Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Diagnostic Considerations in H1N1 Influenza-induced Thrombotic Microangiopathy.

Influenza virus can trigger atypical hemolytic uremic syndrome and present with complement-driven thrombotic microangiopathy (TMA). When administered promptly, complement-blocking therapies can spare organ injury and be lifesaving. However, diagnosing TMA in the setting of a severe viral infection can be challenging, as a significant overlap of symptoms and disease complications exists. This is particularly true in influenza virus infections and more recently, Coronavirus disease 2019 (COVID-19) infections. We present a 16-year-old male with H1N1 influenza-induced atypical hemolytic uremic syndrome who quickly improved with complement-blocking therapy, highlighting an urgent need to include TMA in the differential diagnosis of severe viral infections.

TNFA and IL10 Polymorphisms and IL-6 and IL-10 Levels Influence Disease Severity in Influenza A(H1N1)pdm09 Virus Infected Patients.

Cytokines are key modulators of immune response, and dysregulated production of proinflammatory and anti-inflammatory cytokines contributes to the pathogenesis of influenza A(H1N1)pdm09 virus infection. Cytokine production is impacted by single nucleotide polymorphisms (SNPs) in the genes coding for them. In the present study, SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB were investigated for their association with disease severity and fatality in influenza A(H1N1)pdm09-affected patients with mild disease (n = 293) and severe disease (n = 86). Among those with severe disease, 41 patients had fatal outcomes. In a subset of the patients, levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17 were assayed in the plasma for their association with severe disease. The frequency of TNFA rs1800629 G/A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.95 (1.52-5.73); mild vs. survived severe cases 4.02 (1.84-8.82)). IL10 rs1800896-rs1800872 G-C haplotype was significantly lower (OR with 95% 0.34 (0.12-0.95)), while IL10 rs1800896-rs1800872 G-A haplotype was significantly higher (OR with 95% 12.11 (2.23-76.96)) in fatal cases group compared to that of the mild group. IL-6 and IL-10 levels were significantly higher in fatal cases compared to that of survived severe cases. IL-6 levels had greater discriminatory power than IL-10 to predict progression to fatal outcome in influenza A(H1N1)pdm09 virus-infected patients. To conclude, the present study reports the association of TNFA and IL10 SNPs with severe disease in Influenza A(H1N1)pdm09 virus-infected subjects. Furthermore, IL-6 levels can be a potential biomarker for predicting fatal outcomes in Influenza A(H1N1)pdm09 virus infected subjects.

Influenza co-infection associated with severity and mortality in COVID-19 patients.

BACKGROUND: In COVID-19 patients, undetected co-infections may have severe clinical implications associated with increased hospitalization, varied treatment approaches and mortality. Therefore, we investigated the implications of viral and bacterial co-infection in COVID-19 clinical outcomes. METHODS: Nasopharyngeal samples were obtained from 48 COVID-19 patients (29% ICU and 71% non-ICU) and screened for the presence of 24 respiratory pathogens using six multiplex PCR panels. RESULTS: We found evidence of co-infection in 34 COVID-19 patients (71%). Influenza A H1N1 (n = 17), Chlamydia pneumoniae (n = 13) and human adenovirus (n = 10) were the most commonly detected pathogens. Viral co-infection was associated with increased ICU admission (r = 0.1) and higher mortality (OR 1.78, CI = 0.38-8.28) compared to bacterial co-infections (OR 0.44, CI = 0.08-2.45). Two thirds of COVID-19 critically ill patients who died, had a co-infection; and Influenza A H1N1 was the only pathogen for which a direct relationship with mortality was seen (r = 0.2). CONCLUSIONS: Our study highlights the importance of screening for co-infecting viruses in COVID-19 patients, that could be the leading cause of disease severity and death. Given the high prevalence of Influenza co-infection in our study, increased coverage of flu vaccination is encouraged to mitigate the transmission of influenza virus during the on-going COVID-19 pandemic and reduce the risk of severe outcome and mortality.

