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.

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.

Sample pooling for SARS-CoV-2 RT-PCR screening.

OBJECTIVE: To evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification. METHODS: A total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre. RESULTS: We found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11-98.82), 100%, 100% and 99.79% (95% CI, 99.56-99.90) respectively; accuracy was 99.80% (95% CI, 99.59-99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46-3.28) cycle threshold (Ct) for E gene, 3.36 (95% CI, 2.89-3.85) Ct for the RdRP gene and 2.99 (95% CI, 2.56-3.43) Ct for the N gene. CONCLUSIONS: We found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.

High clinical impact of rapid susceptibility testing on CHROMID ESBL® medium directly from swabs.

BACKGROUND: Antibiotic resistance is a serious public health challenge exacerbated by the widespread use of ß-lactam and glycopeptide antibiotics. The identification of resistances is crucial, and CHROMID ESBL medium has been developed to detect enterobacteria with extended-spectrum ß-lactamases (ESBL). The objective of this study was to evaluate the potential of this medium to detect other types of resistant bacteria. METHODS: Vancomycin, cefoxitin, imipenem, and cefepime disks were used to measure growth on CHROMID ESBL medium of ß-lactam-resistant Gram-negative (83 with ESBL, 57 with carbapenemases, 35 with AmpC and 3 Stenotrophomonas maltophilia) and Gram-positive [37 vancomycin-susceptible (vancoS) microorganisms and 21 vancomycin-resistant (vancoR) Enterococcus faecium] clinical isolates (retrospective study) and colonization by the aforementioned bacteria (prospective study), using 649 rectal swabs, 314 pharyngeal swabs, and 44 swabs from other localizations. RESULTS: Retrospective study: species grown on the medium exhibited different colors. Growth on the medium was observed for: all ESBL enterobacteria, which were susceptible to imipenem and cefoxitin; 95% of isolates with carbapenemases, mostly resistant to imipenem; 80% of those with AmpC; 86% of vancoR E. faecium isolates; and 42% of vancoS E. faecalis isolates, with large growth inhibition halos around the vancomycin disk. Prospective study: vancoR E. faecium, ESBL Klebsiella, Pseudomonas with carbapenemases, A. baumannii (mostly from rectal swabs), S. maltophilia, Achromobacter xylosoxidans, and Burkholderia cenocepacia (mostly from pharyngeal swabs) were isolated from the 246 positive samples. CONCLUSIONS: CHROMID ESBL medium permitted the differential growth of Gram-negative bacteria, many with ESBL and carbapenemases. ESBL enterobacteria were susceptible to imipenem, carbapenemase-producing microorganisms grew around the imipenem disk, and vancoR E. faecium was isolated on the medium. Results of the prospective study demonstrate the potential clinical relevance of this medium. S. maltophilia was more frequently detected with pharyngeal swabs and ESBL Klebsiella, A. baumannii, and Pseudomonas with rectal swabs.

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.

Effects of Lactobacillus coryniformis K8 CECT5711 on the immune response to influenza vaccination and the assessment of common respiratory symptoms in elderly subjects: a randomized controlled trial.

PURPOSE: Elderly people are particularly vulnerable to seasonal influenza. Therefore, vaccination is strongly recommended. However, the vaccine efficacy is lower in the elderly, owing to immunosenescence. The objective of the present study was to evaluate the ability of the probiotic strain Lactobacillus coryniformis K8 CECT5711 to enhance the immune response to the influenza vaccine in the elderly and to assess the effects on symptoms related to respiratory infections. METHODS: A randomized, double-blind, placebo-controlled trial was conducted between November 2015 and April 2016. A total of 98 nursing home residents, more than 65 years of age were randomly assigned to receive L. coryniformis K8 CECT5711 (3 × 109 CFU/day) or a placebo for 2 weeks before influenza vaccination. The primary outcome was the percentage of seroconversion. The secondary outcomes were the incidence of influenza-like illness (ILI) and respiratory symptoms associated with respiratory infections during the 5-month follow-up period. The serum cytokine and immunoglobulin levels were also evaluated. RESULTS: The percentage of responders to vaccination was higher in the probiotic group than in the control group (p = 0.036). L. coryniformis ingestion was associated with a significantly lower incidence of respiratory symptoms commonly associated with respiratory infections (p = 0.007) and lower consumption of analgesics (p = 0.008). CONCLUSION: The administration of L. coryniformis K8 CECT5711 to an elderly population increased the immune response against the influenza vaccine and decreased symptoms associated with respiratory infections. Probiotic administration may be a natural and safe strategy to improve the efficacy of vaccines and to protect against common respiratory infections in susceptible populations.

