Update in Diagnostics of Toscana Virus Infection in a Hyperendemic Region (Southern Spain).

The sandfly fever Toscana virus (TOSV, genus Phlebovirus, family Phenuiviridae) is endemic in Mediterranean countries. In Spain, phylogenetic studies of TOSV strains demonstrated that a genotype, different from the Italian, was circulating. This update reports 107 cases of TOSV neurological infection detected in Andalusia from 1988 to 2020, by viral culture, serology and/or RT-PCR. Most cases were located in Granada province, a hyperendemic region. TOSV neurological infection may be underdiagnosed since few laboratories include this virus in their portfolio. This work presents a reliable automated method, validated for the detection of the main viruses involved in acute meningitis and encephalitis, including the arboviruses TOSV and West Nile virus. This assay solves the need for multiple molecular platforms for different viruses and thus, improves the time to results for these syndromes, which require a rapid and efficient diagnostic approach.

Phylogenetic Analysis of the 2020 West Nile Virus (WNV) Outbreak in Andalusia (Spain).

During recent decades West Nile Virus (WNV) outbreaks have continuously occurred in the Mediterranean area. In August 2020 a new WNV outbreak affected 71 people with meningoencephalitis in Andalusia and six more cases were detected in Extremadura (south-west of Spain), causing a total of eight deaths. The whole genomes of four viruses were obtained and phylogenetically analyzed in the context of recent outbreaks. The Andalusian viral samples belonged to lineage 1 and were relatively similar to those of previous outbreaks which occurred in the Mediterranean region. Here we present a detailed analysis of the outbreak, including an extensive phylogenetic study. As part on this effort, we implemented a local Nextstrain server, which has become a constituent piece of regional epidemiological surveillance, wherein forthcoming genomes of environmental samples or, eventually, future outbreaks, will be included.

Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene.

Toscana virus (TOSV) can cause central nervous system infections in both residents of and travelers to Mediterranean countries. Data mining identified three real-time RT-qPCR assays for detecting TOSV RNA targeting non-overlapping regions in the nucleoprotein gene. Here, they were combined to create a multi-region assay named Trio TOSV RT-qPCR consisting of six primers and three probes. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR, (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one of the reference monoplex assays on clinical samples. In conclusion, the Trio TOSV RT-qPCR assay performs equally or better than the three monoplex assays; therefore, it provides a robust assay that can be used for both research and diagnostic purposes.

A propósito de un nuevo caso de shigelosis por una cepa multirresistente no importada / No disponible

No disponible

A propos of a new case of shigellosis by a non-imported multiresistant strain.

We present the case corresponding to a shigellosis produced by multiresistant S. flexneri in a patient with no recent history of tourism or travel to exotic countries. This case exposes the need to know the distribution of resistant strains, and their emergence not imported in our environment, in the face of choosing the most appropriate type of antibiotic, when appropriate.

Analytical validation of viral CNS Flow Chip kit for detection of acute meningitis and encephalitis.

A new molecular assay (Viral CNS Flow Chip kit, Master Diagnóstica, Spain) has been developed for the detection of eight viruses causing acute meningitis and encephalitis, i.e. herpes simplex viruses 1-2, varicella zoster virus, human enterovirus, human parechovirus, Toscana virus, human cytomegalovirus and Epstein Barr virus. The new assay is a multiplex one-step RT-PCR followed by automatic flow-through hybridization, colorimetric detection and image analysis. The limit of detection was 50 copies/reaction, and 10 copies/reaction for human enterovirus and the other seven viruses, respectively. The analytical validation was performed with nucleic acids extracted from 268 cerebrospinal fluid samples and the results were compared with routine molecular assays. An excellent coefficient of agreement was observed between V-CNS and routine assays [kappa index: 0.948 (95%CI: 0.928-0.968)]. The overall sensitivity and specificity was 95.9% (95%CI: 91.2-98.3%) and 99.9% (95%CI: 99.6-100%), respectively. Viral CNS Flow Chip kit is an efficient multiplex platform for the detection of the main viruses involved in acute meningitis and encephalitis. The inclusion of a TOSV genome target may improve the laboratory diagnosis of viral neurological infections in endemic areas.

