Humoral/Cellular Immune Discordance in Stem Cell Donors: Impact on Cytomegalovirus-Specific Immune Reconstitution after Related Hematopoietic Transplantation.

Cytomegalovirus (CMV) reactivation is an important cause of complications after hematopoietic stem cell transplantation (HSCT). Discrepancies between serologic and cellular CMV-specific immune response have been reported. This study evaluated the impact of lack of CMV-specific CD8+ T cell response in seropositive donors (ie, discordant donors) on the reconstitution of CMV-specific cell-mediated immunity (CMI) after related HSCT in seropositive recipients. CMV-CMI was assessed in donors and recipients using the QuantiFERON-CMV assay (QF). CMV-CMI was prospectively assessed for 1 year in 81 CMV-seropositive HSCT recipients with a haploidentical or matched related donor. A Cox proportional hazard regression analysis was performed. Of the 67 CMV-seropositive donors, 54 (80.6%) were D+QFpos. The remaining 13 CMV-seropositive donors (19.4%) had a QFneg result and thus were classified as discordant donors (D+QFneg). We found that patients with D+QFneg had a significantly higher risk of impaired CMV-CMI reconstitution compared with patients with D+QFpos (log-rank test, P = .001) or D- donors (log-rank test, P = .023). In addition, the D+QFneg group had a higher incidence of single-episode reactivation compared with D+QFpos or D- donors (69.2% versus 44.4% and 28.6%, respectively) but a lower incidence of CMV recurrence compared with the D- group (7.7% versus 57.1%; P = .003). After adjusting for other relevant variables, immune discordance in donors was independently associated with impaired CMV-CMI reconstitution compared with D+QFpos donors (adjusted hazard ratio [HR], 0.18; 95% confidence interval [CI], .06 to .52; P = .001) and D- donors (adjusted HR, .17; 95% CI, .05 to .59; P = .005). Discordant donors were associated with undetectable CMV-CMI during the 12-month follow-up period using the QF assay. The inability of these patients to become QFpos persisted even after CMV reactivation. This might be related to the low frequency of CMV recurrence in this group. CMV-CMI assessment, in conjunction with CMV serostatus, can be of utility to better classify stem cell donors as well as the risk of impaired CMV-CMI reconstitution after HSCT.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

Profilaxis primaria estándar versus prolongada de la infección por citomegalovirus en el trasplante de órgano sólido / Standard versus extended primary cytomegalovirus prophylaxis in solid organ transplantation

Antecedentes y objetivo:Se han comparado la eficacia y seguridad de la profilaxis primaria estándar o prolongada de la infección por citomegalovirus (CMV) en el trasplante de órgano sólido.Materiales y métodos:Estudio retrospectivo de los receptores CMV seronegativos de donante seropositivo (D+/R−) que recibieron profilaxis frente a CMV tras un trasplante de órgano sólido (2007-2017). Se comparó la frecuencia de infección por CMV en los 2 primeros años postrasplante en los receptores que recibieron profilaxis durante más o menos de 100 días. Se evaluó asimismo la mielotoxicidad durante la profilaxis.Resultados:Se analizaron 66 pacientes. De ellos el 43,9% (n=29) presentaron infección por CMV. El 68,2% (n=45) recibieron profilaxis prolongada, sin asociarse su uso con una menor tasa de infección (42,2 vs. 47,6%, p=0,44) ni de enfermedad posprofilaxis (15,6 vs. 19%, p=0,72). La profilaxis prolongada se asoció con una mayor frecuencia de mielotoxicidad (68,9 vs. 42,9%, p<0,05).Conclusiones:La prolongación de la profilaxis primaria más de 100 días no aumenta su efectividad pero sí la toxicidad hematológica. (AU)

Background and objective:We compared the efficacy and safety of standard vs. extended primary cytomegalovirus (CMV) prophylaxis in solid organ transplantation.Materials and methods:Retrospective cohort study of CMV seronegative recipients who received CMV prophylaxis after solid organ transplantation from seropositive donor (D+/R−) (2007–2017). CMV infection in the first two years after transplantation in recipients with prophylaxis longer or shorter than 100 days were compared.Results:CMV infection occurred in 29 of 66 patients (43.9%) with prophylaxis. Forty-five patients (68.2%) received extended prophylaxis. CMV infection and disease rates were not different between patients with extended and standard prophylaxis. However, extended prophylaxis was associated with a higher rate of myelotoxicity (68.9% vs. 42.9%, p<0.05).Conclusions:Extending primary CMV prophylaxis over 100 days did not prevent late-onset infection but it was associated with hematological toxicity. (AU)

Impact of the Acceptance of the Recommendations Made by a Meropenem Stewardship Program in a University Hospital: A Pilot Study.

