Biplex quantitative PCR to detect transcriptionally active human papillomavirus 16 from patient saliva.

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

Metagenomic Analysis for Diagnosis of Hemorrhagic Fever in Minas Gerais, Brazil.

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient's clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.

Paper-based electrochemical biosensors for the diagnosis of viral diseases.

Paper-based electrochemical analytical devices (ePADs) have gained significant interest as promising analytical units in recent years because they can be fabricated in simple ways, are low-cost, portable, and disposable platforms that can be applied in various fields. In this sense, paper-based electrochemical biosensors are attractive analytical devices since they can promote diagnose several diseases and potentially allow decentralized analysis. Electrochemical biosensors are versatile, as the measured signal can be improved by using mainly molecular technologies and nanomaterials to attach biomolecules, resulting in an increase in their sensitivity and selectivity. Additionally, they can be implemented in microfluidic devices that drive and control the flow without external pumping and store reagents, and improve the mass transport of analytes, increasing sensor sensitivity. In this review, we focus on the recent developments in electrochemical paper-based devices for viruses' detection, including COVID-19, Dengue, Zika, Hepatitis, Ebola, AIDS, and Influenza, among others, which have caused impacts on people's health, especially in places with scarce resources. Also, we discuss the advantages and disadvantages of the main electrode's fabrication methods, device designs, and biomolecule immobilization strategies. Finally, the perspectives and challenges that need to be overcome to further advance paper-based electrochemical biosensors' applications are critically presented.

Insights from establishing a high throughput viral diagnostic laboratory for SARS-CoV-2 RT-PCR testing facility: challenges and experiences.

Background: The World Health Organization declared the coronavirus disease 2019 (COVID-19) a global pandemic on 11 March 2020. Identifying the infected people and isolating them was the only measure that was available to control the viral spread, as there were no standardized treatment interventions available. Various public health measures, including vaccination, have been implemented to control the spread of the virus worldwide. India, being a densely populated country, required laboratories in different zones of the country with the capacity to test a large number of samples and report test results at the earliest. The Indian Council of Medical Research (ICMR) took the lead role in developing policies, generating advisories, formulating guidelines, and establishing and approving testing centers for COVID-19 testing. With advisories of ICMR, the National Institute of Cancer Prevention and Research (NICPR) established a high-throughput viral diagnostic laboratory (HTVDL) for RT-PCR-based diagnosis of SARS-CoV-2 in April 2020. HTVDL was established during the first lockdown to serve the nation in developing and adopting rapid testing procedures and to expand the testing capacity using "Real-Time PCR." The HTVDL provided its testing support to the national capital territory of Delhi and western Uttar Pradesh, with a testing capacity of 6000 tests per day. The experience of establishing a high-throughput laboratory with all standard operating procedures against varied challenges in a developing country such as India is explained in the current manuscript which will be useful globally to enhance the knowledge on establishing an HTVDL in pandemic or non-pandemic times.

A robust host-response-based signature distinguishes bacterial and viral infections across diverse global populations.

Limited sensitivity and specificity of current diagnostics lead to the erroneous prescription of antibiotics. Host-response-based diagnostics could address these challenges. However, using 4,200 samples across 69 blood transcriptome datasets from 20 countries from patients with bacterial or viral infections representing a broad spectrum of biological, clinical, and technical heterogeneity, we show current host-response-based gene signatures have lower accuracy to distinguish intracellular bacterial infections from viral infections than extracellular bacterial infections. Using these 69 datasets, we identify an 8-gene signature to distinguish intracellular or extracellular bacterial infections from viral infections with an area under the receiver operating characteristic curve (AUROC) > 0.91 (85.9% specificity and 90.2% sensitivity). In prospective cohorts from Nepal and Laos, the 8-gene classifier distinguished bacterial infections from viral infections with an AUROC of 0.94 (87.9% specificity and 91% sensitivity). The 8-gene signature meets the target product profile proposed by the World Health Organization and others for distinguishing bacterial and viral infections.