Analytical performance of the Alere(TM) i Influenza A&B assay for the rapid detection of influenza viruses / Evaluación del sistema Alere(TM) i Influenza A&B para la detección rápida de virus de la gripe

The analytical performance of the new Alere(TM) i Influenza A&B kit (AL-Flu) assay, based on isothermal nucleic acids amplification, was evaluated and compared with an antigen detection method, SD Bioline Influenza Virus Antigen Test (SDB), and an automated real-time RT-PCR, Simplexa(TM) Flu A/B & VRS Direct assay (SPX), for detection of influenza viruses. An ‘in-house’ RT-PCR was used as the reference method. Sensitivity of AL-Flu, SDB, and SPX was 71.7%, 34.8%, and 100%, respectively. Specificity was 100% for all techniques. The turnaround time was 13min for AL-Flu, 15min for SDB, and 75min for SPX. The Alere(TM) i Influenza A&B assay is an optimal point-of-care assay for influenza diagnosis in clinical emergency settings, and is more sensitive and specific than antigen detection methods (AU)

Se evaluó el nuevo ensayo Alere(TM) i Influenza A&B kit (AL-Flu), basado en la amplificación isotérmica de ácidos nucleicos, y se comparó con un método de detección de antígeno, SD Bioline Influenza Virus Antigen Test (SDB), y con una RT-PCR en tiempo real automática, Simplexa(TM) Flu A/B & VRS Direct assay (SPX), para la detección de virus de la gripe. Se utilizó una RT-PCR en tiempo real casera como método de referencia. La sensibilidad de AL-Flu, SDB y SPX fue del 71,7%, del 34,8% y del 100%, respectivamente. Se obtuvo una especificidad del 100% con todos los métodos. El tiempo de realización fue de 13min para AL-Flu, de 15min para SDB y de 75min para SPX. El ensayo Alere(TM) i Influenza A&B es óptimo para el diagnóstico de gripe en unidades de urgencias, al ser más sensible y específico que las técnicas de detección de antígeno (AU)

Analytical performance of the Alere™ i Influenza A&B assay for the rapid detection of influenza viruses.

The analytical performance of the new Alere™ i Influenza A&B kit (AL-Flu) assay, based on isothermal nucleic acids amplification, was evaluated and compared with an antigen detection method, SD Bioline Influenza Virus Antigen Test (SDB), and an automated real-time RT-PCR, Simplexa™ Flu A/B & VRS Direct assay (SPX), for detection of influenza viruses. An "in-house" RT-PCR was used as the reference method. Sensitivity of AL-Flu, SDB, and SPX was 71.7%, 34.8%, and 100%, respectively. Specificity was 100% for all techniques. The turnaround time was 13min for AL-Flu, 15min for SDB, and 75min for SPX. The Alere™ i Influenza A&B assay is an optimal point-of-care assay for influenza diagnosis in clinical emergency settings, and is more sensitive and specific than antigen detection methods.

Analytical performance of the automated multianalyte point-of-care mariPOC® for the detection of respiratory viruses.

The analytical performance of mariPOC® respi test (ArcDia® Laboratories, Turku, Finland) was evaluated using nucleic acid amplification techniques (NAATs) as the gold standard. The mariPOC assay allows automated detection of antigens from 8 respiratory viruses: influenza A and B viruses, respiratory syncytial virus, adenovirus, human metapneumovirus, and parainfluenza viruses 1-3. Positive results from samples with high viral load are available in 20min. Nasopharyngeal aspirates (n=192) from patients with acute respiratory infection and from previously positive samples were analyzed by mariPOC and NAATs (Simplexa(TM) FluA/FluB & RSV kit [n=118] and Luminex® Respiratory virus panel xTAG® RVP FAST [n=74]). Sensitivity, specificity, positive predictive value, and negative predictive value of mariPOC were 85.4%, 99.2%, 95.9%, and 97%, respectively, and 84.6% of positive results were reported in 20min. The good analytical performance and extended portfolio of mariPOC show this rapid assay as a good alternative for the etiological diagnosis of acute respiratory infection in laboratories that are not equipped with molecular assays.