Spatial mapping of SARS-CoV-2 and H1N1 lung injury identifies differential transcriptional signatures.

Severe SARS-CoV-2 infection often leads to the development of acute respiratory distress syndrome (ARDS), with profound pulmonary patho-histological changes post-mortem. It is not clear whether ARDS from SARS-CoV-2 is similar to that observed in influenza H1N1, another common viral cause of lung injury. Here, we analyze specific ARDS regions of interest utilizing a spatial transcriptomic platform on autopsy-derived lung tissue from patients with SARS-CoV-2 (n = 3), H1N1 (n = 3), and a dual infected individual (n = 1). Enhanced gene signatures in alveolar epithelium, vascular tissue, and lung macrophages identify not only increased regional coagulopathy but also increased extracellular remodeling, alternative macrophage activation, and squamous metaplasia of type II pneumocytes in SARS-CoV-2. Both the H1N1 and dual-infected transcriptome demonstrated an enhanced antiviral response compared to SARS-CoV-2. Our results uncover regional transcriptional changes related to tissue damage/remodeling, altered cellular phenotype, and vascular injury active in SARS-CoV-2 and present therapeutic targets for COVID-19-related ARDS.

Detection of community-acquired respiratory viruses in allogeneic stem-cell transplant recipients and controls-A prospective cohort study.

BACKGROUND: Community-acquired respiratory viruses (CARV) cause upper and lower respiratory tract infections (URTI/LRTI) and may be life-threatening for recipients of an allogeneic stem cell transplantation (allo-SCT). METHODS: In a prospective study encompassing 4 winter-seasons, we collected throat gargles (TG) at random time points from allo-SCT recipients (patients) and controls and followed them up for at least 3 weeks including repetitive sampling and documentation of symptoms. A Multiplex-PCR system to identify 20 CARV and Mycoplasma pneumoniae was used to detect CARV. RESULTS: One hundred ninety-four patients with 426 TG and 273 controls with 549 TG were included. There were more patients with a positive test result (25% vs 11% in the controls), and the patients had a higher number of positive TG (70 = 16%) compared to controls (32 = 6%) (P < .001). Altogether, 115 viruses were detected. Multiple viruses in one TG (11/48, 34%) and prolonged shedding were only observed in patients (13/48, 27%). Patients had more RSV (18/83, 26%) and adenovirus (15/83, 21%) than controls (both viruses 2/32, 6%). Independent risk factors for the detection of CARV included age >40 years (OR 3.38, 95% CI 1.8-6.4, P < .001) and presence of URTI-symptoms (OR 3.22, 95% CI 1.9-5.5, P < .001). No controls developed a LRTI or died whereas 4/48 (8%) patients developed a LRTI (coronavirus in 2, RSV in 1 and influenza A H1N1 in 1 patient). One patient died of CARV (influenza A H1N1). CONCLUSION: Allo-SCT-recipients have more CARV-infections, exhibit a different epidemiology, have more cases of co-infection or prolonged shedding and have a higher rate of LRTI and mortality.

Strengthening timely detection and reporting of unusual respiratory events from health facilities in Yaoundé, Cameroon.

BACKGROUND: The International Health Regulations state that early detection and immediate reporting of unusual health events is important for early warning and response systems. OBJECTIVE: To describe a pilot surveillance program established in health facilities in Yaoundé, Cameroon in 2017 which aimed to enable detection and reporting of public health events. METHODS: Cameroon's Ministry of Health, in partnership with the US Centers for Disease Control and Prevention, Cameroon Pasteur Center, and National Public Health Laboratory, implemented event-based surveillance (EBS) in nine Yaoundé health facilities. Four signals were defined that could indicate possible public health events, and a reporting, triage, and verification system was established among partner organizations. A pre-defined laboratory algorithm was defined, and a series of workshops trained health facilities, laboratory, and public health staff for surveillance implementation. RESULTS: From May 2017 to January 2018, 30 signals were detected, corresponding to 15 unusual respiratory events. All health facilities reported a signal at least once, and more than three-quarters of health facilities reported ≥2 times. Among specimens tested, the pathogens detected included Klebsiella pneumoniae, Moraxella catarrhalis, Streptococcus pneumoniae, Haemophilus influenza, Staphylococcus aureus, Pneumocystis jiroveci, influenza A (H1N1) virus, rhinovirus, and adenovirus. CONCLUSIONS: The events detected in this pilot were caused by routine respiratory bacteria and viruses, and no novel influenza viruses or other emerging respiratory threats were identified. The surveillance system, however, strengthened relationships and communication linkages between health facilities and public health authorities. Astute clinicians can play a critical role in early detection and EBS is one approach that may enable reporting of emerging outbreaks and public health events.

