Infectious Disease Clinical Research Program: Building the Bench.

The role of physicians in the U.S. Armed Forces is diverse, encompassing a wide array of skills and responsibilities to provide superior healthcare to their patients and to advance military medicine. In addition to healthcare delivery and medical education, military physicians are engaged in public health, operational medicine, and cutting-edge medical research. Thus, clinical research is a crucial component of Graduate Medical Education (GME) and supports critical thinking (knowledge, skills, and abilities) and the development of leadership skills among U.S. military physicians. The Infectious Disease Clinical Research Program (IDCRP) education mission was established in 2005 with the overall goal of supporting the development and training of the next generation of clinical researchers in infectious diseases and related public health disciplines in the Armed Forces using several strategies, including didactic learning, mentored research, and research engagement. Through involvement in the IDCRP, infectious disease fellows, residents (e.g., surgical, internal medicine, and pediatrics), and Master of Public Health (MPH) students have continued their education and gained valuable skills related to clinical research. Trainees either conduct research with IDCRP mentors or participate in IDCRP-led practicum experiences, with research projects ranging from epidemiologic studies to microbiological assessments. Consistent with the needs of the Military Health System (MHS), and in accordance with Accreditation Council for Graduate Medical Education goals, the IDCRP provides opportunities for medical and graduate students, residents, and infectious disease fellows to conduct mentored research within the MHS, as well as gain important leadership skills in the conduct of clinical research. Overall, IDCRP continues to further infectious disease research through the support and education of the next generation of active-duty infectious disease researchers in the MHS.

The Acute Respiratory Infection Consortium: A Multi-Site, Multi-Disciplinary Clinical Research Network in the Department of Defense.

INTRODUCTION: Acute respiratory infections (ARI) result in substantial annual morbidity among military personnel and decrease operational readiness. Herein, we summarize the research efforts of the Infectious Disease Clinical Research Program (IDCRP) related to ARIs. METHODS: The ARI Research Area of the IDCRP was established in response to the 2009 emergence of pandemic influenza A/H1N1. That year, IDCRP investigators deployed the ARI Consortium Natural History Study (ARIC NHS), a multi-centered, longitudinal observational study to assess etiology, epidemiology, and clinical characteristics of influenza-like illness (ILI) and severe acute respiratory infections (SARI) in the U.S. military. The success of this initial effort spurred implementation of several new initiatives. These include the FluPlasma trial, designed to evaluate the efficacy of hyperimmune anti-influenza plasma for the treatment of severe influenza; the self-administered live-attenuated influenza vaccine (SNIF) trial, which assessed the immunogenicity and acceptance of a self-administered live-attenuated influenza vaccine in military personnel; the Study to Address Threats of ARI in Congregate Military Populations (ATARI), a prospective study of ILI transmission, etiology and epidemiology in recruits; and the Flu Breath Test (FBT) study, a preliminary study of exhaled volatile organic compounds (VOC) in influenza patients. In addition, the InFLUenza Patient-Reported Outcome (FLU-PRO) survey, a daily diary to measure influenza symptoms during clinical trials, was developed. Lastly, the Pragmatic Assessment of Influenza Vaccine Effectiveness in the DoD (PAIVED) study, a two-year randomized trial designed to compare the effectiveness of the three types of licensed vaccines, launched in Fall 2018. RESULTS: The on-going ARIC NHS has enrolled over 2000 ILI and SARI cases since its inception, providing data on burden and clinical manifestations of ARI in military personnel and their families. The FluPlasma 2 trial concluded subject enrollment in 2018. Preliminary results from ATARI study show a high frequency of respiratory viruses circulating during the first two weeks of recruit training. Based on assessment of FLU-PRO responses, which were found to be reliable and reproducible, the survey may be a useful tool in clinical trials and epidemiological studies. The Flu Breath Study will complete enrollment in 2019. Findings from PAIVED are intended to provide evidence needed for assessing influenza vaccination policy in the military. CONCLUSIONS: The ARI burden in the armed services remains significant every year and the threat is dynamic given emergent and evolving threats, such as influenzas. With strong successes to date, future initiatives of the ARI Research Area will focus on interventional studies, ARI transmission dynamics in congregate military settings, and determinants of risk of pandemic influenza and other emergent respiratory viruses.

