre a correta prescrição de antibióticos na terapia periodontal

Introduction: Periodontal treatment involves procedures aimed to reduce bacterial load, involving or not the use of local or systemic antibiotics. Objective: The purpose of this study was to assess Brazilian dentists' knowledge about antibiotics prescription during periodontal treatment. Material and method: An online questionnaire was available asking for systemic application of local or systemic antibiotics prescription (frequency, and sequence with mechanical periodontal treatment) and in relation to different periodontal diseases. The use of microbial diagnostic services and volunteers' demographic data were also elucidated. Data were statistically analyzed (Binomial Test, p<0.05). Result: Three hundred and ninety-three volunteers answered the questionnaire. Twenty-three percent males and 76.2% females. Average age was 27.7 years. Minority (19.2%) of the volunteers reported to indicate systemic antibiotics to treat gingivitis or periodontitis stages I and II (old slight and moderate chronic periodontitis). Majority of the professionals indicate it to treat periodontal abscess, necrotizing ulcerative gingivitis or periodontitis, periodontitis in severe immunodeficiencies, old aggressive periodontitis, and periodontitis stages III and IV (old severe chronic periodontitis). Rarely (2.5%) the prescription was without the combination with mechanical therapy. Microbiological tests as an auxiliary diagnostic method are rarely (3.1%) requested due to lack of information or cost. Most frequent answers about the number of times professionals reported prescribing systemic antibiotics as part of periodontal treatment per trimester was "at least once", followed by "2 times". Conclusion: It can be concluded that Brazilian dentists have knowledge about the correct prescription of antibiotics in periodontal therapy

Impact of Covid-19 on diabetic adults: systematic review / Impacto da Covid-19 em adultos diabéticos: revisão sistemática

Aim: to identify which complications and prognosis of diabetic patients, hospitalized, who acquired COVID-19, through a systematic review. Methods: a systematic review based on the PRISMA flowchart, including cohort studies, available in Portuguese, English, Spanish, French, and Mandarin, published from 2019 to 2020, using the PICOS strategy, in the databases: PubMed, Web of Science, Scielo, Lilacs, Scopus and Science Direct, which in addition to the inclusion criteria after questionnaires to assess methodological quality and risk of bias. Results: of the 811 articles researched, 6 were included in this research. These studies showed that patients with COVID-19 and higher DM with worse prognosis, spent more time in the ICU, constantly needed indifference, greater complications when related to other comorbidities, high mortality rate, and glycemic control associated with advanced age directly affected patients. Outcomes even of non-diabetic subjects. Conclusion: this review identified the severity of the pathophysiological association is related to older age and biochemical and inflammatory factors linked to the two pathogens and that these subjects are more prone to specialized hospital care, which, however, result in high rates of hospital mortality.

Objetivo: identificar quais complicações e prognósticos dos pacientes diabéticos, internados, que adquiriram COVID-19, por meio de uma revisão sistemática. Métodos: foi realizada uma revisão sistemática baseada no fluxograma PRISMA, incluindo estudos de coorte, disponíveis em português, inglês, espanhol, francês e mandarim, publicados de 2019 a 2020, utilizando a estratégia PICOS, nas bases de dados: PubMed, Web of Science, Scielo, Lilacs, Scopus e Sciece Direct. Além dos critérios de inclusão passam por questionários para avaliar a qualidade metodológica e risco de viés. Resultados: dos 811 artigos pesquisados, 6 foram incluídos nesta pesquisa. Esses estudos mostraram que pacientes com COVID-19 e DM apresentam pior prognóstico, maior permanência em UTI, necessidade constante de ventilação invasiva, maiores complicações quando relacionadas a outras comorbidades, elevado índice de mortalidade, e o controle glicêmico associado à idade avançada afetavam diretamente os desfechos inclusive de pacientes não diabéticos. Conclusão: esta revisão identificou que a gravidade da associação fisiopatológica está relacionada à idade mais avançada e aos fatores bioquímicos e inflamatórios ligados aos dois patógenos e que esses sujeitos são mais propensos ao atendimento hospitalar especializado, o que, no entanto, resulta em altas taxas de mortalidade hospitalar.

Neurological Manifestations Associated With SARS-CoV-2 in Children: A Case Series.

Neurological manifestations of COVID-19 may affect both central and peripheral nervous systems. Unlike in adults, in whom majority of severe cases derive from respiratory complications, neurological involvement is one of the main causes of severe COVID-19 in children. This study aimed to detect viral respiratory pathogens, mainly SARS-CoV-2, in nasopharynx and cerebrospinal fluid samples utilizing qRT-PCR (TaqMan) in a pediatric population in Brazil. We evaluated four children with neurological symptoms and laboratory-confirmed SARS-CoV-2 infection: three presenting with meningoencephalitis and one presenting with Guillain-Barré syndrome. All four patients had mild respiratory symptoms. SARS-CoV-2 RNA was identified in two cerebrospinal fluid samples. SARS-CoV-2 involvement should be considered for differential diagnosis in pediatric cases presenting neurological alterations even if symptoms such as headache, anosmia, or dizziness are absent.

Epidemiologic and clinical investigations during a chikungunya outbreak in Rio Grande do Norte State, Brazil.

The first autochthonous case of chikungunya virus (CHIKV) infection in Brazil was in September 2014 in the State of Amapá, and from there it rapidly spread across the country. The present study was conducted in 2016 in the state of Rio Grande do Norte, and the aims were to describe the epidemiological and the clinical aspects of the CHIKV outbreak. Biological samples from 284 chikungunya suspected cases were screened for CHIKV and Flavivirus (FV) RNA using qRT-PCR. Negative PCR samples were also screened for anti-CHIKV and anti-FVIgM by ELISA. CHIKV RNA were detected in 125 samples mostly occurring from January through March (46%), mainly affecting adults and older adults. We found a gradual decrease in viral RNA over the disease time. Anti-CHIKV IgM was found in 47.5% after negative CHIKV qRT-PCR. Interestingly, 45.0% simultaneously had positive results for CHIKV and FV IgM, suggesting the occurrence of virus co-circulation. The most frequent symptom was fever (91%). Women presented more chance to develop nausea and abdominal pain compared to men. Our data described and allows us to better understand the clinical and epidemiological aspects of the 2016 chikungunya outbreak in Rio Grande do Norte and can help in the early clinical diagnosis of the virus.

Innate immune response in patients with acute Zika virus infection.

Innate immunity receptors (Toll-like receptors/TLRs and RIG-like receptors/RLRs) are important for the initial recognition of Zika virus (ZIKV), modulation of protective immune response, and IFN-α and IFN-ß production. Immunological mechanisms involved in protection or pathology during ZIKV infection have not yet been determined. In this study, we evaluated the mRNA expression of innate immune receptors (TLR3, TLR7, TLR8, TLR9, melanoma differentiation-associated protein 5/MDA-5, and retinoic acid inducible gene/RIG-1), its adapter molecules (Myeloid Differentiation Primary Response Gene 88/Myd88, Toll/IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-ß/TRIF), and cytokines (IL-6, IL-12, TNF-α, IFN-α, IFN-ß, and IFN-γ) in the acute phase of patients infected by ZIKV using real-time PCR in peripheral blood. Patients with acute ZIKV infection had high expression of TLR3, IFN-α, IFN-ß, and IFN-γ when compared to healthy controls. In addition, there was a positive correlation between TLR3 expression compared to IFN-α and IFN-ß. Moreover, viral load is positively correlated with TLR8, RIG-1, MDA-5, IFN-α, and IFN-ß. On the other hand, patients infected by ZIKV showed reduced expression of RIG-1, TLR8, Myd88, and TNF-α molecules, which are also involved in antiviral immunity. Similar expressions of TLR7, TLR9, MDA-5, TRIF, IL-6, and IL-12 were observed between the group of patients infected with ZIKV and control subjects. Our results indicate that acute infection (up to 5 days after the onset of symptoms) by ZIKV in patients induces the high mRNA expression of TLR3 correlated to high expression of IFN-γ, IFN-α, and IFN-ß, even though the high viral load is correlated to high expression of TLR8, RIG-1, MDA-5, IFN-α, and IFN-ß in ZIKV patients.

Polimedicação em Idosos Submetidos a Tratamento Oncológico / Polymedication in Elderly Submitted to Oncological Treatment / Polimedicación en Personas Mayores Recibiendo el Tratamiento Oncológico