Influenza in patients with cancer after 2009 pandemic AH1N1: An 8-year follow-up study in Mexico.

BACKGROUND: Immunosupressed patients are at high risk of influenza-related complications. Influenza AH1N1 has been hypothesized to induce worse outcomes in patients with malignancies, but after the A(H1N1)pdm09 few publications have analyzed the presentation and complications related to influenza afterward. OBJECTIVES: We aimed to describe the characteristics, risk factors, and outcomes of influenza in an oncologic center after the 2009 pandemic and to compare our case distribution to the National community acquired influenza databases in Mexico and the United States. METHODS: We reviewed the cases of confirmed influenza in patients with cancer from an oncological center in Mexico from April 2009 to April 2017. Data on severity and influenza type, malignancy, comorbidities, and outcomes were recorded. We correlated data between the Centers for Disease Control and Prevention (CDC) in the United States and SISVEFLU (Influenza Surveillance Program) in Mexico. RESULTS: One hundred eighty-eight patients were included; 75 (39.9%) had a solid neoplasm and 113 (60.1%) had hematologic malignancies. AH1N1 was the most frequent influenza type (54.2%). Patients with hematologic malignancies had more pneumonia (55% vs 25%, P < .001), needed more hospitalizations (75% vs 39% P < .001), had higher all-cause mortality at 30 days (20% vs 9% P = .048) and influenza-associated mortality (17% vs 7% P = .041). Thirty (16%) patients died within 30 days, and 24 (12.7%) were related to influenza. Influenza type was not associated with worse outcomes. Yearly occurrence of influenza reported by the CDC and SISVEFLU showed a significant correlation (ρ = 0.823, P = .006). CONCLUSIONS: AH1N1 was the dominant serotype. Patients with hematologic malignancies had more severe influenza and presented worse outcomes. Annual SISVEFLU and CDC surveillance information showed a similar distribution of cases along time but influenza serotypes did not match for all seasons.

Identification of SARS-CoV-2 and additional respiratory pathogens cases under the investigation of COVID-19 initial phase in a Brazilian reference laboratory

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

H1N1: perfil clínico-epidemiológico de pacientes atendidos em um hospital / H1N1: clinical and epidemiological profile of patients treated in a hospital

Objetivo: Avaliar casos de suspeita de gripe H1N1, bem como comparar aspectos epidemiológicos e clínicos dos pacientes com gripe H1N1 confirmada em relação àqueles não confirmados; analisar os critérios de gravidade clínica com relação à confirmação (ou não) da gripe H1N1 e seu desfecho (mortalidade); e criar um banco de dados para fins de comparação com a literatura nacional e mundial. Métodos: Estudo retrospectivo de coorte transversal realizado no período sazonal (outono e inverno) no ano de 2016. Foram analisados os prontuários, acessíveis e completos, de pacientes com suspeita clínica de H1N1, além daqueles com resultados definidos na sorologia. A partir dos dados coletados, foi elaborada tabela de análise epidemiológica, com informações clínicas, laboratoriais e sorológicas. Resultados: Destacam-se a média das faixas etárias mais acometidas de 48 anos, além dos sintomas mais comuns que foram dispneia, tosse e mialgia; as comorbidades foram hipertensão arterial sistêmica, cardiopatias, diabetes e doença pulmonar obstrutiva crônica. Conclusão: Este trabalho contribuiu com a caracterização do perfil epidemiológico regional e auxiliou na definição de indicadores de diagnóstico e gravidade, além de agregar à literatura conteúdos de caráter relevante. Este estudo está registrado como CAAE 58664016.2.0000.5515 na Plataforma Brasil. (AU)