Evaluación de la técnica LightCycler ® SeptiFast en recién nacidos y lactantes con sospecha de sepsis / Evaluation of the LightCycler ® SeptiFast test in newborns and infants with clinical suspicion of sepsis

Introducción La sepsis neonatal es una importante causa de morbimortalidad. Un diagnóstico precoz y el inicio rápido del tratamiento son fundamentales en la evolución del recién nacido. El hemocultivo, considerado técnica de referencia para el diagnóstico de sepsis, presenta una baja sensibilidad en este grupo de pacientes. Se planteó evaluar la utilidad de la PCR múltiple LightCycler® SeptiFast (LC-SF) en el diagnóstico de la sepsis neonatal, comparándola con el hemocultivo tradicional. Métodos Se recogieron 42 muestras de sangre correspondientes a 35 recién nacidos con episodios febriles ingresados en la unidad de cuidados intensivos neonatal del Hospital Universitario Virgen de las Nieves. Por cada episodio febril se procesaron 2 muestras de sangre venosa periférica para la realización del ensayo LC-SF y del hemocultivo, respectivamente. Resultados La sensibilidad y la especificidad de LC-SF, comparada con el diagnóstico clínico de sepsis, fueron del 79 y del 87%, respectivamente. La tasa de hemocultivos contaminados fue del 16,7%, detectándose Staphylococcus coagulasa negativa (SCN) y Streptococcus grupo viridans. En LC-SF la tasa de SCN contaminantes fue del 2,4%. La concordancia entre las 2 técnicas diagnósticas fue moderada (índice kappa: 0,369). La técnica LC-SF demostró una mayor concordancia con el diagnóstico clínico final (índice kappa: 0,729) que el hemocultivo (índice kappa: 0,238). Conclusión LC-SF podría ser una herramienta útil, empleada en paralelo con el hemocultivo en recién nacidos, al confirmar o descartar los casos que el hemocultivo no resuelve, además de disminuir el tiempo de obtención de resultado a 7 h( AU)

Introduction Neonatal sepsis is a significant cause of morbidity and mortality. Early diagnosis and prompt antimicrobial therapy are crucial for a favorable outcome of the newborn child. Blood culture, the current “gold standard” method for diagnosing bloodstream infections, has a low sensitivity in newborns. We evaluated the multiplex real-time PCR LightCycler® SeptiFast (LC-SF) for detection of bloodstream infections in newborns, compared with conventional blood culture. Methods A total of 42 blood samples were obtained from 35 subjects presenting with a febrile episode and hospitalized in neonatal intensive care unit at Hospital Universitario Virgen de las Nieves. Two samples were collected during each febrile episode in order to carry out LC-SF assay and blood culture, respectively. Results Sensitivity and specificity of 79% and 87%, respectively, compared with clinical diagnosis, were obtained for LC-SF. Contamination rate of blood cultures was 16.7%, mainly due to coagulase-negative staphylococci (CoNS) and viridans groups of streptococci. Contamination rate of LC-SF by CoNS was 2.4%. Concordance between LC-SF and blood culture was moderate (kappa index: 0.369). LC-SF demonstrated a higher concordance (kappa index: 0.729) with the final clinical diagnosis than blood culture (kappa index: 0.238). Conclusion LC-SF assay could be a useful diagnostic tool, along with a conventional blood culture, in newborn, for confirming or ruling out those cases that blood culture could not determine, shortening the time to result to 7 hours (AU)

Clinic-epidemiologic study of human infection by Granada virus, a new phlebovirus within the sandfly fever Naples serocomplex.

