Laboratory Data Timeliness and Completeness Improves Following Implementation of an Electronic Laboratory Information System in Côte d'Ivoire: Quasi-Experimental Study on 21 Clinical Laboratories From 2014 to 2020.

BACKGROUND: The Ministry of Health in Côte d'Ivoire and the International Training and Education Center for Health at the University of Washington, funded by the United States President's Emergency Plan for AIDS Relief, have been collaborating to develop and implement the Open-Source Enterprise-Level Laboratory Information System (OpenELIS). The system is designed to improve HIV-related laboratory data management and strengthen quality management and capacity at clinical laboratories across the nation. OBJECTIVE: This evaluation aimed to quantify the effects of implementing OpenELIS on data quality for laboratory tests related to HIV care and treatment. METHODS: This evaluation used a quasi-experimental design to perform an interrupted time-series analysis to estimate the changes in the level and slope of 3 data quality indicators (timeliness, completeness, and validity) after OpenELIS implementation. We collected paper and electronic records on clusters of differentiation 4 (CD4) testing for 48 weeks before OpenELIS adoption until 72 weeks after. Data collection took place at 21 laboratories in 13 health regions that started using OpenELIS between 2014 and 2020. We analyzed the data at the laboratory level. We estimated odds ratios (ORs) by comparing the observed outcomes with modeled counterfactual ones when the laboratories did not adopt OpenELIS. RESULTS: There was an immediate 5-fold increase in timeliness (OR 5.27, 95% CI 4.33-6.41; P<.001) and an immediate 3.6-fold increase in completeness (OR 3.59, 95% CI 2.40-5.37; P<.001). These immediate improvements were observed starting after OpenELIS installation and then maintained until 72 weeks after OpenELIS adoption. The weekly improvement in the postimplementation trend of completeness was significant (OR 1.03, 95% CI 1.02-1.05; P<.001). The improvement in validity was not statistically significant (OR 1.34, 95% CI 0.69-2.60; P=.38), but validity did not fall below pre-OpenELIS levels. CONCLUSIONS: These results demonstrate the value of electronic laboratory information systems in improving laboratory data quality and supporting evidence-based decision-making in health care. These findings highlight the importance of OpenELIS in Côte d'Ivoire and the potential for adoption in other low- and middle-income countries with similar health systems.

Ensuring diagnostic testing accuracy for patient care and public health- COVID-19 testing scale-up from an EQA provider's perspective.

In response to the coronavirus pandemic (COVID-19) and scale up of diagnostic testing, the Canadian Microbiology Proficiency Testing program created a new proficiency testing (PT) program for the molecular and antigen detection of SARS-CoV-2. The program was geared to point of care testing (POCT) sites located in each of the eight provincial Health Authorities across British Columbia, Canada, with the intention to monitor testing quality. The PT program consisted of 6 shipments in a year, each containing a set of 4 samples either positive for SARS-CoV-2 virus or negative. The program began with initial 23 sites enrolling in March 2021, expanding to >100 participants by December 2021. After the first two surveys, it was observed that testing performance (accuracy) was consistently acceptable for sites using nucleic acid technology (NAT), however performance by sites using rapid antigen detection (RAD) methods was poor, especially when testing the weakly positive samples. A root cause investigation of poor testing performance revealed gaps in the execution of testing methods and also in results interpretation. These quality issues were most commonly associated with new testers who lacked experience with diagnostic testing. Tester training and mentoring was reinforced as was retraining of personnel; sample processing instructions were modified, and a training video was also created for testing sites. As a result of these interventions, sites improved their testing accuracy and the performance of POCT sites using RAD methods came to more closely match the performance of sites utilizing NAT. Overall, the PT program was highly successfully and improved quality of testing in the province. This work demonstrates the critical value of an external quality assessment (EQA) partner towards improving patient and public health and safety, especially when testing is conducted outside of an accredited medical laboratory setting.

HIV viral load testing and monitoring in Côte d'Ivoire: A survival analysis of viral load testing and suppression, and evaluation of adherence to national recommendations.