Development of a nested real time PCR/high resolution melting assay for human T-cell lymphotropic viruses types 1 and 2 (HTLV-1 and 2) identification.

HTLV-1 and HTLV-2 are present in different high-risk populations, such as sexual workers and injecting drug users (IDUs). HTLV-1 is endemic in areas of Middle East, Southern Japan and Latin America, whereas HTLV-2 infection is endemic among some Native Americans and some Central African tribes. The pathogenic consequences and clinical manifestations of these two viruses differ significantly, demanding an adequate identification; therefore, proper diagnosis of HTLV-1 and 2 infection is crucial. To get a final diagnosis of HTLV-1 or 2 infection, it is recommended that positive serologic samples should be confirmed by PCR assays or western blot (WB) analysis. Thus, the aim of this study was to develop and implement a simple reaction for the rapid identification of HTLV-1 and 2. Nested real-time PCR technique followed by high resolution melting was performed based on the tax/rex sequences of HTLV-1 (M2) and HTLV-2 (MoT) cell lines perfectly discriminating between HTLV-1 from HTLV-2, by distinct melting curve profiles. The sensitivity assay of this method revealed that at least 1 viral copy of HTLV-1 or 1·5 viral copy of HTLV-2 could be amplified. Later, this method was validated using 200 blood samples from corpses. In agreement with previous epidemiological, the HTLV-1 and 2 prevalence was 1·5% (CI 95%: 0·31-4·3) and 0·5% (CI 95%: 0·013-2·75), respectively. The strategy proposed herein has some advantages over other PCR-based tests because it not only reduces considerably time and the costs of the total diagnosis but also allows detection and discrimination of HTLV-1 and 2 in the same reaction.

Major Complex Trait for Early De Novo Programming 'CoV-MAC-TED' Detected in Human Nasal Epithelial Cells Infected by Two SARS-CoV-2 Variants Is Promising to Help in Designing Therapeutic Strategies.

BACKGROUND: Early metabolic reorganization was only recently recognized as an essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels connected to increased aerobic fermentation, which is linked to alpha-tubulin-based cell restructuring and control of cell cycle progression, were identified as a major complex trait for early de novo programming ('CoV-MAC-TED') during SARS-CoV-2 infection. This trait was highlighted as a critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named 'ReprogVirus', which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the 'ReprogVirus Platform'. This platform is currently under development. However, so far, an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing the data collection of early SARS-CoV-2 infection responses has not been defined. RESULTS: Here, we demonstrate transcriptome-level profiles of the most critical 'ReprogVirus' gene sets for identifying 'CoV-MAC-TED' in cultured human nasal epithelial cells infected by two SARS-CoV-2 variants differing in disease severity. Our results (a) validate 'Cov-MAC-TED' as a crucial trait for early SARS-CoV-2 reprogramming for the tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. CONCLUSION: In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the 'ReprogVirus Platform'. Thus, this cell system is highly promising to advance integrative data analyses with the help of artificial intelligence methodologies for designing anti-SARS-CoV-2 strategies.

Nano-therapeutic strategies to target coronavirus.

The coronaviruses have caused severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS), and the more recent coronavirus pneumonia (COVID-19). The global COVID-19 pandemic requires urgent action to develop anti-virals, new therapeutics, and vaccines. In this review, we discuss potential therapeutics including human recombinant ACE2 soluble, inflammatory cytokine inhibitors, and direct anti-viral agents such as remdesivir and favipiravir, to limit their fatality. We also discuss the structure of the SARS-CoV-2, which is crucial to the timely development of therapeutics, and previous attempts to generate vaccines against SARS-CoV and MERS-CoV. Finally, we provide an overview of the role of nanotechnology in the development of therapeutics as well as in the diagnosis of the infection. This information is key for computational modeling and nanomedicine-based new therapeutics by counteracting the variable proteins in the virus. Further, we also try to effectively share the latest information about many different aspects of COVID-19 vaccine developments and possible management to further scientific endeavors.

[Early Detection of COVID-19 in Portugal: Use of Clinical Records]. / Deteção Precoce de COVID-19 em Portugal: Uso de Registos Clínicos.

INTRODUCTION: Syndromic surveillance allows early detection of changes in the population's morbidity pattern. The aim of this study is to evaluate the usefulness of indicators related to access to healthcare services, in COVID-19 surveillance. MATERIAL AND METHODS: A time series analysis was performed using the weekly incidence rate of COVID-19 in Mainland Portugal, between weeks 14/2020 (March 30 to April 5) and 25/2020 (June 15 to 21), and six indicators: 1) COVID-19 consultations in primary healthcare; 2) number of COVID-19 emergency department visits; 3) number of emergency department visits due to viral pneumonia; 4) number of hospitalizations due to viral pneumonia; 5) proportion of emergency department visits due to viral pneumonia; and 6) proportion of hospitalizations for viral pneumonia. Pearson correlation and cross-correlations were computed. RESULTS: A strong correlation was found between the weekly incidence rate of COVID-19 and all indicators. [(1) 0.76; (2) 0.82; (3) 0.77; (4) 0.84; (5) 0.86; e (6) 0.90]. Emergency department visits and hospitalizations for viral pneumonia detect variations in the frequency of the disease with a one week lag compared to the incidence rate of COVID-19, in one week. COVID-19 consultations in primary healthcare and emergency department visits trail behind the incidence rate of COVID-19, in one week. The proportion of viral pneumonias in emergency department visits, or hospitalizations, is temporally aligned with the weekly incidence rate of COVID-19. DISCUSSION: The delay found in the COVID-19 primary healthcare consultations and emergency department visits, may be related to changes in access to healthcare services and clinical coding. Emergency department visits and hospitalizations for viral pneumonia may be useful in the early detection of COVID-19. Viral pneumonia may have been coded as being of unknown origin. Future monitoring of these indicators is necessary to ascertain whether the incidence of COVID-19 is significantly influenced by changes in testing strategies. The indicators described in this study will be an asset for the optimization of testing strategies, allocation of healthcare resources to the communities that are most vulnerable to severe morbidity and assessing vaccination impact. As such, surveillance systems based on clinical data will be a valuable complementary tool to SINAVE. CONCLUSION: The indicators under analysis could be used regularly, with special attention to viral pneumonias, to detect outbreaks of COVID-19. Information on pneumonia of unknown etiology may be considered in the surveillance of COVID-19.

Introdução: A vigilância sindrómica permite a identificação precoce de alterações no padrão de morbilidade da população. Este estudo tem como objetivo avaliar a utilidade de indicadores relativos a cuidados de saúde primários e hospitalares, na vigilância da COVID-19.Material e Métodos: Foi realizada uma análise de séries temporais utilizando a taxa de incidência semanal de COVID-19 em Portugal Continental, entre as semanas 14/2020 (30 março a 05 abril) e 25/2020 (15 a 21 junho), e seis indicadores: 1) consultas em cuidados de saúde primários por COVID-19; 2) número de episódios de urgência por COVID-19; 3) número de episódios de urgência por pneumonia vírica; 4) número de internamentos por pneumonia vírica; 5) proporção de episódios de urgência por pneumonia vírica face ao total de episódios de urgência por pneumonia; e 6) proporção de internamentos por pneumonia vírica face ao total de internamentos por pneumonia. Foram calculadas correlações de Pearson e correlações cruzadas.Resultados: Foi encontrada uma correlação forte entre a taxa de incidência semanal de COVID-19 e todos os indicadores [(1) 0,76; (2) 0,82; (3) 0,77; (4) 0,84; (5) 0,86; e (6) 0,90]. Os episódios de urgência e internamento por pneumonias víricas detetam variações na frequência da doença, com uma semana de antecedência. As consultas em cuidados de saúde primários e urgências por COVID-19 registam uma semana de atraso relativamente à evolução da taxa de incidência. A proporção de pneumonias víricas face ao número de pneumonias em episódios de urgência, ou internamentos, encontra-se alinhada temporalmente com a evolução da taxa de incidência semanal de COVID-19.Discussão: O atraso encontrado no padrão de evolução de consultas em CSP, e de episódios de urgência por COVID-19 face à incidência de COVID-19, poderá estar relacionado com a reorganização dos serviços de saúde e criação de códigos específicos para estas consultas. Episódios de urgência e internamentos por pneumonia vírica poderão ser úteis para a deteção precoce de possíveis surtos de COVID-19. Pneumonias víricas poderão ter sido classificadas como pneumonias de causa indeterminada. A monitorização futura destes indicadores é necessária de modo a averiguar se a incidência de COVID-19 é influenciada significativamente por alterações na estratégia de testagem. Os indicadores deste trabalho serão uma mais valia para a adequação de estratégias de testagem, alocação de recursos de saúde a comunidades mais vulneráveis à morbilidade severa e avaliação de programas de vacinação. Como tal, os sistemas de vigilância com base em registos de saúde serão um complemento valioso ao SINAVE.Conclusão: Sugere-se que os indicadores em análise sejam utilizados de forma regular, com especial atenção à informação relativa a pneumonias víricas, como forma de detetar precocemente surtos de COVID-19. A informação relativa a pneumonias de causa indeterminada poderá ser considerada na monitorização da COVID-19.

A comparison of swab types on sample adequacy, suspects comfort and provider preference in COVID-19.

AIM: This study was aimed to compare the virological, suspect reported outcomes and provider preferences during COVID-19 swab taking procedure used for sampling. METHODS: The COVID-19 suspects are subjected to nasopharyngeal (NP) and oropharyngeal (OP) swabs for testing. Two types of swabs (Nylon and Dacron) are used for sample collection. Prospectively each suspect's response is collected and assessed for self-reported comfort level. The provider's experience with each suspect and virological outcomes recorded separately. The sample adequacy was compared based on swab types and demographic characteristics. RESULTS: A total of 1008 COVID-19 suspects were considered for comparison of various outcomes. Dacron and flocked Nylon swab sticks are used for taking 530 and 478 samples, respectively. Suspects who underwent the procedure using Nylon swabs were six times more likely to have pain/discomfort compared to when Dacron swab was used (Adj RR (95% CI: 6.76 (3.53 to 13, p=0.0001))). The providers perceived six times more resistance with the Nylon swabs compared to Dacron Swabs (Adj RR (95% CI: 5.96 (3.88 to 9.14, p=0.0001))). The pediatric population had a higher rate of blood staining in Dacron swab [Dacron 66 (80.5%); Nylon 51 (54.8%) p=0.0001]. The sample adequacy rate and laboratory positivity rate were not significantly different from each other. CONCLUSIONS: Given the comparable virological outcomes, the difference in suspect and providers comfort should drive swab selection based on characteristics of the suspects. The bulbous Nylon swab caused more pain/discomfort in adults compared to Dacron.

[Advances in molecular biology research on human parvovirus B19].

Human parvovirus B19 (B19 virus) is one of the two parvoviruses that cause human diseases. As an important pathogen to humans, it causes infectious erythema in children, acute aplastic anemia, fetal edema and death. In this review, we focus on the recent advances in the molecular virology of B19V, such as viral genotypes, viral receptor, genomic features and viral replication, viral transcription and post-transcription regulation, viral nonstructural and structural protein features and functions, viral diagnosis and antiviral agents, to provide reference for further study of B19 pathogenesis mechanisms, treatment and diagnostic strategies.