Antimicrobial stewardship programs (ASP) promote appropriate antimicrobial use. We present a 4-year retrospective study that evaluated the clinical impact of the acceptance of the recommendations made by a meropenem-focused ASP. A total of 318 meropenem audits were performed. The ASP team (comprising infectious disease physicians, pharmacists and microbiologists) considered meropenem use in 96 audits (30.2%) to be inappropriate. The reasons to consider these uses inappropriate were the possibility of de-escalating to a narrower-spectrum antibiotic, in 66 (68.7%) audits, and unnecessary meropenem use, in 30 (31.3%) audits. The ASP team recommended de-escalation in 66 audits (68.7%) and discontinuation of meropenem in 30 audits (31.3%). ASP interventions were stratified according to whether or not recommendations were followed. The group in which recommendations were accepted and followed (i.e., accepted audit, AA) included 66 audits (68.7%) and the group in which recommendations were not followed (i.e., rejected audit, RA) included 30 (31.3%) audits. The comorbidity of the AA group (Charlson score) was higher than in the RA group (7.0 (5.0-9.0) vs. 6.0 (4.0-7.0), p = 0.02). Discontinuation of meropenem was recommended in 83.3% of audits in the AA group vs. 62.2% in the RA group (OR 3.05 (1.03-8.99), p = 0.04). Ertapenem de-escalation resulted in a 100% greater rate of follow-up compared with the non-carbapenem option (100% vs. 51.9%, OR 1.50 (1.21-1.860), p = 0.001). Significant differences were observed in the AA group when cultures were taken before antibiotic prescription-98.5% vs. 83.3% (p = 0.01, OR 13.0 (1.45-116.86))-or when screening cultures were taken-45.5% vs. 19.2% (p = 0.03, OR 3.5 (1.06-11.52)). There were no differences between the groups in terms of overall mortality and 30-day mortality, length of stay, Clostridiodes difficile infection, 30-day readmission or hospitalization costs. In conclusion, meropenem ASP recommendations contributed to a decrease in meropenem prescription without worsening clinical and economic outcomes.

[Standard versus extended primary cytomegalovirus prophylaxis in solid organ transplantation]. / Profilaxis primaria estándar versus prolongada de la infección por citomegalovirus en el trasplante de órgano sólido.

BACKGROUND AND OBJECTIVE: We compared the efficacy and safety of standard vs. extended primary cytomegalovirus (CMV) prophylaxis in solid organ transplantation. MATERIALS AND METHODS: Retrospective cohort study of CMV seronegative recipients who received CMV prophylaxis after solid organ transplantation from seropositive donor (D+/R-) (2007-2017). CMV infection in the first two years after transplantation in recipients with prophylaxis longer or shorter than 100 days were compared. RESULTS: CMV infection occurred in 29 of 66 patients (43.9%) with prophylaxis. Forty-five patients (68.2%) received extended prophylaxis. CMV infection and disease rates were not different between patients with extended and standard prophylaxis. However, extended prophylaxis was associated with a higher rate of myelotoxicity (68.9% vs. 42.9%, p<0.05). CONCLUSIONS: Extending primary CMV prophylaxis over 100 days did not prevent late-onset infection but it was associated with hematological toxicity.

HIV-1 diagnosis using dried blood spots from patients in Kinshasa, DRC: a tool to detect misdiagnosis and achieve World Health Organization 2030 targets.

INTRODUCTION: Currently, only 54% of the population of the Democratic Republic of the Congo (DRC) know their HIV status. The aim of this study was to detect HIV misdiagnosis from rapid diagnostic tests (RDT) and to evaluate serological immunoassays using dried blood spots (DBS) from patients in Kinshasa, DRC. METHODS: Between 2016 and 2018, 365 DBS samples were collected from 363 individuals and shipped to Spain. The samples were from people with a new HIV positive (n = 123) or indeterminate (n = 23) result, known HIV-positive patients (n = 157), and a negative control group (n = 62). HIV serology was performed using Elecsys HIV combi PT (Roche), VIDAS HIV Duo Quick (BioMérieux), and Geenius (Bio-Rad). In addition, HIV RNA detection was performed in all samples using the COBAS AmpliPrep/COBAS Taqman HIV-1 Test 2.0 (Roche). RESULTS: Overall, 272 samples were found to be positive and 93 to be negative for HIV serology. The sensitivity was 100% for both Elecsys and VIDAS techniques, but specificity was slightly higher for the VIDAS test: 100% (96.1-100%) vs 98.9% (94.1-99.9%). Of the 23 indeterminate cases using RDT, only three cases were true-positives with a detectable viral load. Eleven samples out of the 280 classified as positive by RDT corresponded to nine patients who had received a false diagnosis of HIV through RDT (3.9%); six of them had been on antiretroviral therapy for at least 2 years. CONCLUSIONS: Elecsys HIV combi PT and VIDAS HIV Duo Quick immunoassays showed high sensitivity and specificity when using DBS. RDT-based serological diagnosis can lead to HIV misdiagnosis with personal and social consequences in sub-Saharan Africa.

Immunological Biomarkers of Fatal COVID-19: A Study of 868 Patients.

Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients.

HCV Diagnosis and Sequencing Using Dried Blood Spots from Patients in Kinshasa (DRC): A Tool to Achieve WHO 2030 Targets.

The World Health Organization has established an elimination plan for hepatitis C virus (HCV) by 2030. In Sub-Saharan Africa (SSA) access to diagnostic tools is limited, and a number of genotype 4 subtypes have been shown to be resistant to some direct-acting antivirals (DAAs). This study aims to analyze diagnostic assays for HCV based on dried blood spots (DBS) specimens collected in Kinshasa and to characterize genetic diversity of the virus within a group of mainly HIV positive patients. HCV antibody detection was performed on 107 DBS samples with Vidas® anti-HCV and Elecsys anti-HCV II, and on 31 samples with INNO-LIA HCV. Twenty-six samples were subjected to molecular detection. NS3, NS5A, and NS5B regions from 11 HCV viremic patients were sequenced. HCV seroprevalence was 12.2% (72% with detectable HCV RNA). Both Elecsys Anti-HCV and INNO-LIA HCV were highly sensitive and specific, whereas Vidas® anti-HCV lacked full sensitivity and specificity when DBS sample was used. NS5B/NS5A/NS3 sequencing revealed exclusively GT4 isolates (50% subtype 4r, 30% 4c and 20% 4k). All 4r strains harbored NS5A resistance-associated substitutions (RAS) at positions 28, 30, and 31, but no NS3 RAS was detected. Elecsys Anti-HCV and INNO-LIA HCV are reliable methods to detect HCV antibodies using DBS. HCV subtype 4r was the most prevalent among our patients. RASs found in subtype 4r in NS5A region confer unknown susceptibility to DAA.

The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: A pilot, double-blind, placebo-controlled, randomized clinical trial.

BACKGROUND: Ivermectin inhibits the replication of SARS-CoV-2 in vitro at concentrations not readily achievable with currently approved doses. There is limited evidence to support its clinical use in COVID-19 patients. We conducted a Pilot, randomized, double-blind, placebo-controlled trial to evaluate the efficacy of a single dose of ivermectin reduce the transmission of SARS-CoV-2 when administered early after disease onset. METHODS: Consecutive patients with non-severe COVID-19 and no risk factors for complicated disease attending the emergency room of the Clínica Universidad de Navarra between July 31, 2020 and September 11, 2020 were enrolled. All enrollments occurred within 72 h of onset of fever or cough. Patients were randomized 1:1 to receive ivermectin, 400 mcg/kg, single dose (n = 12) or placebo (n = 12). The primary outcome measure was the proportion of patients with detectable SARS-CoV-2 RNA by PCR from nasopharyngeal swab at day 7 post-treatment. The primary outcome was supported by determination of the viral load and infectivity of each sample. The differences between ivermectin and placebo were calculated using Fisher's exact test and presented as a relative risk ratio. This study is registered at ClinicalTrials.gov: NCT04390022. FINDINGS: All patients recruited completed the trial (median age, 26 [IQR 19-36 in the ivermectin and 21-44 in the controls] years; 12 [50%] women; 100% had symptoms at recruitment, 70% reported headache, 62% reported fever, 50% reported general malaise and 25% reported cough). At day 7, there was no difference in the proportion of PCR positive patients (RR 0·92, 95% CI: 0·77-1·09, p = 1·0). The ivermectin group had non-statistically significant lower viral loads at day 4 (p = 0·24 for gene E; p = 0·18 for gene N) and day 7 (p = 0·16 for gene E; p = 0·18 for gene N) post treatment as well as lower IgG titers at day 21 post treatment (p = 0·24). Patients in the ivermectin group recovered earlier from hyposmia/anosmia (76 vs 158 patient-days; p < 0.001). INTERPRETATION: Among patients with non-severe COVID-19 and no risk factors for severe disease receiving a single 400 mcg/kg dose of ivermectin within 72 h of fever or cough onset there was no difference in the proportion of PCR positives. There was however a marked reduction of self-reported anosmia/hyposmia, a reduction of cough and a tendency to lower viral loads and lower IgG titers which warrants assessment in larger trials. FUNDING: ISGlobal, Barcelona Institute for Global Health and Clínica Universidad de Navarra.

Histopathological findings in fatal COVID-19 severe acute respiratory syndrome: preliminary experience from a series of 10 Spanish patients.

In December 2019, an outbreak of severe acute respiratory syndrome associated to SARS-CoV2 was reported in Wuhan, China. To date, little is known on histopathological findings in patients infected with the new SARS-CoV2. Lung histopathology shows features of acute and organising diffuse alveolar damage. Subtle cellular inflammatory infiltrate has been found in line with the cytokine storm theory. Medium-size vessel thrombi were frequent, but capillary thrombi were not present. Despite the elevation of biochemical markers of cardiac injury, little histopathological damage could be confirmed. Viral RNA from paraffin sections was detected at least in one organ in 90% patients.

Non-pigmented rapidly growing mycobacteria smooth and rough colony phenotypes pathogenicity evaluated using in vitro and experimental models.

Non-pigmented rapidly growing mycobacteria (NPRGM) are widely distributed in water, soil and animals. It has been observed an increasing importance of NPRGM related-infections, particularly due to the high antimicrobial resistance. NPRGM have rough and smooth colony phenotypes, and several studies have showed that rough colony variants are more virulent than smooth ones. However, other studies have failed to validate this observation. In this study, we have performed two models, invitro and in vivo, in order to assess the different pathogenicity of these two phenotypes. We used collection and clinical strains of Mycobacteriumabscessus, Mycobacterium fortuitum and Mycobacteriumchelonae. On the invitro model (macrophages), phagocytosis was higher for M. abscessus and M. fortuitum rough colony variant strains when compared to smooth colony variants. However, we did not find differences with colonial variants of M. chelonae. Survival of Galleriamellonella larvae in the experimental model was lower for M. abscessus and M. fortuitum rough colony variants when compared with larvae infected with smooth colony variants. We did not find differences in larvae infected with M. chelonae.Results of our in vivo study correlated well with the experimental model. This fact could have implications on the interpretation of the clinical significance of the NPRGM isolate colonial variants.

Utility Of POC Xpert HIV-1 Tests For Detection-Quantification Of Complex HIV Recombinants Using Dried Blood Spots From Kinshasa, D. R. Congo.

Point-of-Care (POC) molecular assays improve HIV infant diagnosis and viral load (VL) quantification in resource-limited settings. We evaluated POC performance in Kinshasa (Democratic Republic of Congo), with high diversity of HIV-1 recombinants. In 2016, 160 dried blood samples (DBS) were collected from 85 children (60 HIV-, 18 HIV+, 7 HIV-exposed) and 75 HIV+ adults (65 treated, 10 naive) at Monkole Hospital (Kinshasa). We compared viraemia with Cepheid-POC-Xpert-HIV-1VL and the non-POC-COBAS®AmpliPrep/COBAS®TaqMan®HIV-1-Testv2 in all HIV+, carrying 72.4%/7.2% HIV-1 unique/complex recombinant forms (URF/CRF). HIV-1 infection was confirmed in 14 HIV+ children by Cepheid-POC-Xpert-HIV-1Qual and in 70 HIV+ adults by both Xpert-VL and Roche-VL, identifying 8 false HIV+ diagnosis performed in DRC (4 adults, 4 children). HIV-1 was detected in 95.2% and 97.6% of 84 HIV+ samples by Xpert-VL and Roche-VL, respectively. Most (92.9%) HIV+ children presented detectable viraemia by both VL assays and 74.3% or 72.8% of 70 HIV+ adults by Xpert or Roche, respectively. Both VL assays presented high correlation (R2 = 0.89), but showing clinical relevant ≥0.5 log VL differences in 15.4% of 78 cases with VL within quantification range by both assays. This is the first study confirming the utility of Xpert HIV-1 tests for detection-quantification of complex recombinants currently circulating in Kinshasa.

Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance.

Influenza viruses cause annual seasonal epidemics and occasional pandemics. It is important to elucidate the stringency of bottlenecks during transmission to shed light on mechanisms that underlie the evolution and propagation of antigenic drift, host range switching or drug resistance. The virus spreads between people by different routes, including through the air in droplets and aerosols, and by direct contact. By housing ferrets under different conditions, it is possible to mimic various routes of transmission. Here, we inoculated donor animals with a mixture of two viruses whose genomes differed by one or two reverse engineered synonymous mutations, and measured the transmission of the mixture to exposed sentinel animals. Transmission through the air imposed a tight bottleneck since most recipient animals became infected by only one virus. In contrast, a direct contact transmission chain propagated a mixture of viruses suggesting the dose transferred by this route was higher. From animals with a mixed infection of viruses that were resistant and sensitive to the antiviral drug oseltamivir, resistance was propagated through contact transmission but not by air. These data imply that transmission events with a looser bottleneck can propagate minority variants and may be an important route for influenza evolution.

Effects of previous episodes of influenza and vaccination in preventing laboratory-confirmed influenza in Navarre, Spain, 2013/14 season.

We estimated whether previous episodes of influenza and trivalent influenza vaccination prevented laboratory-confirmed influenza in Navarre, Spain, in season 2013/14. Patients with medically-attended influenza-like illness (MA-ILI) in hospitals (n = 645) and primary healthcare (n = 525) were included. We compared 589 influenza cases and 581 negative controls. MA-ILI related to a specific virus subtype in the previous five seasons was defined as a laboratory-confirmed influenza infection with the same virus subtype or MA-ILI during weeks when more than 25% of swabs were positive for this subtype. Persons with previous MA-ILI had 30% (95% confidence interval (CI): -7 to 54) lower risk of MA-ILI, and those with previous MA-ILI related to A(H1N1)pdm09 or A(H3N2) virus, had a, respectively, 63% (95% CI: 16-84) and 65% (95% CI: 13-86) lower risk of new laboratory-confirmed influenza by the same subtype. Overall adjusted vaccine effectiveness in preventing laboratory-confirmed influenza was 31% (95% CI: 5-50): 45% (95% CI: 12-65) for A(H1N1)pdm09 and 20% (95% CI: -16 to 44) for A(H3N2). While a previous influenza episode induced high protection only against the same virus subtype, influenza vaccination provided low to moderate protection against all circulating subtypes. Influenza vaccine remains the main preventive option for high-risk populations.

Effectiveness of subunit influenza vaccination in the 2014-2015 season and residual effect of split vaccination in previous seasons.

BACKGROUND: In Navarra, Spain, subunit vaccine was first used in the 2014-2015 season, whereas trivalent split-virion influenza vaccines had been used in previous seasons. We estimate the effectiveness of the subunit vaccine in the current season and split vaccine in the two previous seasons against laboratory-confirmed influenza in the 2014-2015 season. METHODS: Patients with influenza-like illness hospitalized or attended by sentinel general practitioners were swabbed for influenza testing. The previous and current vaccine status of laboratory-confirmed cases was compared to test-negative controls. RESULTS: Among 1213 patients tested, 619 (51%) were confirmed for influenza virus: 52% influenza A(H3N2), 46% influenza B, and 2% A(H1N1)pdm09. The overall effectiveness for subunit vaccination in the current season was 19% (95% confidence interval [CI]: -13 to 42), 2% (95%CI: -47 to 35) against influenza A(H3N2) and 32% (95%CI: -4 to 56) against influenza B. The effectiveness against any influenza was 67% (95%CI: 17-87) for 2012-2013 and 2013-2014 vaccination only, 42% (95%CI: -31 to 74) for 2014-2015 vaccination only, and 38% (95%CI: 8-58) for vaccination in the 2012-2013, 2013-2014 and 2014-2015 seasons. The same estimates against influenza A(H3N2) were 47% (95%CI: -60 to 82), -54% (95%CI: -274 to 37) and 28% (95%CI: -17 to 56), and against influenza B were 82% (95%CI: 19-96), 93% (95%CI: 45-99) and 43% (95%CI: 5-66), respectively. CONCLUSION: These results suggest a considerable residual protection of split vaccination in previous seasons, low overall effectiveness of current season subunit vaccination, and possible interference between current subunit and previous split vaccines.

Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

UNLABELLED: The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE: Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.

Influencia del criterio de selección de pacientes para la toma de frotis en la estimación de la efectividad de la vacuna antigripal / Influence of distinct criteria for selecting patients for swabbing on estimation of the effectiveness of the influenza vaccine

Objetivo: estimar la efectividad de la vacuna antigripal según el criterio de selección en la toma de frotis. Método: estudio de casos y controles de casos confirmados (n = 909) y controles negativos para gripe (n = 732) en las temporadas 2010-2011 a 2012-2013 en Navarra. La efectividad ajustada de la vacuna se estimó incluyendo todos los frotis de pacientes con síndrome gripal y seleccionando sólo los dos primeros por médico y semana. Resultados: los dos primeros pacientes por médico y semana estaban menos vacunados (7,9% frente a 12,5%, p = 0,021) y se confirmaron menos para gripe (53,6% frente a 66,4%, p <0,001), diferencias que se redujeron al ajustar por covariables. La efectividad de la vacuna calculada con todos los frotis fue del 49% (intervalo de confianza del 95% [IC95%]: 23-66%) y del 55% (IC95%: 27-72%) al analizar los dos primeros frotis semanales. Conclusión: la selección de los primeros pacientes semanales puede sesgar la efectividad de la vacuna antigripal, aunque en las temporadas analizadas este sesgo fue pequeño

Objective: to estimate the effectiveness of the influenza vaccine under different criteria for selecting patients for swabbing. Method: a case-control study was performed of laboratory-confirmed cases (n = 909) and negative controls for influenza (n = 732) in the 2010-2011 to 2012-2013 seasons in Navarre (Spain). The adjusted vaccine effectiveness was estimated by including all swabs from patients with influenza-like-illness and selecting only the first two cases per physician and week. Results: the first two patients per physician and week were less frequently vaccinated against influenza (7.9% vs. 12.5%, p = 0.021) and less often received confirmation of influenza (53.6% vs. 66.4%, p <0.001) than subsequent patients. These differences decreased after adjustment for covariates. The effectiveness of the influenza vaccine was 49% (95% CI: 23-66%) when all swabs were included and was 55% (95% CI: 27-72%) when we selected the first two swabs per week and physician. Conclusion: the selection of the first two patients per physician and week may bias assessment of the effectiveness of the influenza vaccine, although this bias was small in the seasons analyzed

[Influence of distinct criteria for selecting patients for swabbing on estimation of the effectiveness of the influenza vaccine]. / Influencia del criterio de selección de pacientes para la toma de frotis en la estimación de la efectividad de la vacuna antigripal.

OBJECTIVE: To estimate the effectiveness of the influenza vaccine under different criteria for selecting patients for swabbing. METHOD: A case-control study was performed of laboratory-confirmed cases (n=909) and negative controls for influenza (n=732) in the 2010-2011 to 2012-2013 seasons in Navarre (Spain). The adjusted vaccine effectiveness was estimated by including all swabs from patients with influenza-like-illness and selecting only the first two cases per physician and week. RESULTS: The first two patients per physician and week were less frequently vaccinated against influenza (7.9% vs. 12.5%, p=0.021) and less often received confirmation of influenza (53.6% vs. 66.4%, p <0.001) than subsequent patients. These differences decreased after adjustment for covariates. The effectiveness of the influenza vaccine was 49% (95% CI: 23-66%) when all swabs were included and was 55% (95% CI: 27-72%) when we selected the first two swabs per week and physician. CONCLUSION: The selection of the first two patients per physician and week may bias assessment of the effectiveness of the influenza vaccine, although this bias was small in the seasons analyzed.

Influencia de los tiempos entre el inicio de síntomas, la toma de frotis nasofaríngeo y su procesamiento en laboratorio en la detección de gripe / Effect of the time between symptom onset, swabbing and testing on the detection of influenza virus

Introducción La vigilancia epidemiológica de la gripe requiere la recogida de frotis nasofaríngeos en atención primaria para su análisis en laboratorios de referencia. Evaluamos la influencia en el resultado de laboratorio, de los tiempos transcurridos desde el inicio de síntomas hasta la recogida del frotis (TSF) y desde entonces hasta su procesamiento en laboratorio (TFL).Métodos Analizamos las muestras recogidas en la red centinela de gripe de Navarra en la temporada 2009-2010. Los hisopados se conservaron refrigerados hasta su estudio mediante RT-PCR y cultivo viral. Se analizó el porcentaje de positividad a gripe en función del TSF y del TFL mediante regresión logística. Resultados Se analizaron 937 frotis y 373 (40%) fueron positivos para gripe mediante RT-PCR. El TSF osciló entre 0-15 días. En el análisis ajustado por periodo, laboratorio y edad, la detección del virus de la gripe descendió a menos de la mitad en el cultivo cuando el TSF era de 4-5 días (OR = 0,47; IC 95% 0,24-0,94), y en la RT-PCR, cuando el TSF era mayor de 5 días (OR = 0,24; IC 95% 0,09-0,65). El TFL no afectó de forma significativa al resultado de muestras procesadas por RT-PCR (OR por día transcurrido = 0,96; IC 95% 0,88-1,04), ni por cultivo viral (OR por día transcurrido = 0,97; IC 95% 0,89-1,06).Conclusiones Un TSF superior a 3 días redujo la probabilidad de confirmación de gripe, afectando más al cultivo que a la PCR. El TFL dentro de un rango de dos semanas no afectó de forma relevante al resultado de la RT-PCR ni del cultivo (AU)

Background Influenza surveillance requires the collection of nasopharyngeal swabs in Primary Care for testing in reference laboratories. We evaluated the influence on the laboratory results of the time since the onset of symptoms to swabbing (TSS) and from then until laboratory processing (TSL).Methods We analysed swabs collected in the Sentinel Network of Navarra during the 2009-2010 influenza season. The samples were kept refrigerated until analysed by RT-PCR and viral culture. We analysed the percentage of positive swabs to influenza virus in accordance with the TSS and TSL by logistic regression. Results From a total of 937 swabs, 373 (40%) were positive for influenza by RT-PCR. The TSS ranged from 0-15 days. In the adjusted analysis by period, laboratory and age, having a positive influenza culture decreased to less than half when the TSS was 4-5 days (OR=0.47; 95% CI, 0.24-0.94), and having a positive RT-PCR decreased when the TSS was 5 days or more (OR=0.24, 95% CI, 0.09-0.65). TSL does not significantly affect the result of the RT-PCR (OR by each day=0.96; 95% CI, 0.88-1.04), or the result of the viral culture (OR by each day=0.97, 95% CI, 0.89-1.06).Conclusions A TSS over 3 days reduced the likelihood of confirmation of influenza, affecting the viral culture more than the RT-PCR. A TSL within a range of two weeks had no significant effect on the results of the RT-PCR or the viral culture (AU)

[Effect of the time between symptom onset, swabbing and testing on the detection of influenza virus]. / Influencia de los tiempos entre el inicio de síntomas, la toma de frotis nasofaríngeo y su procesamiento en laboratorio en la detección de gripe.

BACKGROUND: Influenza surveillance requires the collection of nasopharyngeal swabs in Primary Care for testing in reference laboratories. We evaluated the influence on the laboratory results of the time since the onset of symptoms to swabbing (TSS) and from then until laboratory processing (TSL). METHODS: We analysed swabs collected in the Sentinel Network of Navarra during the 2009-2010 influenza season. The samples were kept refrigerated until analysed by RT-PCR and viral culture. We analysed the percentage of positive swabs to influenza virus in accordance with the TSS and TSL by logistic regression. RESULTS: From a total of 937 swabs, 373 (40%) were positive for influenza by RT-PCR. The TSS ranged from 0-15 days. In the adjusted analysis by period, laboratory and age, having a positive influenza culture decreased to less than half when the TSS was 4-5 days (OR=0.47; 95% CI, 0.24-0.94), and having a positive RT-PCR decreased when the TSS was 5 days or more (OR=0.24, 95% CI, 0.09-0.65). TSL does not significantly affect the result of the RT-PCR (OR by each day=0.96; 95% CI, 0.88-1.04), or the result of the viral culture (OR by each day=0.97, 95% CI, 0.89-1.06). CONCLUSIONS: A TSS over 3 days reduced the likelihood of confirmation of influenza, affecting the viral culture more than the RT-PCR. A TSL within a range of two weeks had no significant effect on the results of the RT-PCR or the viral culture.