Routine viral load (VL) monitoring is the standard of care in Côte d'Ivoire and allows for effective treatment guidance for people living with human immunodeficiency virus (HIV) to reach viral load suppression (VLS). For VL monitoring to be effective in reducing the impact of HIV, it must be provided in accordance with national guidance. This study aimed to evaluate VL testing, VLS rates and adherence to national guidance for VL testing using data collected from three national laboratories. We collected data on VL testing between 2015-2018 from OpenELIS (OE), an open-source electronic laboratory information system. We merged data by unique patient ID for patients (0-80 years old) who received multiple VL tests to calculate time between tests. We defined VLS as HIV RNA ≤1,000 copies/mL based on Côte d'Ivoire national and WHO guidance at the time of data collection. We used the Kaplan-Meier survival estimator to estimate time between ART (antiretroviral therapy) initiation and the first VL test, time between subsequent VL tests, and to estimate the proportion of people living with HIV (PLHIV) who were virally suppressed within 12 months of ART initiation. At the first documented VL test, 79.6% of patients were virally suppressed (95% CI: 78.9-80.3). Children under 15 were the least likely to be virally suppressed (55.2%, 95% CI: 51.5-58.8). The median time from ART initiation to the first VL sample collection for testing was 7.8 months (IQR:6.2-13.4). 72.4% of patients were virally suppressed within one year of treatment initiation (95% CI:71.5-73.3). Approximately 30% of patients received a second VL test during the 4-year study period. The median time between the first and second VL tests was 24.9 months (IQR: 4.7->40). Most PLHIV received their first VL test within the recommended 12 months of ART initiation but did not receive subsequent VL monitoring tests within the recommended time frame, reducing the benefits of VL monitoring. While VLS was fairly high, children were least likely to be virally suppressed. Our findings highlight the importance of regular VL monitoring after the first VL test, especially for children.

Contexts for developing of national essential diagnostics lists. Lessons from a mixed-methods study of existing documents, stakeholders and decision making on tier-specific essential in-vitro diagnostics in African countries.

Since 2019, the WHO recommends the development and implementation of National Essential Diagnostics List (NEDL) to facilitate availability of In-Vitro Diagnostics (IVDs) across the various tiers of the healthcare pyramid, facilities with or without a laboratory on-site. To be effective, the development of NEDL should take into consideration the challenges and opportunities associated with current modalities for organization of tier specific testing services in-country. We conducted a mixed-methods analysis set out to explore available national policies, guidelines and decision-making processes that affect accessibility of diagnostics in African countries; 307 documents from 48 African countries were reviewed and 28 in-depth (group) interviews with 43 key-informants in seven countries were conducted between June and July 2022. Of the 48 countries, Nigeria was the only one with formal NEDL. Twenty-five countries had national test menus (63% outdated, from 2015 or earlier) all specifying tests by laboratory tier (5 including the "community tier"), with additional details on equipment (20), consumables (12), and personnel requirements (11). The most popular criteria to select essential IVDs in the quantitative analysis relate to specificities of the tests, whereas in the qualitative study most mentioned were health care and laboratory contextual factors. Quality assurance and waste management for tests at "community tier" were highlighted as concerns by all the respondents. Additional barriers to implementation included the low decision-making power of Laboratory Directorates within the Ministry of Health, as well as the chronic budgetary gaps for clinical laboratory services and policy and strategic plan development outside of vertical programmes. Four countries out of seven would rather revise their test menus by updating them and add ''community tier", than developing a separate NEDL, the former being considered more operational. This study provides a unique set of practical recommendations to the process of development and effective implementation on NEDL in Africa.

Development and national scale implementation of an open-source electronic laboratory information system (OpenELIS) in Côte d'Ivoire: Sustainability lessons from the first 13 years.

PURPOSE: Côte d'Ivoire has a tiered public health laboratory system of 9 reference laboratories, 77 laboratories at regional and general hospitals, and 100 laboratories among 1,486 district health centers. Prior to 2009, nearly all of these laboratories used paper registers and reports to collect and report laboratory data to clinicians and national disease monitoring programs. PROJECT: Since 2009 the Ministry of Health (MOH) in Côte d'Ivoire has sought to implement a comprehensive set of activities aimed at strengthening the laboratory system. One of these activities is the sustainable development, expansion, and technical support of an open-source electronic laboratory information system (OpenELIS), with the long-term goal of Ivorian technical support and managerial sustainment of the system. This project has addressed the need for a comprehensive, customizable, low- to no-cost, open-source LIS to serve the public health systems with initial attention to HIV clients and later expansion to cover the general population. This descriptive case study presents the first published summary of original work which has been ongoing since 2009 in Côte d'Ivoire to transform the laboratory information management systems and processes nationally. IMPACT: OpenELIS is now in use at 106 laboratories across Côte d'Ivoire. This article describes the iterative planning, design, and implementation process of OpenELIS in Côte d'Ivoire, and the evolving leadership, ownership, and capacity of the Ivorian MOH in sustaining the system. This original work synthesizes lessons learned from this 13-year experience towards strengthening laboratory information systems in other low resource settings.