Granada virus (GRV), a new phlebovirus within the Naples serocomplex, has been recently described in phlebotomine sandflies from Spain. The presence of anti-GRV immunoglobulin G (IgG) antibodies was investigated by indirect fluorescence assay (IFA) and neutralization test (NT) in 920 serum samples from the Granada population. By IFA, an overall GRV seroprevalence of 15.8% (N = 145) was observed, significantly increasing up to 65 years. NT was positive in 18% of anti-GRV IFA-positive samples. IgG antibodies against Toscana virus (TOSV), a hyperendemic phlebovirus within Granada province, were detected in 40% of anti-GRV-positive cases. Anti-GRV IgM antibodies were detected in 36 (6.6%) of 547 acute-phase serum samples from individuals with febrile illness, exanthema, and/or acute respiratory infection. All positives were anti-TOSV IgM-negative. GRV may infect humans, with most cases being asymptomatic. The codetection of anti-GRV and anti-TOSV IgG antibodies could be attributable to cross-reactivity or exposure to the same transmission vector.

[Evaluation of the LightCycler® SeptiFast test in newborns and infants with clinical suspicion of sepsis]. / Evaluación de la técnica LightCycler® SeptiFast en recién nacidos y lactantes con sospecha de sepsis.

INTRODUCTION: Neonatal sepsis is a significant cause of morbidity and mortality. Early diagnosis and prompt antimicrobial therapy are crucial for a favorable outcome of the newborn child. Blood culture, the current "gold standard" method for diagnosing bloodstream infections, has a low sensitivity in newborns. We evaluated the multiplex real-time PCR LightCycler(®) SeptiFast (LC-SF) for detection of bloodstream infections in newborns, compared with conventional blood culture. METHODS: A total of 42 blood samples were obtained from 35 subjects presenting with a febrile episode and hospitalized in neonatal intensive care unit at Hospital Universitario Virgen de las Nieves. Two samples were collected during each febrile episode in order to carry out LC-SF assay and blood culture, respectively. RESULTS: Sensitivity and specificity of 79% and 87%, respectively, compared with clinical diagnosis, were obtained for LC-SF. Contamination rate of blood cultures was 16.7%, mainly due to coagulase-negative staphylococci (CoNS) and viridans groups of streptococci. Contamination rate of LC-SF by CoNS was 2.4%. Concordance between LC-SF and blood culture was moderate (kappa index: 0.369). LC-SF demonstrated a higher concordance (kappa index: 0.729) with the final clinical diagnosis than blood culture (kappa index: 0.238). CONCLUSION: LC-SF assay could be a useful diagnostic tool, along with a conventional blood culture, in newborn, for confirming or ruling out those cases that blood culture could not determine, shortening the time to result to 7 hours.

Laboratory detection of respiratory viruses by automated techniques.

Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection.

Association of Human bocavirus with Respiratory Infections in Outpatients and in Patients Attended at a Reference Hospital.

The role of Human bocavirus (HBoV) in human infectious disease is unclear due to the frequent detection of this virus in association with other respiratory viruses with a recognized pathogenic role in acute respiratory infection. We have analyzed the impact of HBoV in outpatients and in patients requiring hospitalisation or emergency attention for acute respiratory infections. Respiratory viruses were investigated by real-time PCR, direct antigen detection and/or viral culture by shell-vial assay. Nasopharyngeal aspirates, BAL and/or sputum samples from patients attended at a reference hospital, and nasal/throat swabs from outpatients were used. Respiratory viruses were detected in 660 samples (47%). HBoV detection rate was 12.6%, only preceded by respiratory syncytial virus (25%). Co-detections were observed in 12.9% of samples, and HBoV was present in 81% of them. Similar detection rates of HBoV were obtained in individuals with positive and negative results for other respiratory viruses (12.5% and 12.7%, respectively). The crossing point value was taken as a measure of HBoV viral load. Higher HBoV loads were observed in children, and in patients from the hospital. HBoV viral load was not associated with symptoms of upper respiratory tract infection or lower respiratory tract disease. Although HBoV is frequently detected in respiratory specimens, there is a poor association between HBoV-positive specimens and clinical parameters. A clinical impact of HBoV in respiratory infection probably occurs in few cases.

Development and preliminary evaluation of a rapid oligochromatographic assay for specific detection of new human influenza A H1N1 virus.

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments.