Immune response to co-administration of measles, mumps, and rubella (MMR), and yellow fever vaccines: a randomized non-inferiority trial among one-year-old children in Argentina.

BACKGROUND: In yellow fever (YF) endemic areas, measles, mumps, and rubella (MMR), and YF vaccines are often co-administered in childhood vaccination schedules. Because these are live vaccines, we assessed potential immune interference that could result from co-administration. METHODS: We conducted an open-label, randomized non-inferiority trial among healthy 1-year-olds in Misiones Province, Argentina. Children were randomized to one of three groups (1:1:1): Co-administration of MMR and YF vaccines (MMR1YF1), MMR followed by YF vaccine four weeks later (MMR1YF2), or YF followed by MMR vaccine four weeks later (YF1MMR2). Blood samples obtained pre-vaccination and 28 days post-vaccination were tested for immunoglobulin G antibodies against measles, mumps, and rubella, and for YF virus-specific neutralizing antibodies. Non-inferiority in seroconversion was assessed using a -5% non-inferiority margin. Antibody concentrations were compared with Kruskal-Wallis tests. RESULTS: Of 851 randomized children, 738 were correctly vaccinated, had ≥ 1 follow-up sample, and were included in the intention-to-treat population. Non-inferior seroconversion was observed for all antigens (measles seroconversion: 97.9% in the MMR1YF1 group versus 96.3% in the MMR1YF2 group, a difference of 1.6% [90% CI -1.5, 4.7]; rubella: 97.9% MMR1YF1 versus 94.7% MMR1YF2, a difference of 3.3% [-0.1, 6.7]; mumps: 96.7% MMR1YF1 versus 97.9% MMR1YF2, a difference of -1.3% [-4.1, 1.5]; and YF: 96.3% MMR1YF1 versus 97.5% YF1MMR2, a difference of -1.2% [-4.2, 1.7]). Rubella antibody concentrations and YF titers were significantly lower following co-administration; measles and mumps concentrations were not impacted. CONCLUSION: Effective seroconversion was achieved and was not impacted by the co-administration, although antibody levels for two antigens were lower. The impact of lower antibody levels needs to be weighed against missed opportunities for vaccination to determine optimal timing for MMR and YF vaccine administration. TRIAL REGISTRATION: The study was retrospectively registered in ClinicalTrials.gov (NCT03368495) on 11/12/2017.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient.

Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA) protein of influenza A(H3N2) viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78%) were A(H3N2) strains, 244 (21.3%) were type B strains, and 8 (0.7%) were A(H1N1)pdm09 strains. Out of 888 A(H3N2) samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2) samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients.

Lidocaine reinforces the anti-inflammatory action of dexamethasone on myeloid and epithelial cells activated by inflammatory cytokines or SARS-CoV-2 infection.

BACKGROUND: Severe cases of Coronavirus Disease 2019 (COVID-19) that require admission to the Intensive Care Unit (ICU) and mechanical ventilation assistance show a high mortality rate with currently few therapeutic options available. Severe COVID-19 is characterized by a systemic inflammatory condition, also called "cytokine storm", which can lead to various multi-organ complications and ultimately death. Lidocaine, a safe local anesthetic that given intravenously is used to treat arrhythmias, has long been reported to have an anti-inflammatory and pro-homeostatic activity. METHODS: We studied the capacity of lidocaine to modulate cytokine secretion of mouse and human myeloid cell lines activated by different cytokines or Toll Like Receptor (TLR) ligands (flagellin (FliC), Lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (Poly I:C) and N-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl (Pam3Cys-SKKKK)) or by Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection to epithelial cells. Reporter cell lines were used to study modulation of lidocaine of specific signaling pathways. RESULTS: Lidocaine used in combination with dexamethasone, had an additive effect in the modulation of cellular inflammatory response triggered by Tumoral Necrosis Factor alpha (TNFα), Interleukin 1 beta (IL-1ß) as well as different TLR ligands. We also found that lidocaine in combination with dexamethasone modulates the Nuclear factor kappa B (NF-κB) pathway, inflammasome activation as well as interferon gamma receptor (IFNγR) signaling without affecting the type I interferons (Type I IFNs) pathway. Furthermore, we showed that lidocaine and dexamethasone treatment of epithelial cells infected with SARS-CoV-2 modulated the expression of chemokines that contribute to pro-inflammatory effects in severe COVID. CONCLUSIONS: We reported for the first time in vitro anti-inflammatory capacity of lidocaine on SARS-CoV-2 triggered immune pathways. These results indicated the potential of lidocaine to treat COVID-19 patients and add tools to the therapeutic options available for these concerning cases.

Cross-protection and cross-neutralization capacity of ancestral and VOC-matched SARS-CoV-2 adenoviral vector-based vaccines.

COVID-19 vaccines were originally designed based on the ancestral Spike protein, but immune escape of emergent Variants of Concern (VOC) jeopardized their efficacy, warranting variant-proof vaccines. Here, we used preclinical rodent models to establish the cross-protective and cross-neutralizing capacity of adenoviral-vectored vaccines expressing VOC-matched Spike. CoroVaxG.3-D.FR, matched to Delta Plus Spike, displayed the highest levels of nAb to the matched VOC and mismatched variants. Cross-protection against viral infection in aged K18-hACE2 mice showed dramatic differences among the different vaccines. While Delta-targeted vaccines fully protected mice from a challenge with Gamma, a Gamma-based vaccine offered only partial protection to Delta challenge. Administration of CorovaxG.3-D.FR in a prime/boost regimen showed that a booster was able to increase the neutralizing capacity of the sera against all variants and fully protect aged K18-hACE2 mice against Omicron BA.1, as a BA.1-targeted vaccine did. The neutralizing capacity of the sera diminished in all cases against Omicron BA.2 and BA.5. Altogether, the data demonstrate that a booster with a vaccine based on an antigenically distant variant, such as Delta or BA.1, has the potential to protect from a wider range of SARS-CoV-2 lineages, although careful surveillance of breakthrough infections will help to evaluate combination vaccines targeting antigenically divergent variants yet to emerge.

Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005-2008.

OBJECTIVE: To describe the virological characteristics of the influenza strains circulating in Argentina in 2005-2008 and to assess the prevalence of antiviral resistance. METHODS: On the basis of their geographical spread and prevalence, influenza A and B isolates grown in Madin-Darby canine kidney cells were selected after antigenic and genomic characterization to be analyzed for antiviral resistance by enzymatic assay and pyrosequencing. Amantadine susceptibility was evaluated by pyrosequencing for known resistance markers on 45 strains of influenza A. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay of 67 influenza A and 46 influenza B strains, some of which were further analyzed by sequencing the neuraminidase gene. RESULTS: Resistance to amantadine was observed only on A(H3N2) strains (29/33); all of them carried the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 12 (34.3%) of the 35 A(H1N1) strains from 2008; all of them carried the mutation H275Y in their neuraminidase sequence. All these viruses remained sensitive to zanamivir. CONCLUSIONS: This study describes a high incidence of amantadine-resistant influenza A(H3N2) viruses since 2006 and an unprecedented increase in oseltamivir resistance detected only in influenza A(H1N1) viruses isolated in 2008. Influenza A and B viruses were more sensitive to oseltamivir than to zanamivir, and influenza A viruses were more sensitive to both neuraminidase inhibitors than the influenza B viruses. The national data generated and analyzed in this study may help increase knowledge about influenza antiviral drug resistance, which is a problem of global concern.

Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina.

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization-World Health Organization-International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

Molecular characterization of severe and mild cases of influenza A (H1N1) 2009 strain from Argentina.

While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide.

Genetic evolution of influenza viruses among selected countries in Latin America, 2017-2018.

OBJECTIVE: Since the 2009 influenza pandemic, Latin American (LA) countries have strengthened their influenza surveillance systems. We analyzed influenza genetic sequence data from the 2017 through 2018 Southern Hemisphere (SH) influenza season from selected LA countries, to map the availability of influenza genetic sequence data from, and to describe, the 2017 through 2018 SH influenza seasons in LA. METHODS: We analyzed influenza A/H1pdm09, A/H3, B/Victoria and B/Yamagata hemagglutinin sequences from clinical samples from 12 National Influenza Centers (NICs) in ten countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Ecuador, Mexico, Paraguay, Peru and Uruguay) with a collection date from epidemiologic week (EW) 18, 2017 through EW 43, 2018. These sequences were generated by the NIC or the WHO Collaborating Center (CC) at the U.S Centers for Disease Control and Prevention, uploaded to the Global Initiative on Sharing All Influenza Data (GISAID) platform, and used for phylogenetic reconstruction. FINDINGS: Influenza hemagglutinin sequences from the participating countries (A/H1pdm09 n = 326, A/H3 n = 636, B n = 433) were highly concordant with the genetic groups of the influenza vaccine-recommended viruses for influenza A/H1pdm09 and influenza B. For influenza A/H3, the concordance was variable. CONCLUSIONS: Considering the constant evolution of influenza viruses, high-quality surveillance data-specifically genetic sequence data, are important to allow public health decision makers to make informed decisions about prevention and control strategies, such as influenza vaccine composition. Countries that conduct influenza genetic sequencing for surveillance in LA should continue to work with the WHO CCs to produce high-quality genetic sequence data and upload those sequences to open-access databases.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells.