Delivering Modern Global Health Learning Requires New Obligations and Approaches.

Introduction: The COVID-19 pandemic has forced a new look (or modernization) for both the obligations and approaches to achieve best-practices in global health learning. These best-practices have moved beyond traditional, face-to-face (F2F), classroom-based didactics to the use of innovative online, asynchronous and synchronous instructional design and the information and communication technology (ICT) tools to support it. But moving to this higher level of online in-service and pre-service training, key obligations (e.g., stopping neocolonialization, cultural humility, reversing brain drain, gender equity) must guide the modernization of instructional design and the supporting ICT. To positively impact global health training, educators must meet the needs of learners where they are. Purpose: We describe a set of multi-communication methods, e-Learning principles, strategies, and ICT approaches for educators to pivot content delivery from traditional, F2F classroom didactics into the modern era. These best-practices in both the obligations and approaches utilize thoughtful, modern strategies of instructional design and ICT. Approach: We harnessed our collective experiences in global health training to present thoughtful insights on the guiding principles, strategies, and ICT environment central to develop learning curricula that meet trainee needs and how they can be actualized. Specifically, we describe five strategies: 1. Individualized learning; 2. Provide experiential learning; 3. Mentor … Mentor … Mentor; 4. Reinforce learning through assessment; and 5. Information and communication technology and tools to support learning. Discussion: We offer a vision, set of guiding principles, and five strategies for successful curricula delivery in the modern era so that global health training can be made available to a wider audience more efficiently and effectively.

Adaptation of an electronic dashboard to monitor HIV viral load testing in Côte d'Ivoire.

BACKGROUND: The Ministère de le Santé et de l'Hygiène Publique in Côte d'Ivoire and the international community have invested in health information systems in Côte d'Ivoire since 2009, including electronic laboratory information systems. These systems have been implemented in more than 80 laboratories to date and capture all test results produced from these laboratories, including HIV viral load (VL) testing. In 2018 the national HIV programme in Côte d'Ivoire requested international support to develop real-time tools such as dashboards to aggregate and display test-specific data such as HIV VL testing to support the country's programmatic response to HIV. INTERVENTION: The VL dashboard was adapted in 2018 using source software code obtained from the Kenyan Ministry of Health and modified for the Ivorian context. The dashboard enables users to assess relevant clinical data from all Ivoirians living with HIV who undergo VL testing through dashboard data visualisations, including the number of VL tests, kinds of samples tested, and VL levels stratified by demographics and geographic location. LESSONS LEARNT: The VL dashboard enables rapid analysis of VL testing data from across the country and enables the national HIV programme, donors and partners to respond rapidly to issues pertaining to access, turn-around times and others. RECOMMENDATIONS: Adapting existing open-source software is an effective and efficient way to implement transformative tools such as dashboards. The VL dashboard will likely be an essential tool for Côte d'Ivoire to meet the United Nations Programme on HIV/AIDS 90-90-90 targets.

Improving laboratory quality and capacity through leadership and management training: Lessons from Zambia 2016-2018.

BACKGROUND: Competent leadership and management are imperative for delivering quality laboratory services; however, few laboratory managers receive job-specific training in organisational management and leadership. OBJECTIVE: To develop and evaluate participants' competencies in organisational leadership and management as measured through learner and laboratory quality improvement assessments. METHODS: This professional development programme employed a mentored, blended learning approach, utilising in-person didactic and online training, with the practical application of a capstone project in the laboratories. Programme impact was evaluated through a series of pre- and post-laboartory assessments using the Stepwise Laboratory Improvement Process Towards Accreditation checklist, as well as learner-competency assessments through online quizzes and discussions. RESULTS: From 2016 to 2018, 31 managers and quality officers from 16 individual laboratories graduated from the programme having completed capstone projects addressing areas in the entire laboratory testing process. Laboratories increased their compliance with the International Organization for Standardization 15189 standard and all but two laboratories significantly increased their accreditation scores. Two laboratories gained three stars, two laboratories gained two stars, and five laboratories gained one star. Five laboratories subsequently achieved International Organization for Standardization 15189 accreditation in 2019. CONCLUSION: This programme taught leadership theory to laboratory managers and allowed them to implement leadership and management practices in the laboratory setting. Programmes such as this complement existing laboratory quality management training programmes such as Strengthening Laboratory Management Toward Accreditation.