False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors / Resultado falso negativo no diagnóstico molecular de SARS-CoV-2 em amostras com inibidores de amplificação

ABSTRACT Introduction: Although reverse transcription-polymerase chain reaction (rRT-PCR) is the gold standard method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), some factors, such as the presence of amplification inhibitors, lead to false-negative results. Objective: Here we describe the differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to the presence of amplification inhibitors. Material and method: Viral ribonucleic acid (RNA) from samples of nasopharyngeal swabs from 20 patients previously detected as "Negative" and 21 patients detected as "Positive" for SARS-CoV-2 was performed with the EasyExtract DNA-RNA kit (Interprise®). The rRT-PCR was performed with the OneStep/COVID-19 kit (IBMP), with normal and diluted (80 µl of H2O RNAse free) samples, totaling 82 tests. Results: The results indicate that there is an average variation (a < 0.05) delaying the Cq between the results of amplification of the internal control (IC), N gene (NG), and ORF1ab (OF), 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. Discussion: The extraction kit does not completely purify the inhibitor compounds; therefore, no amplified product result may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution; this process reduces the efficiency of rRT-PCR to 29.8% in detecting SARS-CoV-2. Conclusion: Knowing the rRT-PCR standards of diluted samples can assist in the identification of false-negative cases and, consequently, avoid incorrect diagnosis.

RESUMEN Introducción: Aunque la reacción en cadena de la polimerasa con transcriptasa reversa en tiempo real (rRT-PCR) sea el método de referencia para detección del coronavirus tipo 2 del síndrome respiratorio agudo grave (Sars-CoV-2), algunos factores como la presencia de inhibidores de amplificación conducen a resultados falsos negativos. Objetivo: Describimos las diferencias entre los resultados de rRT-PCR para infección por Sars-CoV-2 en muestras normales y diluidas, simulando la necesidad de dilución debido a la presencia de inhibidores de amplificación. Material y método: La extracción de ácido ribonucleico (ARN) viral de muestras de hisopos nasofaríngeos de 20 pacientes previamente detectados como "negativos" y 21 pacientes detectados como "positivos" para Sars-CoV-2 se realizó con el kit Easy Extract DNA-RNA (Interprise®). La rRT-PCR se realizó con el kit OneStep/Covid-19 (IBMP), con muestras normales y diluidas (80 µl de H2O libre de ARNasa), totalizando 82 pruebas. Resultados: Los resultados indican que hay una variación media (a < 0,05) retrasando el ciclo de cuantificación (Cq) entre los resultados de amplificación del control interno (CI), gen N (GN) y ORF1ab (OF) de 1,811 Cq, 3,840 Cq y 3,842 Cq. Discusión: El kit de extracción no purifica completamente los compuestos inhibidores; por lo tanto, puede ocurrir no amplificación. Obtuvimos un diagnóstico falso negativo de 19,04% después de la dilución de la muestra; ese proceso reduce la eficiencia de la rRT-PCR hacia 29,8% en la detección de Sars-CoV-2. Conclusión: Conocer los patrones de la rRT-PCR de muestras diluidas puede ayudar en la identificación de casos falsos negativos y, por consiguiente, evitar un diagnóstico equivocado.

RESUMO Introdução: Embora a reação em cadeia da polimerase de transcrição reversa (rRT-PCR) seja o método padrão-ouro para detecção de coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), alguns fatores como a presença de inibidores de amplificação levam a resultados falso negativos. Objetivo: Descrevemos as diferenças entre os resultados de rRT-PCR para infecção por SARS-CoV-2 em amostras normais e diluídas, simulando a necessidade de diluição devido à presença de inibidores de amplificação. Material e método: A extração de ácido ribonucleico (RNA) viral de amostras de suabes nasofaríngeos de 20 pacientes previamente detectados como "negativos" e 21 pacientes detectados como "positivos" para SARS-CoV-2 foi realizada com kit o EasyExtract DNA-RNA (Interprise®). A rRT-PCR foi realizada com o kit OneStep/COVID-19 (IBMP), com amostras normais e diluídas (80 µl de H2O RNAse-free), totalizando 82 testes. Resultados: Os resultados indicam que existe uma variação média (a < 0,05) atrasando o Cq entre os resultados de amplificação do controle interno (CI), gene N (GN) e ORF1ab (OF) de 1,811 Cq, 3,840 Cq e 3,842 Cq, respectivamente. Discussão: O kit de extração não purifica completamente os compostos inibidores, portanto, pode ocorrer não amplificação. Obtivemos um diagnóstico falso negativo de 19,04% após a diluição da amostra; esse processo reduz a eficiência da rRT-PCR para 29,8% na detecção de SARS-CoV-2. Conclusão: Conhecer os padrões da rRT-PCR de amostras diluídas pode auxiliar na identificação de casos falso negativos e, consequentemente, evitar um diagnóstico incorreto.

Factores asociados al desarrollo de la bronquiolitis / Associated factors with the development of the bronchiolitis

RESUMEN Fundamento: la bronquiolitis aguda es una enfermedad de etiología viral caracterizada por obstrucción de la pequeña vía aérea que afecta a los niños menores de 24 meses. Objetivo: determinar los factores asociados al desarrollo de la bronquiolitis aguda. Métodos: se realizó un estudio de casos y controles en el municipio de Guáimaro, provincia Camagüey, entre junio de 2016 a diciembre de 2017. Los casos fueron 37 niños menores de dos años con diagnóstico clínico de bronquiolitis e igual cantidad de niños sin este diagnóstico conformaron los controles. La información se obtuvo mediante un cuestionario a los padres, donde se obtuvo información sobre: la edad del niño, el sexo, la severidad, edad gestacional al nacer, vía de nacimiento, peso al nacer, lactancia materna exclusiva, historia de atopia familiar, concurrir a circulo infantil, hermanos en edad escolar, madre fumadora en el embarazo y exposición al humo del tabaco en el domicilio. Resultados: hubo predominio de los menores de seis meses de sexo masculino y clasificado de leves. Las condiciones que mostraron asociación estadística con la aparición de la bronquiolitis fueron: madre fumadora en el embarazo, ausencia de lactancia materna exclusiva, la historia familiar de atopia y la exposición al humo del tabaco en el domicilio. Conclusiones: fueron los factores de riesgo para el desarrollo de la bronquiolitis: el fumar en el embarazo, ausencia de lactancia materna exclusiva, la historia familiar de atopia y la exposición al humo del tabaco en el domicilio.

ABSTRACT Background: the acute bronchiolitis is a disease caused by viruses that produced obstruction of the airways in children less than two years old. Objective: to determine the factors associated with the development of acute bronchiolitis. Methods: a case-control study was made in Guáimaro, Camagüey province, Cuba from June 2015 to January 2017. The cases were 37 children under two years old with diagnostic of bronchiolitis and the same number of children without diagnostic were the controls. The information was obtained through a questionnaire applied to parents of children. The variables analyzed were age, gender, severity, weight at birth, gestational age at birth, childbirth, breastfeeding, family history of atopic disease, siblings in scholar age, nursery assistance, smoker mother during pregnancy and exposition to tobacco smoke in the home. Results: there was predominance of the minors of six months, of masculine sex and classified of light. There was significant relationships between bronchiolitis and the smoker mother during pregnancy, absence of exclusive breastfeeding, family history of atopic disease and exposition to tobacco smoke in the home. Conclusions: were factors for the development of bronchiolitis: mother smoked during pregnancy, absence of exclusive breastfeeding, family history of atopic disease and exposition to tobacco smoke in the home.

Priorities in applied research to ensure programmatic success in the global elimination of canine rabies.

The elimination of human rabies mediated by dogs is attainable in concept, based upon current sensitive and specific diagnostic methods, existing safe and effective human and veterinary vaccines and a sound virological, pathological and epidemiological understanding of the disease. Globally, all developed countries achieved this goal. Regionally, major progress occurred throughout the Americas. However, less advancement is evident in Africa and Asia. Our objective was to concentrate upon those salient improvements to extant tools and methods over the next five years which could assist and simplify the task for both those developing countries that have already begun the process, as well as other localities in the earlier stages of consideration. We considered several categories of applied research which could be accomplished in the short term, based upon the available scientific evidence and recent recommendations from subject matter experts and key opinion leaders, focused upon perceived major limitations to prior program success. Areas of concentration included: laboratory-based surveillance, pathogen detection and characterization; human rabies prophylaxis; veterinary biologics; implementation of canine vaccination; and oral vaccination of free-ranging community dogs. Further real-time application in these core areas with proven techniques and technology would simplify attaining not only the global goal focused subtly upon human mortality, but the actual elimination of canine rabies as well.

Future management of viral diseases: role of new technologies and new approaches in microbial interactions.

BACKGROUND: New technologies allow rapid detecting and counting of virus genomes in clinical specimens, defining susceptibility to specific antivirals, pinpointing molecular sequences correlated to virulence traits, and identifying viral and host factors driving resolution or chronicity of infections. As a result, during the past three decades the diagnostic virology laboratory has become crucial for patient care and an integral component of the multifarious armamentarium for patient management. This change in paradigm has caused obsolescence of methods once considered the reference standard of infectious disease diagnosis that were used to detect whole or specific components of virions in the specimen. OBJECTIVES: This review provides an overview of standard and novel technologies applied to molecular diagnosis of viral infections and illustrates some crucial points for correcting interpretation of the laboratory data. SOURCES: Peer-reviewed literature of topics pertinent to this review. CONTENT AND IMPLICATIONS: New technologies are reinventing the way virologic diagnoses are made, with a conversion to new, simpler-to-use platforms. Although indicated for the same purpose, not all methods are equal and can yield different results. Further, tests identifying multiple analytes at once can detect microorganisms present or activated as a result of pathologic processes triggered by other pathogens or noninfectious causes. Thus, new directions will have to be taken in the way in which the diagnoses of viral diseases are performed. This represents a breakthrough in the clinical virology laboratory.

Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety.

Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer-scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme-linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. In this context, the "catch-all" properties of TEM make it possible to document the presence of viruses or virus-like particles in these products.

Emerging technologies for the detection of viral infections.

Viruses represent one of the major environmental agents that cause human illness and disease. However, the ability to diagnose viral infections is limited by detection capability and scope. Here we describe several emerging technologies that provide rapid and/or high-quality viral diagnostic information. Two technologies, novel CRISPR-based diagnostics and a portable DNA sequencing instrument, are uniquely suited to increase the number of viral agents analyzed, even in point of care settings. We also discuss a phage-based method for generating comprehensive viral profiles of previous exposure/infection and a fluid-phase immunoassay that yields highly quantitative viral antibody analyses. Future applications of these approaches will accelerate on-site clinical diagnosis of viral infections and provide insights into the role viruses play in complex diseases.

Detection of Influenza A and B Viruses and Respiratory Syncytial Virus by Use of Clinical Laboratory Improvement Amendments of 1988 (CLIA)-Waived Point-of-Care Assays: a Paradigm Shift to Molecular Tests.

An accurate laboratory diagnosis of influenza, respiratory syncytial virus (RSV), and other respiratory viruses can help to guide patient management, antiviral therapy, infection prevention strategies, and epidemiologic monitoring. Influenza has been the primary driver of rapid laboratory testing due to its morbidity and mortality across all ages, the availability of antiviral therapy, which must be given early to have an effect, and the constant threat of new pandemic strains. Over the past 30 years, there has been an evolution in viral diagnostic testing, from viral culture to rapid antigen detection, and more recently, to highly sensitive nucleic acid amplification tests (NAAT), as well as a trend to testing at the point of care (POC). Simple rapid antigen immunoassays have long been the mainstay for POC testing for influenza A and B viruses and respiratory syncytial virus (RSV) but have been faulted for low sensitivity. In 2015, the first POC NAAT for the detection of influenza was approved by the Food and Drug Administration (FDA), ushering in a new era. In 2017, the FDA reclassified rapid influenza diagnostic tests (RIDTs) from class I to class II devices with new minimum performance standards and a requirement for annual reactivity testing. Consequently, many previously available RIDTs can no longer be purchased in the United States. In this review, recent developments in Clinical Laboratory Improvement Amendments of 1988 (CLIA)-waived testing for respiratory virus infections will be presented, with the focus on currently available FDA-cleared rapid antigen and molecular tests primarily for influenza A and B viruses and RSV.