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.

West Nile virus outbreak in humans and epidemiological surveillance, west Andalusia, Spain, 2016.

In Andalusia, Spain, West Nile virus (WNV) surveillance takes place from April to November, during the active vector period. Within this area seroconversion to this virus was evidenced in wild birds in 2004, affecting horses and two humans for the first time in 2010. Since 2010, the virus has been isolated every year in horses, and national and regional surveillance plans have been updated with the epidemiological changes found. WNV is spreading rapidly throughout southern Europe and has caused outbreaks in humans. Here we describe the second WNV outbreak in humans in Andalusia, with three confirmed cases, which occurred between August and September 2016, and the measures carried out to control it. Surveillance during the transmission season is essential to monitor and ensure prompt identification of any outbreaks.

Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

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.

Multilocus Sequence Typing analysis of human Campylobacter coli in Granada (Spain) / Tipificación mediante secuencias multilocus de cepas humanas de Campylobacter coli en Granada (España)

Introduction. Different subtypes of Campylobacter spp. have been associated with diarrhoea and a Multilocus Sequence Typing (MLST) method has been performed for subtyping. In the present work, MLST was used to analyse the genetic diversity of eight strains of Campylobacter coli. Material and methods. Nineteen genetic markers were amplified for MLST analysis: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. After comparing the obtained sequences with the Campylobacter MLST database, the allele numbers, sequence types (STs) and clonal complexes (CCs) were assigned. Results. The 8 C. coli isolates yielded 4 different STs belonging to 2 CCs. Seven isolates belong to ST-828 clonal complex and only one isolate belong to ST-21. Two samples came from the same patient, but were isolated in two different periods of time. Conclusions. MLST can be useful for taxonomic characterization of C. coli isolates (AU)

Introducción. Diferentes subtipos de Campylobacter spp. se han asociado con diarrea y la técnica de tipado mediante análisis de secuencias de múltiples locus (MLST) se ha empleado para la tipificación genética. En el presente trabajo, la técnica MLST se utilizó para analizar la diversidad genética de ocho cepas de Campylobacter coli. Material y métodos. 19 marcadores genéticos fueron amplificados mediante el análisis MLST: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. Después de comparar las secuencias obtenidas con la base de datos MLST para Campylobacter, se asignaron el número de los alelos, los secuenciotipos (STs) y los complejos clonales (CCs). Resultados. Las 8 cepas de C. coli aisladas mostraron 4 STs diferentes pertenecientes a 2 CCs. Siete aislamientos pertenecieron al complejo clonal ST-828 y sólo un aislado perteneció al ST-21. Dos aislados pertenecieron al mismo paciente, pero fueron obtenidos en diferentes periodos de tiempo. Conclusiones. La técnica MLST puede ser útil para la caracterización taxonómica de aislados de C. coli (AU)

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.

Comparison of epidemiology and clinical characteristics of infections by human parechovirus vs. those by enterovirus during the first month of life.

UNLABELLED: Human parechoviruses (HPeV) have been recently recognized as important viral agents in paediatric infections. The aims of this study were to investigate the HPeV infection prevalence in infants <1 month in Spain and, secondly, to analyse the clinical and epidemiological characteristics of the infected patients compared with those infected by enterovirus (EV). Infants <1 month with neurological or systemic symptoms were included in a multicentre prospective study. EV and HPeV detection by RT-PCR and genotyping were performed in cerebrospinal fluids (CSF), sera or throat swabs. Out of the total of 84 infants studied during 2013, 32 were EV positive (38 %) and 9 HPeV positive (11 %). HPeV-3 was identified in eight cases and HPeV-5 in one. Mean age of HPeV-positive patients was 18 days. Diagnoses were fever without source (FWS) (67 %), clinical sepsis (22 %) and encephalitis (11 %). Leukocytes in blood and CSF were normal. Pleocytosis (p = 0.03) and meningitis (p = 0.001) were significantly more frequent in patients with EV infections than with HPeV. CONCLUSIONS: Although HPeV-3 infections were detected less frequently than EV, they still account for approximately 10 % of the cases analysed in infants younger than 1 month. HPeV-3 was mainly associated with FWS and without leukocytosis and pleocytosis in CSF. In these cases, HPeV screening is desirable to identify the aetiologic agent and prevent unnecessary treatment and prolonged hospitalization.

Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.

Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples. Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs. The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses. In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants.

Infección por citomegalovirus humano / Infection by human cytomegalovirus

La infección por citomegalovirus humano (CMV) tiene una altísima prevalencia mundial. Tras la infección primaria, el virus pasa a un estado de latencia, pudiendo aparecer recurrencias por reinfección con una cepa nueva o por reactivación de la replicación del CMV latente. Los cuadros clínicos más graves se dan en infección congénita y en pacientes inmunodeprimidos, en los que se comporta como patógeno oportunista. Las técnicas serológicas son de elección en la infección primaria y para determinar el estado inmune frente a CMV en el donante y receptor de órganos. Aunque faltan estudios estandarizados, la reciente comercialización de métodos de medida de la respuesta inmune celular ofrece buenas perspectivas para predecir el riesgo de enfermedad por CMV en inmunodeprimidos. Las técnicas moleculares, que han ido sustituyendo al cultivo y/o detección de antígeno, son actualmente los procedimientos más utilizados en el diagnóstico de rutina y control de la infección por CMV

Prevalence of human cytomegalovirus infection is very high worldwide. Following primary infection, the virus remains latent, being able to cause recurrences either by reinfection with a new strain or by reactivation of the replication of the latent virus. The most severe disease is seen in congenital infection and in immunosuppressed patients, in whom the virus act as an opportunistic pathogen. Serological techniques are the methods of choice in primary infection and to determine the immune status against CMV in organ donor and receptor. Although well-standardized studies are lacking, the recent commercial availability of methods that measure cellular immune response are promising to predict the risk of CMV disease in immunosuppressed individuals. Molecular assays, that have gradually been substituting viral culture and/or antigen detection, are the most widely used methods for the diagnosis and control of CMV infection

Infección por citomegalovirus humano / Infection by human cytomegalovirus

La infección por citomegalovirus humano (CMV) tiene una altísima prevalencia mundial. Tras la infección primaria, el virus pasa a un estado de latencia, pudiendo aparecer recurrencias por reinfección con una cepa nueva o por reactivación de la replicación del CMV latente. Los cuadros clínicos más graves se dan en infección congénita y en pacientes inmunodeprimidos, en los que se comporta como patógeno oportunista. Las técnicas serológicas son de elección en la infección primaria y para determinar el estado inmune frente a CMV en el donante y receptor de órganos. Aunque faltan estudios estandarizados, la reciente comercialización de métodos de medida de la respuesta inmune celular ofrece buenas perspectivas para predecir el riesgo de enfermedad por CMV en inmunodeprimidos. Las técnicas moleculares, que han ido sustituyendo al cultivo y/o detección de antígeno, son actualmente los procedimientos más utilizados en el diagnóstico de rutina y control de la infección por CMV (AU)

Prevalence of human cytomegalovirus infection is very high worldwide. Following primary infection, the virus remains latent, being able to cause recurrences either by reinfection with a new strain or by reactivation of the replication of the latent virus. The most severe disease is seen in congenital infection and in immunosuppressed patients, in whom the virus act as an opportunistic pathogen. Serological techniques are the methods of choice in primary infection and to determine the immune status against CMV in organ donor and receptor. Although well-standardized studies are lacking, the recent commercial availability of methods that measure cellular immune response are promising to predict the risk of CMV disease in immunosuppressed individuals. Molecular assays, that have gradually been substituting viral culture and/or antigen detection, are the most widely used methods for the diagnosis and control of CMV infection (AU)

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.