BACKGROUND: Two new subclades of influenza A(H3N2) viruses became prominent during the 2014-2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes previously associated with alterations in receptor-binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK-SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage. METHODS: Gene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK-SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage-acquired amino acid substitutions and polymorphisms affected virus antigenicity. RESULTS: Viruses were able to infect MDCK-SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK-SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK-SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage. CONCLUSIONS: Current H3N2 viruses should be cultured in the MDCK-SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Antigenic and genomic relation between human influenza viruses that circulated in Argentina in the period 1995-1999 and the corresponding vaccine components.

BACKGROUND: The analysis of epidemic influenza virus has been focused on antigenic and genomic characterization of the hemagglutinin (HA) glycoprotein in order to detect new variants for the recommendation of the vaccine strains in each season. Since October 1998, WHO organized a second meeting to evaluate the vaccine formula for the southern hemisphere. OBJECTIVES: (a) Present the antigenic and genomic characterization of influenza strains obtained from the Argentina surveillance network, (b) compare between strains collected in Argentina and other countries with the vaccine formula strains used in each season. STUDY DESIGN: Influenza strains were collected during a 5-year period (1995-1999). Initially, laboratory diagnosis was done by immunofluorescence (IF) assay on clinical samples, followed by viral isolation in Madin-Darby canine kidney (MDCK) cells. The isolates were characterized antigenically by hemagglutination-inhibition (HI) assay with post-infection ferret antisera. The genomic characterization consisted on RT-PCR followed by sequencing of the HA1 portion of the HA gene. The comparison between reference and circulating strains was analyzed by the construction of phylogenetic trees. RESULTS: The H3N2 circulating strains matched the corresponding vaccine component only in 1999, the first year when a vaccine recommended specifically for the southern hemisphere was used. Besides, H1N1 circulating strains matched the corresponding vaccine component only in 1998. Regarding to influenza B, only in 1995, the circulating strains showed no match to the B vaccine component. CONCLUSIONS: The results showed the usefulness of an intensified influenza laboratory surveillance to access the correct vaccine and the importance of having the necessary tools to characterize the circulating strains.

Antigenic and genomic characterization of human influenza A and B viruses circulating in Argentina after the introduction of influenza A(H1N1)pdm09.

This study was conducted as part of the Argentinean Influenza and other Respiratory Viruses Surveillance Network, in the context of the Global Influenza Surveillance carried out by the World Health Organization (WHO). The objective was to study the activity and the antigenic and genomic characteristics of circulating viruses for three consecutive seasons (2010, 2011 and 2012) in order to investigate the emergence of influenza viral variants. During the study period, influenza virus circulation was detected from January to December. Influenza A and B, and all current subtypes of human influenza viruses, were present each year. Throughout the 2010 post-pandemic season, influenza A(H1N1)pdm09, unexpectedly, almost disappeared. The haemagglutinin (HA) of the A(H1N1)pdm09 viruses studied were segregated in a different genetic group to those identified during the 2009 pandemic, although they were still antigenically closely related to the vaccine strain A/California/07/2009. Influenza A(H3N2) viruses were the predominant strains circulating during the 2011 season, accounting for nearly 76 % of influenza viruses identified. That year, all HA sequences of the A(H3N2) viruses tested fell into the A/Victoria/208/2009 genetic clade, but remained antigenically related to A/Perth/16/2009 (reference vaccine recommended for this three-year period). A(H3N2) viruses isolated in 2012 were antigenically closely related to A/Victoria/361/2011, recommended by the WHO as the H3 component for the 2013 Southern Hemisphere formulation. B viruses belonging to the B/Victoria lineage circulated in 2010. A mixed circulation of viral variants of both B/Victoria and B/Yamagata lineages was detected in 2012, with the former being predominant. A(H1N1)pdm09 viruses remained antigenically closely related to the vaccine virus A/California/7/2009; A(H3N2) viruses continually evolved into new antigenic clusters and both B lineages, B/Victoria/2/87-like and B/Yamagata/16/88-like viruses, were observed during the study period. The virological surveillance showed that the majority of the circulating strains during the study period were antigenically related to the corresponding Southern Hemisphere vaccine strains except for the 2012 A(H3N2) viruses.

Rol del virus sincicial respiratorio en una cohorte de adultos mayores / Respiratory Syncytial Virus in the elderly adults

El rol de los virus respiratorios distintos de influenza en las infecciones respiratorias agudas en los adultos mayores ha sido probablemente subestimado. En los últimos años, los avances en técnicas moleculares de diagnóstico han hecho posible la identificación rápida del virus sincicial respiratorio humano (HRSV). Realizamos un estudio prospectivo observacional para evaluar el rol del HRSV en mayores de 65 años que se hospitalizaron por infecciones respiratorias en nuestra institución, ubicada en la ciudad de La Plata, provincia de Buenos Aires. Fueron reclutados 124 pacientes y el HRSV se detectó en 13, influenza B en 9 e influenza A en 8. La presentación clínica más frecuente de los The role of respiratory viruses other than influenza in acute respiratory tract infections among elderly adults has probably been underestimated. Recent advances in molecular diagnosis have made the rapid identification of human respiratory syncitial virus HRSV infection possible. We conducted a prospective observational study to evaluate the role of HRSV in elderly patients (>65 years of age) hospitalized for acute respiratory infections. A total of 124 patients were recruited, HRSV infection was identified in 13 patients, Influenza B in 9 patients and influenza A in 8 patients. The most frequent clinical presentation was bronchospasm and the infection was prevalent in patients with comorbidities. HRSV infections accounted for an important number of hospital admissions and has been associated with high mortality rates (23%). pacientes con HRSV fue el broncoespasmo y afectó principalmente a personas con comorbilidades. HRSV fue responsable de un número importante de internaciones por enfermedad respiratoria aguda en mayores de 65 años en nuestra institución y se asoció a mortalidad elevada (23%).

The role of respiratory viruses other than influenza in acute respiratory tract infections among elderly adults has probably been underestimated. Recent advances in molecular diagnosis have made the rapid identification of human respiratory syncitial virus HRSV infection possible. We conducted a prospective observational study to evaluate the role of HRSV in elderly patients (>65 years of age) hospitalized for acute respiratory infections. A total of 124 patients were recruited, HRSV infection was identified in 13 patients, Influenza B in 9 patients and influenza A in 8 patients. The most frequent clinical presentation was bronchospasm and the infection was prevalent in patients with comorbidities. HRSV infections accounted for an important number of hospital admissions and has been associated with high mortality rates (23%).

Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient

Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA) protein of influenza A(H3N2) viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78%) were A(H3N2) strains, 244 (21.3%) were type B strains, and 8 (0.7%) were A(H1N1)pdm09 strains. Out of 888 A(H3N2) samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2) samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients.

Addition of the immunostimulatory oligonucleotide IMT504 to a seasonal flu vaccine increases hemagglutinin antibody titers in young adult and elder rats, and expands the anti-hemagglutinin antibody repertoire.

Flu vaccines are partially protective in infants and elder people. New adjuvants such as immunostimulatory oligonucleotides (ODNs) are strong candidates to solve this problem, because a combination with several antigens has demonstrated effectiveness. Here, we report that IMT504, the prototype of a major class of immunostimulatory ODNs, is a potent adjuvant of the influenza vaccine in young adult and elderly rats. Flu vaccines that use virosomes or whole viral particles as antigens were combined with IMT504 and injected in rats. Young adult and elderly animals vaccinated with IMT504-adjuvated preparations reached antibody titers 20-fold and 15-fold higher than controls, respectively. Antibody titers remained high throughout a 120 day-period. Animals injected with the IMT504-adjuvated vaccine showed expansion of the anti-hemagglutinin antibody repertoire and a significant increase in the antibody titer with hemagglutination inhibition capacity when confronted to viral strains included or not in the vaccine. This indicates that the addition of IMT504 in flu vaccines may contribute to the development of significant cross-protective immune response against shifted or drifted flu strains.

Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005–2008 / Vigilancia virológica y resistencia a los antivíricos del virus de la gripe humana en la Argentina, 2005–2008

Objective. To describe the virological characteristics of the influenza strains circulating in Argentina in 2005–2008 and to assess the prevalence of antiviral resistance. Methods. On the basis of their geographical spread and prevalence, influenza A and B isolates grown in Madin–Darby canine kidney cells were selected after antigenic and genomic characterization to be analyzed for antiviral resistance by enzymatic assay and pyrosequencing. Amantadine susceptibility was evaluated by pyrosequencing for known resistance markers on 45 strains of influenza A. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay of 67 influenza A and 46 influenza B strains, some of which were further analyzed by sequencing the neuraminidase gene. Results. Resistance to amantadine was observed only on A(H3N2) strains (29/33); all of them carried the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 12 (34.3%) of the 35 A(H1N1) strains from 2008; all of them carried the mutation H275Y in their neuraminidase sequence. All these viruses remained sensitive to zanamivir. Conclusions. This study describes a high incidence of amantadine-resistant influenza A(H3N2) viruses since 2006 and an unprecedented increase in oseltamivir resistance detected only in influenza A(H1N1) viruses isolated in 2008. Influenza A and B viruses were more sensitive to oseltamivir than to zanamivir, and influenza A viruses were more sensitive to both neuraminidase inhibitors than the influenza B viruses. The national data generated and analyzed in this study may help increase knowledge about influenza antiviral drug resistance, which is a problem of global concern.

Objetivo. Describir las características virológicas de las cepas de virus de la gripe que circulaban en la Argentina entre el 2005 y el 2008, y evaluar la prevalencia de la resistencia a los antivíricos. Métodos. Según su diseminación geográfica y su prevalencia, se seleccionaron aislados de gripe A y B cultivados en células renales caninas de Madin-Darby después de su caracterización antigénica y genómica, y se analizó su resistencia a los antivíricos mediante análisis enzimático y pirosecuenciación. La sensibilidad a la amantadina se evaluó por pirosecuenciación para los marcadores conocidos de resistencia en 45 cepas de gripe A. La sensibilidad al oseltamivir y al zanamivir se evaluó mediante análisis enzimático de 67 cepas de gripe A y 46 cepas de gripe B, algunas de las cuales se analizaron en mayor profundidad mediante la secuenciación del gen de la neuraminidasa. Resultados. Se observó resistencia a la amantadina solo en las cepas de gripe A (H3N2) (29/33); todas ellas tenían la mutación S31N en su secuencia de M2. Se observó resistencia al oseltamivir en 12 (34,3%) de las 35 cepas de gripe A (H1N1) aisladas en el 2008; todas ellas tenían la mutación H275Y en su secuencia de neuraminidasa. Todos estos virus conservaron su sensibilidad al zanamivir. Conclusiones. En este estudio se describe una incidencia elevada del virus de la gripe A (H3N2) resistente a la amantadina desde el 2006 y un aumento sin precedentes de la resistencia al oseltamivir detectada solo en los virus de la gripe A (H1N1) aislados en el 2008. Los virus de la gripe A y B fueron más sensibles al oseltamivir que al zanamivir y los virus de la gripe A fueron más sensibles a ambos inhibidores de la neuraminidasa que los virus de la gripe B. Los datos nacionales generados y analizados en este estudio pueden ayudar a aumentar los conocimientos acerca de la resistencia a los fármacos antivíricos dirigidos contra el virus de la gripe, lo que es un motivo de preocupación mundial.

Molecular characterization of severe and mild cases of influenza A (H1N1) 2009 strain from Argentina / Caracterización molecular de cepas de influenza A (H1N1) 2009 de casos leves y graves de la Argentina

While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide.

Mientras que la tasa de letalidad (CFR) para (H1N1)pdm en todo el mundo era del 0.4%, en la Argentina la mortalidad observada fue de 4.5%. La secuenciación del genoma completo de 26 cepas de virus argentinos de influenza A (H1N1)pdm de casos leves y graves y de 8 cepas secuenciadas parcialmente no mostró evidencia de que la elevada tasa de letalidad se pueda atribuir directamente a cambios en el virus. No se encontraron hallazgos de recombinación, de mutaciones asociadas con la resistencia a los medicamentos antivirales ni de variaciones genéticas que puedan contribuir a la virulencia observada. Si bien la mutación D225G asociada con la gravedad, comunicada en informes procedentes de Ucrania y Noruega, no se ha encontrado en las cepas argentinas estudiadas, se ha observado un cambio aminoacídico en la región (S206T) en torno al dominio del sitio de unión al receptor en la HA, el mismo hallado en cepas distribuidas alrededor del mundo.