Objective: To evaluate cases of suspected H1N1 flu, as well as to compare epidemiological and clinical aspects of patients with confirmed H1N1 influenza to those who were not confirmed; to analyze the criteria of clinical severity regarding the confirmation (or not) of H1N1 influenza, and its outcome (mortality); and to create a database to be compared with the national and world literature. Methods: This is a cross-sectional retrospective cohort study, carried out in the seasonal period ( fall/winter) of 2016. Accessible and complete medical records of patients with clinical suspicion of H1N1 were analyzed along with those with defined serology results. Based on the collected data, a table of epidemiological analysis was elaborated with clinical, laboratory and serological information. Results: The mean age of the most affected age groups was 48 years; the most common symptoms were dyspnea, cough and myalgia; and the comorbidities were systemic arterial hypertension, cardiopathies, diabetes, and chronic obstructive pulmonary disease. Conclusion: This work contributed to the characterization of the regional epidemiological profile, and helped in the definition of indicators of diagnosis and severity, besides adding relevant content to the literature. This study is registered as CAAE 58664016.2.0000.5515 at Plataforma Brasil. (AU)

Macrophage migration inhibitory factor enhances influenza-associated mortality in mice.

Influenza-associated mortality continues to occur annually despite available antiviral therapies. New therapies that improve host immunity could reduce influenza virus disease burden. Targeting macrophage migration inhibitory factor (MIF) has improved the outcomes of certain inflammatory diseases, but its role in influenza viral infection is unclear. Here, we showed that, during influenza viral infection, Mif-deficient mice have less inflammation, viral load, and mortality compared with WT control mice; conversely, Tg mice, overexpressing Mif in alveolar epithelial cells, had higher inflammation, viral load, and mortality. Antibody-mediated blockade of MIF in WT mice during influenza viral infection improved their survival. Mif-deficient murine lungs showed reduced levels of parkin, a mitophagy protein that negatively regulates antiviral signaling, prior to infection and augmented antiviral type I/III IFN levels in the airspaces after infection as compared with WT lungs. Additionally, in vitro assays with human lung epithelial cells showed that treatment with recombinant human MIF increased the percentage of influenza virus-infected cells. In conclusion, our study reveals that MIF impairs antiviral host immunity and increases inflammation during influenza infection and suggests that targeting MIF could be therapeutically beneficial during influenza viral infection.

Serial Section Array Scanning Electron Microscopy Analysis of Cells from Lung Autopsy Specimens following Fatal A/H1N1 2009 Pandemic Influenza Virus Infection.

A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally "stitching" together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu.IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia.

Magnetic SERS Strip for Sensitive and Simultaneous Detection of Respiratory Viruses.

Rapid and early diagnosis of respiratory viruses is key to preventing infections from spreading and guiding treatments. Here, we developed a sensitive and quantitative surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-based LFIA) strip for simultaneous detection of influenza A H1N1 virus and human adenovirus (HAdV) by using Fe3O4@Ag nanoparticles as magnetic SERS nanotags. The new type of Fe3O4@Ag magnetic tags, which were conjugated with dual-layer Raman dye molecules and target virus-capture antibodies, performs the following functions: specific recognition and magnetic enrichment of target viruses in the solution and SERS detection of the viruses on the strip. Based on this strategy, the magnetic SERS strip can directly be used for real biological samples without any sample pretreatment steps. The limits of detection for H1N1 and HAdV were 50 and 10 pfu/mL, respectively, which were 2000 times more sensitive than those from the standard colloidal gold strip method. Moreover, the proposed strip is easy to operate, rapid, stable, and can achieve high throughput and is thus a potential tool for early detection of virus infection.