Utility of Point of Care and Rapid Diagnostics in Humanitarian Emergencies.

BACKGROUND: In 2019, there were 70.8 million forcibly displaced people worldwide. Among the top causes of morbidity and mortality were measles, diarrhea, respiratory illness, and malaria. Availability of accurate diagnostics that are of low complexity, affordable, and produce timely results on site without the need for expensive laboratory equipment, extensive training, or distant transport of samples, are essential tools in the response to humanitarian emergencies (HE). Early detection of infectious diseases with epidemic potential and coordinated outbreak response, can result in significant decrease in morbidity and mortality. CONTENT: This review explores the utility of point of care and rapid diagnostic tests (POCT/RDTs) in HE and presents a review and analysis of the low complexity, availability, and ease of use of these diagnostic modalities that make them helpful tools in these settings, despite the generally lower test performance metrics associated with them over conventional laboratory-based assays. We review the literature to understand how POCT/RDTs have been used in HE response to produce lifesaving information without the need for a robust system for transporting test samples to more sophisticated laboratories, as this is often prohibitive in areas affected by conflict or natural disasters. SUMMARY: We propose that POCT/RDTs be considered essential healthcare tools provided to countries following a HE and suggest that UN agencies and vulnerable countries include effective RDTs in their essential diagnostics as part of their national preparedness and response plans.

Remote Mentorship Using Video Conferencing as an Effective Tool to Strengthen Laboratory Quality Management in Clinical Laboratories: Lessons From Cambodia.

BACKGROUND: Providing professional development opportunities to staff working in clinical laboratories undergoing quality improvement programs can be challenged by limited funding, particularly in resource-limited countries such as Cambodia. Using innovative approaches such as video conferencing can connect mentors with practitioners regardless of location. This study describes and evaluates the methods, outputs, and outcomes of a quality improvement program implemented in 12 public hospital laboratories in Cambodia between January 2018 and April 2019. The program used mixed intervention methods including both in-person and remote-access training and mentorship. METHODS: Training outputs were quantified from the activity reports of program trainers and mentors. Program outcomes were measured by pre- and postimplementation audits of laboratory quality management system conformity to international standards. Variations in improved outcomes were assessed in relation to the time spent by laboratory personnel in video conference training and mentoring activity. An additional cross-sectional comparison described the difference in final audit scores between participating and nonparticipating laboratories. RESULTS: Laboratories significantly improved their audit scores over the project period, showing significant improvement in all sections of the ISO 15189 standard. Pre- and postaudit score differences and laboratory personnel participation time in remote mentoring activities showed a strong monotonic relationship. Average input per laboratory was 6,027±2,454 minutes of participation in video conference activities with mentors. Audit scores of participating laboratories were significantly higher than those of laboratories with no quality improvement program. CONCLUSION: Laboratories improved significantly in ISO 15189 conformity following structured laboratory quality management systems training supported by remote and on-site mentoring. The correlation of laboratory participation in video conference activities highlights the utility of remote video conferencing technology to strengthen laboratories in resource-limited settings and to build communities of practice to address quality improvement issues in health care. These findings are particularly relevant in light of the COVID-19 pandemic.

Strengthening the clinical laboratory workforce in Cambodia: a case study of a mixed-method in-service training program to improve laboratory quality management system oversight.