Introdução: O paciente idoso com câncer possui risco potencial de polimedicação pelo aumento de comorbidade e complexidade do tratamento do câncer que, muitas vezes, inclui múltiplos antineoplásicos e medicamentos de suporte. Objetivo: Identificar a frequência de polimedicação, o uso de medicamentos potencialmente inapropriados e a interação medicamentosa em pacientes idosos com câncer. Método: Coletaram-se informações sociodemográficas, farmacológicas e clínicas de 20 pacientes idosos (≥65 anos) com câncer, polimedicados (≥cinco medicamentos), de julho a agosto de 2016, em um ambulatório em São Luís ­ MA, por meio de prontuário eletrônico e questionários aplicados em entrevistas. Para análise dos dados, utilizaram-se as ferramentas: Classificação Anatômica Terapêutica Química, Micromedex, Drugs, Lexicomp e o Manual de fitoterápicos da Anfarmag. O Beers Criteria 2015 foi utilizado para identificação dos medicamentos potencialmente inapropriados para idosos. Resultados: Entre os pacientes, 70% eram do sexo masculino, com idade média de 73 anos (DP=7,9). Os tipos de câncer mais prevalentes foram os de próstata e mama. Quanto às comorbidades, as cardiovasculares e as endócrinas foram as mais relatadas. Foram identificados 134 medicamentos prescritos e de automedicação, dos quais, 41 eram antineoplásicos. Oitenta por cento dos pacientes estavam expostos a risco potencial de interação medicamentosa, totalizando 90 (63,2% de gravidade moderada, 21,2% importante e 8,8% menor). Detectaram-se quatro interações envolvendo plantas medicinais e sete medicamentos inapropriados para idosos em 20% e 65% dos pacientes, respectivamente. Conclusão: Entre os pacientes incluídos neste estudo, verificou-se a ocorrência de polimedicação, de interações medicamentosas potenciais e do uso de medicamentos potencialmente inapropriados para idosos.

Introduction: The elderly patient with cancer has potential risk of polymedication due to the increase of comorbidity and complexity of the cancer treatment, which often includes multiple antineoplastic and supportive medications. Objective: The objective is to identify the frequency of polypharmacy, potentially inappropriate medication use and drug interaction in elderly patients with cancer. Method: It were collected sociodemographic, pharmacological and clinical information from 20 elderly patients (≥ 65 years) with cancer, polymedicated (≥five drugs), from July to August 2016, in an outpatient clinic in São Luís, MA through electronic chart and questionnaires applied in interviews. To analyze the data, it were used the: Anatomical Therapeutic Chemical (ATC), Micromedex, Drugs, Lexicomp and the Anfarmag Phytotherapy Guideline. Beers Criteria 2015 was used to identify potentially inappropriate medication for the elderly. Results: Among the patients, 70% were males, with a mean age of 73 years (SD=7.9). The most prevalent types of cancer were prostate cancer and breast. Cardiovascular and endocrine comorbidities were the most reported. A total of 134 drugs prescribed and self-medication were identified, of which 41 were antineoplastic. Eighty percent of the patients were exposed to potential risk of drug interaction, totaling 90 (63.2% of moderate, 21.2% of significant and 8.8% of mild severity). There were 4 interactions involving medicinal plants and seven inappropriate medications in use by 20% e 65% of the elderly patients, respectively. Conclusion: Among the patients included in this study, it was verified the occurrence of polypharmacy, potential drug interactions and the use of potentially inappropriate medications for the elderly.

Introducción: Un paciente con cáncer de edad avanzada tiene riesgo potencial de polmedicación debido a mayor comorbilidad y complejidad del tratamiento del cáncer, que incluye múltiples medicamentos antineoplásicos y de soporte. Objectivo: Identificar la frecuencia de polifarmacia, de uso de medicamentos potencialmente inapropiados y interacciónes medicamentosa en personas mayores con cancro. Método: En el período de julio a agosto de 2016, en ambulatorio en São Luís­MA, se recolectó informaciones sociodemográficas, farmacológicas y clínicas de 20 pacientes ancianos (≥ 65 años) cancro, polimedicados (≥ cinco medicamentos), por medio de recolección en registro médico electrónico y cuestionarios. Para análisis de datos, se utilizaron herramientas como Clasificación Anatómica Terapéutica Química, Micromedex, Drugs, Lexicomp y Manual de fitoterápicos de la Anfarmag. Beers Criteria 2015 se utilizó para identificar los medicamentos potencialmente inapropiados para ancianos. Resultados: Entre los pacientes, 70% eran del sexo masculino, con edad promedio de 73 años (DP=7,9). Los tipos de cancro más prevalentes fueron de próstata y mama. En cuanto a las comorbilidades, las cardiovasculares y endocrinas fueron las más relatadas. Se identificaron 134 prescripciones y automedicación, de los cuales 41 antineoplásicos. 80% de pacientes estaban expuestos a interacción medicamentosa, totalizando 90 (63,2% de gravedad moderada, 21,2% importantes y 8,8% leves). Se detectaron 4 interacciones que involucra plantas medicinales y siete medicamentos inapropriados para ancianos en el 20% y 65% de los pacientes, respectivamente. Conclusión: Entre los pacientes incluidos en estudio, verificamos aparición de polifarmacia, riesgos de posibles interacciones medicamentosas y el uso de medicamentos potencialmente inapropiados para los ancianos.

Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus.

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.

Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.