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.

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.

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.

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.

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.

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.

[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.

Lymphocytic choriomeningitis virus-associated meningitis, southern Spain.

Lymphocytic choriomeningitis virus (LCMV) was detected in 2 patients with acute meningitis in southern Spain within a 3-year period. Although the prevalence of LCMV infection was low (2 [1.3%] of 159 meningitis patients), it represents 2.9% of all pathogens detected. LCMV is a noteworthy agent of neurologic illness in immunocompetent persons.

Circulation of other respiratory viruses and viral co-infection during the 2009 pandemic influenza.

Coinciding with the pandemic wave of the influenza A(H1N1)pdm09 virus, other respiratory viruses have co-circulated in our area and were responsible for many acute respiratory infections and influenza-like illness (ILI). Apart from the pandemic virus that was responsible for most ILI cases, incidence rates of other viruses have varied among geographical areas. In general, human rhinovirus was the most frequent among individuals from the community, and respiratory syncytial virus among hospitalized patients. Detection rates of other respiratory viruses such as human metapneumovirus, adenovirus or parainfluenza viruses have been much lower. On the basis of an interference mechanism, human rhinovirus may contribute to modulate the pandemic wave, although available data are not conclusive to support this hypothesis. In contrast, the epidemic wave of respiratory syncytial virus during 2009-2010 was similar to previous seasons. Overall, incidence rates of respiratory viruses other than influenza did not change significantly during the pandemic season compared to other seasons. No association has been found between coinfection of pandemic influenza and other respiratory viruses with the prognosis of patients with influenza. The involvement of clinical virology laboratories in the etiological diagnosis of ILI cases has improved and has optimized diagnostic procedures.

[West Nile virus infection]. / Infección por virus West Nile.

West Nile virus (WNV) is an arbovirus usually transmitted by mosquitoes. The main reservoirs are birds, although the virus may infect several vertebrate species, such as horses and humans. Up to 80% of human infections are asymptomatic. The most frequent clinical presentation is febrile illness, and neuroinvasive disease can occur in less than 1% of cases. Spain is considered a high-risk area for the emergence of WNV due to its climate and the passage of migratory birds from Africa (where the virus is endemic). These birds nest surrounding wetlands where populations of possible vectors for the virus are abundant. Diagnosis of human neurological infections can be made by detection of IgM in serum and/or cerebrospinal fluid samples, demonstration of a four-fold increase in IgG antibodies between acute-phase and convalescent-phase serum samples, or by detection of viral genome by reverse transcription-polymerase chain reaction (especially useful in transplant recipients). Since WNV is a biosafety level 3 agent, techniques that involve cell culture are restricted to laboratories with this level of biosafety, such as reference laboratories. The National Program for the Surveillance of WNV Encephalitis allows the detection of virus circulation among birds and vectors in areas especially favorable for the virus, such as wetlands, and provides information for evaluation of the risk of disease in horses and humans.

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.

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.

[Influenza a (H1N1) virus infection in humans: review to 30th October 2009]. / Infección en humanos por virus de la gripe A(H1N1): revisión al 30 de octubre de 2009.

Since human infection by a novel influenza virus A H1N1 of swine origin was reported in April 2009, the virus has spread worldwide causing a pandemic. In the Southern Hemisphere, the first pandemic wave has taken place, coinciding with Austral Winter. In the Northern Hemisphere, transmission has been sustained under the basal level of epidemic until the first weeks of October, when incidence rates have risen up to the pidemic level in some countries, including Spain. This work reviews the differential characteristics of this novel virus in terms of pathogenicity, clinical syndrome and epidemiology, as well as the diagnostic, prophylactic and therapeutic procedures available; information we consider relevant to minimize the impact of this new pandemic in our area.

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.

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.

Genetic diversity of Toscana virus.

Distribution of Toscana virus (TOSV) is evolving with climate change, and pathogenicity may be higher in nonexposed populations outside areas of current prevalence (Mediterranean Basin). To characterize genetic diversity of TOSV, we determined the coding sequences of isolates from Spain and France. TOSV is more diverse than other well-studied phleboviruses (e.g.,Rift Valley fever virus).

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.

Reverse transcription, real-time PCR assay for detection of Toscana virus.

BACKGROUND: The arthropod-borne Toscana virus is a common cause of acute neurological infection in the Mediterranean basin. Recently, a new lineage, highly divergent from the Italian prototype, has been reported in Spain. OBJECTIVE: We describe a reverse transcription, real-time PCR assay for detection of both Toscana virus genotypes. The real-time PCR uses a TaqMan probe and an internal control to identify false negative results. STUDY DESIGN: A conserved region of the two known lineages of Toscana virus, located at the 3' end of the small segment of their genomes, was chosen to design both the primers and the probe. RESULTS: The sensitivity of the assay was 0.0158 TICD(50) per reaction of Toscana virus, equivalent to seven copies of cDNA. No other phleboviruses or RNA viruses were amplified by this specific real-time PCR. CONCLUSIONS: The assay seems to be sensitive, reliable and easy to be applied in the diagnosis of autochthonous and/or imported suspected cases of Toscana virus infection.

[Respiratory viruses: old and new. Review of diagnostic methods]. / Virus respiratorios: viejos y nuevos virus. Revisión de métodos diagnósticos.

Acute respiratory infections (ARI) of viral origin are one of the main causes of morbidity and mortality worldwide. In addition to traditional viruses, such as the influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza viruses 1 to 4, and adenovirus, other viruses such as metapneumovirus, new coronaviruses (human coronavirus NL63 and HKU1 and severe acute respiratory syndrome [SARS]-coronavirus), and recently bocaviruses, have been identified as causal agents of ARI. Although most of these viral infections follow a benign and selflimiting course in healthy adults, the consequences for the health care systems increase when they involve children, the elderly, immunosuppressed individuals, or those with chronic underlying diseases. These viral infections are an important cause of hospitalization and death, mainly during the cold months of the year, and, from a social-health perspective, ARI are a drain on economic resources and a frequent cause of work absenteeism. Occasionally, some of these viruses may cause emergent world health problems, as has occurred with the influenza virus pandemic strain and SARScoronavirus. While classical diagnostic methods based on culture and antigen detection remain useful for traditional respiratory viruses, recently described viruses are diagnosed mainly by molecular amplification techniques.