Surface-Independent and Oriented Immobilization of Antibody via One-Step Polydopamine/Protein G Coating: Application to Influenza Virus Immunoassay.

For the construction of high-performance biosensor, it is important to interface bioreceptors with the sensor surface densely and in the optimal orientation. Herein, a simple surface modification method that can optimally immobilize antibodies onto various kinds of surfaces is reported. For the surface modification, a mixture of polydopamine (PDA) and protein G was employed. PDA is a representative mussel-inspired polymer, and protein G is an immunoglobulin-binding protein that enables an antibody to have an optimal orientation. The surface characteristics of PDA/Protein G mixture-coated substrates are analyzed and the PDA/protein G ratio is optimized to maximize the antibody binding efficiency. Moreover, the antibody-immobilized substrates are applied to the detection of influenza viruses with the naked eye, providing a detection limit of 2.9 × 103 pfu mL-1 . Importantly, the several substrates (glass, SiO2 , Si, Al2 O3 , polyethylene terephthalate, polyethylene, polypropylene, and paper) can be modified by simple incubation with the mixture of PDA/protein G, and then the anti-influenza A H1N1 antibodies can be immobilized on the substrates successfully. Regardless of the substrate, the influenza viruses are detectable after the sandwich immunoreaction and silver enhancement procedure. It is anticipated that the developed PDA/protein G coating method will extend the range of applicable materials for biosensing.

Antiviral treatment and virological monitoring of oseltamivir-resistant influenza virus A(H1N1)pdm09 in a patient with chronic B lymphocytic leukemia.

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.

Hospital-acquired influenza in an Australian tertiary Centre 2017: a surveillance based study.

BACKGROUND: In 2017, Australia experienced its highest levels of influenza virus activity since the 2009 pandemic. This allowed detailed comparison of the characteristics of patients with community and hospital-acquired influenza, and infection control factors that contributed to influenza spread. METHODS: A surveillance based study was conducted on hospitalised patients with laboratory-confirmed influenza at the Canberra Hospital during April-October 2017. Differences between the hospital-acquired and community-acquired patient characteristics and outcomes were assessed by univariate analysis. Epidemiologic curves were developed and cluster distribution within the hospital was determined. RESULTS: Two hundred and ninety-two patients were included in the study. Twenty-eight (9.6%) acquired influenza in hospital, representing a higher proportion than any of the previous 5 years (range 0.9-5.8%). These patients were more likely to have influenza A (p = 0.021), had higher rates of diabetes (p = 0.015), malignancy (p = 0.046) and chronic liver disease (p = 0.043). Patients acquiring influenza in hospital met clinical criteria for influenza like illness in 25% of cases, compared with 64.4% for community-acquired cases (p < 0.001). Hospital-acquired influenza cases occurred in two distinct clusters. Patients were moved an average of 5 times after diagnosis. Mean length of stay following diagnosis was 13 days compared to 5 days for community-acquired cases (p < 0.001). Of the patients with hospital-acquired influenza, 22 were in shared rooms during their incubation period and 9 were not isolated in single rooms following diagnosis. Treatment was initiated within the recommended 48 h period following symptom onset for 62.5% of hospital-acquired cases compared with 39.8% of community-acquired cases (p = 0.033). CONCLUSIONS: Our results show that clinical presentation differed between patients with hospital-acquired influenza compared with those who acquired influenza in the community. Cases occurred in two clusters suggesting intra-hospital transmission rather than random importation from the community, highlighting the importance of infection control measures to limit influenza spread. Patients with hospital-acquired influenza may present without classical features of an influenza-like illness and this should promote earlier diagnostic testing and isolation to limit spread. Movement of patients after diagnosis is likely to facilitate spread within the hospital.

Identification of Newly Emerging Influenza Viruses by Detecting the Virally Infected Cells Based on Surface Enhanced Raman Spectroscopy and Principal Component Analysis.

Rapid diagnosis and quarantine of influenza virus mutant-infected people is critical to contain the fatal viral infection spread because effective antiviral drugs are normally not available. Conventional methods, however, cannot be used for the diagnosis because these methods need predefined labels, likely also unavailable for just emerging viruses. Here, we propose label-free identification of cells infected with different influenza viruses based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). Viral envelope proteins that are displayed on the surface of cells after infection of influenza viruses were targeted for this identification. Cells that expressed the envelope proteins of A/WSN/33 H1N1 or A/California/04/2009 H1N1 influenza viruses produced distinct SERS signals. Cells that displayed combinations of the envelope proteins from these two viral variants, an indication of emergence of a new virus, also generated characteristic SERS patterns. However, the cell's own surface proteins often hindered the identification of virally infected cells by producing SERS peaks similar to viral ones. PCA of the obtained SERS patterns could effectively capture the virus-specific signal components from the jumbled SERS peaks. Our study demonstrates a potential of combination of SERS and PCA to identify newly emerging influenza viruses through sensing the cells infected with the viruses.

Application of an immunoglobulin Y-alkaline phosphatase bioconjugate as a diagnostic tool for influenza A virus.

The diagnosis of influenza A virus is essential since it can be confused with influenza A like illness and lead to inaccurate drug prescription. In this study, the M2e peptide, a strategic antigen that is conserved in all virus subtypes, was used as a diagnostic marker of influenza A. For the first time, M2e-specific IgY antibody was covalently conjugated to alkaline phosphatase (ALP) enzyme in the presence of glutaraldehyde. The antibody-enzyme bioconjugate was characterized by fluorescence and Fourier-transform infrared spectroscopy. Subsequently, the diagnostic value of this bioconjugate was evaluated by direct sandwich ELISA using nasopharyngeal swab samples positive/negative for H1N1 and H3N2, which were previously analyzed by rRT-PCR for influenza. In conclusion, the M2e-specific IgY-ALP bioconjugate demonstrated positive results for Influenza A in samples that were diagnosed as Influenza A via the RT-PCR method.

Respiratory Pathogens in Infants Diagnosed with Acute Lower Respiratory Tract Infection in a Tertiary Care Hospital of Western India Using Multiplex Real Time PCR.

OBJECTIVE: To determine the frequency of respiratory pathogens in infants diagnosed with acute lower respiratory tract infections. METHODS: A prospective cross-sectional observational study was conducted in infants hospitalized with a diagnosis of acute lower respiratory tract infection (ALRTI), in a tertiary care hospital in a metropolitan city of Western India. Nasopharyngeal swabs were analyzed by multiplex real time polymerase chain reaction, for 18 viruses and 3 bacteria (H. influenzae type b, C. pneumoniae and M. pneumoniae). The entire data was entered in Microsoft excel sheet and frequencies were determined. RESULTS: One hundred eligible infants were enrolled. Pathogens were detected in 82 samples, which included Respiratory syncytial viruses (RSV) A / B (35.4%), Human rhinovirus (25.6%), Adenovirus (22%), Human Parainfluenza viruses (11%), Human bocavirus (9.8), Human metapneumovirus A / B (8.5%), Influenza A (H1N1) pdm 09 (6.1%), Parechovirus (3.7%), Human coronaviruses (3.66%), Haemophilus influenzae type b (6.1%), Chlamydia pneumoniae (2.4%) and Mycoplasma pneumoniae (2.4%). Influenza A (other than H1N1), Influenza B, Human Coronavirus 229E and Enterovirus were not detected. The rate of coinfection was 34% and rhinovirus was the most common of the multiple pathogens. CONCLUSIONS: Spectrum of viral etiologies of ALRTI is highlighted. Etiological diagnosis of ALRTI would enable specific antiviral therapy, restrict antibiotic use and help in knowing burden of disease.