Antibody contributes to heterosubtypic protection against influenza A-induced tachypnea in cotton rats.

BACKGROUND: Influenza virus infection or vaccination evokes an antibody response to viral hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, which results in immunity against influenza A viruses of the same HA and NA subtype. A heterosubtypic immune response that offers some protection against different influenza A subtypes has been suggested from epidemiologic studies in human influenza outbreaks, and has been induced in experimental animal models. Original studies of such cross-protection showed that cytotoxic T lymphocytes (CTL) protect H3N2-immune mice from a lethal H1N1 infection. More recent studies in mice demonstrate that antibodies also contribute to heterosubtypic immunity (HSI). We previously demonstrated that HSI in cotton rats (Sigmodon hispidus) is characterized by protection of H3N2-immune animals from influenza H1N1-induced increase in respiratory rate (tachypnea). Alternatively, H1N1-immune animals are protected from H3N2-induced tachypnea. The experiments described in this report were designed to elucidate the immune mechanism that prevents this very early sign of disease. RESULTS: Our results show that cotton rats provided with H1N1-immune serum prior to challenge with an H3N2 virus were protected from influenza-associated tachypnea, with the degree of protection correlating with the antibody titer transferred. Immunization with an inactivated preparation of virus delivered intramuscularly also provided some protection suggesting that CTL and/or mucosal antibody responses are not required for protection. Antibodies specific for conserved epitopes present on the virus exterior are likely to facilitate this protection since prophylactic treatment of cotton rats with anti-M2e (the extracellular domain of M2) but not anti-nucleoprotein (NP) reduced virus-induced tachypnea. CONCLUSION: In the cotton rat model of heterosubtypic immunity, humoral immunity plays a role in protecting animals from influenza-induced tachypea. Partial protection against respiratory disease caused by different influenza A subtypes can be attained with either live virus administered intranasally or inactivated virus delivered intramuscularly suggesting that either vaccine regimen may provide some protection against potential pandemic outbreaks in humans.

History of U.S. military contributions to the study of respiratory infections.

History reveals a tremendous impact of respiratory pathogens on the U.S. military, dating back to the time of the Revolutionary and Civil Wars, during which 90% of casualties were for nonbattle injury, including several respiratory illnesses such as measles, whooping cough, and complicated pneumonia. The devastating impact of the influenza pandemic at the end of World War I led to a more proactive approach to research into the etiologies and potential preventive measures for such diseases. The development of the Armed Forces Epidemiological Board, with its subordinate commissions, coincided with the massive mobilization for World War II. Efforts of the board during and after the war led to significant progress against many common pathogens, such as the landmark studies of group A Streptococcus among young trainees at Warren Air Force Base, which led to the development of highly effective prophylactic and therapeutic strategies to prevent rheumatic fever. Military pediatricians contributed greatly to this work, as well as subsequent investigations into both the pathogenesis of and prophylactic therapy for a variety respiratory pathogens, including pertussis and respiratory syncytial virus. The momentum of this work continues to this day, among researchers from all three military branches.

WITHDRAWN: Abstract.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.cppeds.2015.06.007. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Utility of complete blood count and blood culture screening to diagnose neonatal sepsis in the asymptomatic at risk newborn.