BACKGROUND: Laboratory diagnostic testing service delivery and compliance with international standards for laboratory quality are directly influenced by laboratory workforce competency. Many hospital laboratories in constrained resource settings such as Cambodia struggle to cope with the training needs of laboratory professionals in an environment of competing healthcare development priorities. Resource-limited countries need an adaptable and effective approach to provide laboratory professionals with job-specific quality oversight training to ensure the accuracy, timeliness, and reliability of diagnostic services. CASE PRESENTATION: Here, we describe the results of an in-service training and mentoring program conducted with the Cambodia Ministry of Health at 12 tertiary-level hospital laboratories to drive improvements in laboratory quality management systems toward ISO 15189 accreditation, which demonstrated significant progress between baseline and outcome audits in a concurrent study. This case study describes the program, and evaluates how the four primary activities, including actionable gap assessments and planning, centralized and in situ training curriculum, in-person mentoring, and remote tele-mentoring via video communication technologies, contributed towards quality improvement in the participating laboratories. We evaluated participant responses to Likert scale and free response questions from program and training evaluation surveys, and we used thematic analysis to develop a model of best practices within the program's four primary activities to inform future training approaches. Of these activities, participants agreed most highly that in-person visits and planning based on gap assessments contributed to their learning and ability to improve laboratory operations. Tele-mentoring was rated lowest by participants, who were critical of excessive group dialogue and distraction during web-conferencing; however, feedback suggests both in-person and remote mentoring contribute to continuing education, accountability to action, and peer collaboration and problem solving to improve workforce efforts toward improved quality management systems. CONCLUSIONS: We recommend here a package of in-service training activities for laboratory quality management system improvement initiatives in resource constrained settings that includes needs-based curricula and personalized action plans for participants; interactive and on-site training workshops; and in-person mentoring, complemented with well managed and regular tele-mentoring that focuses on knowledge retention, accountability to goals, and collaborative problem solving. Our model presents an adaptable approach to human resource development for quality improvement in medical laboratories.

Implementation research: a mentoring programme to improve laboratory quality in Cambodia.

OBJECTIVE: To implement a mentored laboratory quality stepwise implementation (LQSI) programme to strengthen the quality and capacity of Cambodian hospital laboratories. METHODS: We recruited four laboratory technicians to be mentors and trained them in mentoring skills, laboratory quality management practices and international standard organization (ISO) 15189 requirements for medical laboratories. Separately, we trained staff from 12 referral hospital laboratories in laboratory quality management systems followed by tri-weekly in-person mentoring on quality management systems implementation using the LQSI tool, which is aligned with the ISO 15189 standard. The tool was adapted from a web-based resource into a software-based spreadsheet checklist, which includes a detailed action plan and can be used to qualitatively monitor each laboratory's progress. The tool - translated into Khmer - included a set of quality improvement activities grouped into four phases for implementation with increasing complexity. Project staff reviewed the laboratories' progress and challenges in weekly conference calls and bi-monthly meetings with focal points of the health ministry, participating laboratories and local partners. We present the achievements in implementation from September 2014 to March 2016. FINDINGS: As of March 2016, the 12 laboratories have completed 74-90% of the 104 activities in phase 1, 53-78% of the 178 activities in phase 2, and 18-26% of the 129 activities in phase 3. CONCLUSION: Regular on-site mentoring of laboratories using a detailed action plan in the local language allows staff to learn concepts of quality management system and learn on the job without disruption to laboratory service provision.

GLA-AF, an emulsion-free vaccine adjuvant for pandemic influenza.

The ongoing threat from Influenza necessitates the development of new vaccine and adjuvant technologies that can maximize vaccine immunogenicity, shorten production cycles, and increase global vaccine supply. Currently, the most successful adjuvants for Influenza vaccines are squalene-based oil-in-water emulsions. These adjuvants enhance seroprotective antibody titers to homologous and heterologous strains of virus, and augment a significant dose sparing activity that could improve vaccine manufacturing capacity. As an alternative to an emulsion, we tested a simple lipid-based aqueous formulation containing a synthetic TLR4 ligand (GLA-AF) for its ability to enhance protection against H5N1 infection. GLA-AF was very effective in adjuvanting recombinant H5 hemagglutinin antigen (rH5) in mice and was as potent as the stable emulsion, SE. Both adjuvants induced similar antibody titers using a sub-microgram dose of rH5, and both conferred complete protection against a highly pathogenic H5N1 challenge. However, GLA-AF was the superior adjuvant in ferrets. GLA-AF stimulated a broader antibody response than SE after both the prime and boost immunization with rH5, and ferrets were better protected against homologous and heterologous strains of H5N1 virus. Thus, GLA-AF is a potent emulsion-free adjuvant that warrants consideration for pandemic influenza vaccine development.

Inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice.

Highly pathogenic influenza A viruses, including avian H5N1 viruses and the 1918 pandemic virus, cause severe respiratory disease in humans and animals. Virus infection is followed by intense pulmonary congestion due to an extensive influx of macrophages and neutrophils, which can release large quantities of reactive oxygen species potentially contributing to the pathogenesis of lung disease. Here, the role of nitric oxide (NO), a potent signaling molecule in inflammation, was evaluated following highly pathogenic influenza virus challenge in mice. We observed higher levels of NO in mice infected with H5N1 and 1918 viruses as compared to a seasonal H1N1 virus. Mice deficient in inducible NO synthase (NOS2(-/-)) exhibited reduced morbidity, reduced mortality, and diminished cytokine production in lung tissue following H5N1 and 1918-virus challenge, compared with wild-type control mice. Furthermore, systemic treatment of mice with the NOS inhibitor NG-monomethyl-l-arginine delayed weight loss and death among 1918 virus infected mice compared to untreated control animals. This study demonstrates that NO contributes to the pathogenic outcome of H5N1 and 1918 viral infections in the mouse model.

Mice lacking both TNF and IL-1 receptors exhibit reduced lung inflammation and delay in onset of death following infection with a highly virulent H5N1 virus.

BACKGROUND: Highly pathogenic avian influenza viruses of the H5N1 subtype continue to cross the species barrier to infect humans and cause severe disease. It has been suggested that an exaggerated immune response contributes to the pathogenesis of H5N1 virus infection in mammals. In particular, H5N1 virus infections are associated with a high expression of the proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α). METHODS: We investigated the compounding affects of both cytokines on the outcome of H5N1 virus disease by using triple mutant mice deficient in 3 signaling receptors, TNF-R1, TNF-R2, and IL-1-RI. RESULTS: Triple mutant mice exhibited reduced morbidity and a significant delay in mortality following lethal challenge with a lethal H5N1 virus, whereas no such differences were observed with the less virulent A/PR/8/34 (H1N1) virus. H5N1-infected triple mutant mice displayed diminished cytokine production in lung tissue and a quantifiable decrease of macrophages and neutrophils in the lungs postinfection. Moreover, morphometric analysis of airway sections revealed less extensive inflammation in H5N1-infected triple mutant mice, compared with infected wild-type mice. CONCLUSIONS: The combined signaling from the TNF or IL-1 receptors promotes maximal lung inflammation that may contribute to the severity of disease caused by H5N1 virus infection.

P58(IPK): a novel "CIHD" member of the host innate defense response against pathogenic virus infection.

To support their replication, viruses take advantage of numerous cellular factors and processes. Recent large-scale screens have identified hundreds of such factors, yet little is known about how viruses exploit any of these. Influenza virus infection post-translationally activates P58(IPK), a cellular inhibitor of the interferon-induced, dsRNA-activated eIF2alpha kinase, PKR. Here, we report that infection of P58(IPK) knockout mice with influenza virus resulted in increased lung pathology, immune cell apoptosis, PKR activation, and mortality. Analysis of lung transcriptional profiles, including those induced by the reconstructed 1918 pandemic virus, revealed increased expression of genes associated with the cell death, immune, and inflammatory responses. These experiments represent the first use of a mammalian infection model to demonstrate the role of P58(IPK) in the antiviral response. Our results suggest that P58(IPK) represents a new class of molecule, a cellular inhibitor of the host defense (CIHD), as P58(IPK) is activated during virus infection to inhibit virus-induced apoptosis and inflammation to prolong host survival, even while prolonging viral replication.

Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge.

Influenza vaccines capable of inducing cross-reactive or heterotypic immunity could be an important first line of prevention against a novel subtype virus. Influenza virus-like particles (VLPs) displaying functional viral proteins are effective vaccines against replication-competent homologous virus, but their ability to induce heterotypic immunity has not been adequately tested. To measure VLP vaccine efficacy against a known influenza pandemic virus, recombinant VLPs were generated from structural proteins of the 1918 H1N1 virus. Mucosal and traditional parenteral administrations of H1N1 VLPs were compared for the ability to protect against the reconstructed 1918 virus and a highly pathogenic avian H5N1 virus isolated from a fatal human case. Mice that received two intranasal immunizations of H1N1 VLPs were largely protected against a lethal challenge with both the 1918 virus and the H5N1 virus. In contrast, mice that received two intramuscular immunizations of 1918 VLPs were only protected against a homologous virus challenge. Mucosal vaccination of mice with 1918 VLPs induced higher levels of cross-reactive immunoglobulin G (IgG) and IgA antibodies than did parenteral vaccination. Similarly, ferrets mucosally vaccinated with 1918 VLPs completely survived a lethal challenge with the H5N1 virus, while only a 50% survival rate was observed in parenterally vaccinated animals. These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates heterotypic immunity against an influenza virus strain with threatening pandemic potential.

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice.

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.