BACKGROUND: In May 1996, the CDC recommended obtaining a complete blood count and blood culture (BC) from all asymptomatic "at risk" newborns; those > or =35 weeks gestation born to mothers with group B streptococcal vaginal colonization or those with maternal fever, premature rupture of membranes or previous infant with group B streptococcal disease; who did not receive adequate intrapartum antibiotic prophylaxis. DESIGN/METHODS: During the study period (May 1996 to July 1999), a complete blood count and BC were obtained within 4 h from all asymptomatic at risk newborns of > or =35 weeks gestation. White blood cell count (WBC) and BC results and prevalence of clinical or culture-proven sepsis were obtained by chart review. We determined the sensitivity/specificity and likelihood ratios of an abnormal WBC (total >30 000 or <5000/mm3; absolute neutrophil count <1500/mm3, or a band form-polymorphonuclear cell ratio of >0.2) to distinguish between clinically septic vs. nonseptic term at risk newborns. RESULTS: Of 20 554 deliveries 1665 were initially asymptomatic at risk newborns; 17 (1.0%) developed early onset sepsis, all within 48 h. WBC was abnormal in 7 of 17 (41%) and in 447 of 1648 (27%) who remained nonseptic. None of the 1665 term at risk newborns had a positive BC. The sensitivity and specificity of an abnormal WBC in predicting which at risk newborns would develop sepsis were 41 and 73%, respectively. The positive likelihood ratio was 1.52, whereas the negative likelihood ratio was 0.81, with an odds ratio of 1.88. CONCLUSIONS: Since the implementation of the CDC guidelines for maternal intrapartum antibiotic prophylaxis, culture-proved sepsis has become rare at our institution. Although BC did not aid in the diagnosis of sepsis among asymptomatic at risk newborns, close observation in the first 24 h remained critically important.

Age-dependent replication of respiratory syncytial virus in the cotton rat.

Despite the documented disease burden of respiratory syncytial virus (RSV) in the elderly, little is known about the underlying risk factors or pathogenesis of RSV in a geriatric population. This report describes an age-dependent change of RSV clearance in the lung and nose of the cotton rat. Six days postinfection with RSV, lung and nose viral titers were significantly higher in all older age groups as compared with 4- to 6-week old cotton rats (P < 0.05). When comparing the 4- to 6-week old animals to the 15- to 16-month old animals 6 days postinfection, there was over an 800- and 100-fold increase in lung and nose viral titers, respectively. The cotton rat may prove to be a useful model in eliciting mechanisms of severe RSV disease in the elderly.

Co-infection of the cotton rat (Sigmodon hispidus) with Staphylococcus aureus and influenza A virus results in synergistic disease.

Bacterial super-infection of influenza patients is the primary cause of excess mortality during influenza pandemics, with Staphylococcus aureus (S. aureus) having the highest fatality rate. The cotton rat (Sigmodon hispidus) is an excellent model for both influenza and S. aureus pathogenesis, and therefore a potential tool to model co-infection. We compared physiologic and pathologic changes in cotton rats infected with both S. aureus and influenza A/Wuhan/359/95 (H3N2), with animals infected with each pathogen alone. Co-infected cotton rats demonstrated significantly higher mortality, lower temperatures on 2 and 3 days post-inoculation (p.i.), higher levels of bacteremia and pulmonary bacterial load 4 days p.i., and worse pathology 7 days p.i. Early indicators of exacerbated disease coincided with higher pulmonary mRNA levels for IL-1beta, IL-6, IL-10 and IFNy, supporting the idea that these may contribute to disease severity. Our results demonstrate that the cotton rat is a good model of influenza and S. aureus co-infection, with increased mortality and hypothermia as well as prolonged bacterial duration indicative of synergistic disease that may be the result of increased induction of both pro- and anti-inflammatory cytokines.

Evidence of a cross-protective immune response to influenza A in the cotton rat model.

Epidemiologic evidence suggests that cross-protective immune responses to influenza A viruses that have different hemagglutinin and neuraminidase subtypes occur in humans. This study characterized this heterosubtypic immunity in cotton rats (Sigmodon hispidus). Animals were infected with influenza A/PR/8/34 (H1N1) or A/Wuhan/359/95 (H3N2), and then challenged with A/Wuhan/359/95(H3N2) virus 4 weeks later. Viral titers, respiratory rates, and pathology of the respiratory tract following primary and secondary infection were compared. Cross-protection from heterosubtypic influenza A challenge in cotton rats was characterized by enhanced viral clearance, protection from tachypnea, a vigorous early cellular recall response, and a reduction in bronchiolar epithelial cell damage. Cross-protection was retained in steroid treated animals, in which the inflammatory recall response was minimal. Identification of the mechanisms that contribute to cross-protection in cotton rats may lead to the development of influenza vaccine strategies that are broadly protective.

Prospects of antiviral and anti-inflammatory therapy for respiratory syncytial virus infection.

Respiratory syncytial virus is the leading viral cause of death in children less than 2 years of age, and is an increasing cause of morbidity and mortality in transplant patients and the elderly. Respiratory syncytial virus causes upper and lower respiratory tract infections, which can lead to severe bronchiolitis and pneumonia. High-risk groups for severe respiratory syncytial virus infection include infants with a history of premature birth with or without chronic lung disease, children with congenital heart disease, children with cystic fibrosis or chronic lung diseases, and immunosuppressed patients or patients with immunodeficiency. However, the majority of infants who have severe respiratory syncytial virus disease are born at full term and are otherwise healthy. It is unclear why children, the elderly and the immunosuppressed are at much higher risk for severe disease; however, a respiratory syncytial virus-induced immune pathologic mechanism has long been suspected. Attempts to develop a safe and effective vaccine against respiratory syncytial virus have failed. Antirespiratory syncytial virus immunotherapy, although effective prophylactically, does not provide any beneficial clinical outcome when administered therapeutically, indicating that respiratory syncytial virus-induced pathology is most likely the result of the inflammatory response to infection, rather than a direct viral cytopathic effect. Thus, a combined antiviral and anti-inflammatory therapy may represent the safest and most efficient treatment for acute respiratory syncytial virus infection. In this review, the current knowledge that has set the rationale for the development of such therapy is summarized.

The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis.

Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral 'adaptation'. It was previously demonstrated that animals infected intranasally with 10(7) TCID50 of a recent H3N2 influenza, A/Wuhan/359/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day 3. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly 3 weeks. Interleukin 1 alpha (IL1alpha), alpha interferon (IFN-alpha), IL6, tumour necrosis factor alpha (TNF-alpha), GROalpha and MIP-1beta mRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-gamma, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.

Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy.

Cyclooxygenases (COXs) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. COX-2 is the inducible isoform that is up-regulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. The roles of cyclooxygenases and their products, PGs, have not been evaluated during respiratory syncytial virus (RSV) infection. In this study we demonstrate that COX-2 is induced by RSV infection of human lung alveolar epithelial cells with the concomitant production of PGs. COX-2 induction was dependent on the dose of virus and the time postinfection. PG production was inhibited preferentially by NS-398, a COX-2-specific inhibitor, and indomethacin, a pan-COX inhibitor, but not by SC-560, a COX-1-specific inhibitor. In vivo, COX-2 mRNA expression and protein production were strongly induced in the lungs and cells derived from bronchioalveolar lavage of cotton rats infected with RSV. The pattern of COX-2 expression in vivo in lungs is cyclical, with a final peak on day 5 that correlates with maximal histopathology. Treatment of cotton rats with indomethacin significantly mitigated lung histopathology produced by RSV. The studies described in this study provide the first evidence that COX-2 is a potential therapeutic target in RSV-induced disease.

Influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor.

Influenza viruses are one of the leading causes of morbidity and mortality during winter months. Increased respiratory rate (tachypnea) is a sign of increasing lower respiratory disease during influenza infection and is frequently observed in hospitalized patients. We investigated this clinical sign in influenza virus-infected cotton rats (Sigmodon hispidus) and the efficacy of antiviral and anti-inflammatory therapy in reducing symptomatic disease. Cotton rats infected intranasally with A/Wuhan/359/95 (H3N2) had increased respiratory rates from 1 to 4 days postinfection that correlated with the dose of virus used to inoculate the animal but not the amount of virus recovered from the lung. In addition, evaluation of sequential lung tissue pathology revealed that extensive epithelial cell destruction of small airways correlated with tachypnea. Increased respiratory rate was not observed in immune animals, supporting results that demonstrated a requirement for exposure to, and infection by, large amounts of live virus for induction of tachypnea. A variety of therapeutic approaches proved ineffective in reducing tachypnea, including anti-inflammatory therapy with systemic triamcinolone acetonide, bronchodilatory therapy with levalbuterol, or antiviral therapy with zanamivir. These results, together with the pathologic observations, suggest that early disruption of the lower respiratory tract epithelium is a major component of the pathophysiology of influenza infection. Therapeutic approaches need to be tailored to clear airway obstruction and restore an intact epithelium.