Establishment of the Clinician-Scientist Investigator Opportunity Network to Develop Military Medical Research Leaders.

INTRODUCTION: The ability of military clinicians to conduct military medical research is often limited because of competing priorities and a lack of research mentorship. The Clinician-Scientist Investigator Opportunity Network (CSION) was developed with the intent of training clinicians how to engage in requirements-driven research within the DoD. MATERIALS AND METHODS: Three to five academic medical faculties were selected from a pool of applicants each year to participate in a 2-year research fellowship. To be eligible for the CSION program, applicants had to meet the following criteria: (1) Completed residency graduate medical education training, (2) not be currently enrolled as a graduate medical education trainee, and (3) obtained permission from their department leadership to focus 25% of their duty hours on CSION participation to include didactic and research efforts. The remaining 75% of fellows' time was dedicated to clinical duties. Monthly didactics, intensive mentorship, and consistent support were offered to each fellow by the CSION leadership team. Metrics were recorded to include both research and clinical productivity. RESULTS: Between January 2019 and December 2022, 12 CSION fellows graduated from the program (four in the class of 2020, three in 2021, and five in 2022). From 2019 to 2021, the 12 CSION fellows initiated 204 research protocols, generated 489 publications/presentations, and secured 33 research grants. All graduates of the program remain active in clinical research with multiple graduates currently assigned to research positions. CONCLUSIONS: The CSION research education program is a 2-year additional duty research fellowship producing clinician-scientists conducting military-relevant medical research and publications and may be considered a low-cost/highly efficient alternative to achieve the reported benefits of the MD-PhD tract. The expansion of the CSION program may improve the quality of military medical research and health care.

C1 Inhibitor Limits Organ Injury and Prolongs Survival in Swine Subjected to Battlefield Simulated Injury.

Complement system activation is recognized as a deleterious component of the mammalian physiological response to traumatic injury with severe hemorrhage (TH). Female Yorkshire swine were subjected to a simulated austere prehospital battlefield scenario. Each animal underwent controlled hemorrhage of 22 mL/kg at 100 mL/min rate for approximately 10 min followed by soft tissue injury, femur fracture, and spleen injury. Subsequent blood loss was uncontrolled. Twenty-eight minutes postinjury the animals were randomized into treatment or no treatment with recombinant human C1 esterase inhibitor (C1INH) (500 IU/kg, n = 11) and into receiving or not permissive hypotensive resuscitation (n = 14) with infusion of 45 mL/kg lactated Ringer's solution (2× blood lost). Observation and animal maintenance continued for 6 h at which time the animals had either expired or were euthanized. Heart, lung, and small intestine tissue samples were collected. Pharmacokinetic, hemodynamic, and metabolic parameters as well as survival time, plasma complement activity and tissue deposition, cytokine levels, and tissue injury were determined. We found that administration of C1INH protected tissues from damage, reduced the levels of inflammatory cytokines, and improved blood chemistry. Immunohistochemical analyses revealed that C1INH administration following TH markedly reduced complement activation and deposition in tissues. Importantly, C1INH administration prolonged survival of animals particularly in those which received resuscitation fluid infusion. Our data urge early administration of C1INH to limit organ damage and prolong survival of those injured in the battlefield.

Tranexamic acid and trauma: current status and knowledge gaps with recommended research priorities.

A recent large civilian randomized controlled trial on the use of tranexamic acid (TXA) for trauma reported important survival benefits. Subsequently, successful use of TXA for combat casualties in Afghanistan was also reported. As a result of these promising studies, there has been growing interest in the use of TXA for trauma. Potential adverse effects of TXA have also been reported. A US Department of Defense committee conducted a review and assessment of knowledge gaps and research requirements regarding the use of TXA for the treatment of casualties that have experienced traumatic hemorrhage. We present identified knowledge gaps and associated research priorities. We believe that important knowledge gaps exist and that a targeted, prioritized research effort will contribute to the refinement of practice guidelines over time.

Real-time RT-PCR assays for type and subtype detection of influenza A and B viruses.

Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of 'type,' i.e., influenza virus A and B, and 'subtype,' i.e., H1, H3, and H5, specific, single-step/reaction vessel format, real-time RT-PCR assays using total RNA from archived reference strains, shell-vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. 'Type' and 'subtype' specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field-deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.

Influenza A (H3N2) outbreak, Nepal.

In July 2004, an outbreak of influenza A (H3N2) was detected at 3 Bhutanese refugee camps in southeastern Nepal. Hemagglutination inhibition showed that approximately 40% of the viruses from this outbreak were antigenically distinct from the A/Wyoming/3/03 vaccine strain. Four amino acid differences were observed in most of the 26 isolates compared with the A/Wyoming/3/2003 vaccine strain. All 4 substitutions are located within or adjacent to known antibody-binding sites. Several isolates showed a lysine-to-asparagine substitution at position 145 (K145N) in the hemagglutinin molecule, which may be noteworthy since position 145 is located within a glycosylation site and adjacent to an antibody-binding site. H3N2 viruses continue to drift from the vaccine strain and may remain as the dominant strains during the 2005-2006 influenza season. Thus, the 2005-2006 Northern Hemisphere vaccine strain was changed to A/California/7/2004, a virus with all 4 amino acid substitutions observed in these Nepalese isolates.

Rapid identification of dengue virus by reverse transcription-polymerase chain reaction using field-deployable instrumentation.

Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.

Identification of Aedes aegypti and its respective life stages by real-time polymerase chain reaction.

An Aedes aegypti-specific, fluorogenic probe hydrolysis (Taq-Man), polymerase chain reaction assay was developed for real-time screening using a field-deployable thermocycler. Laboratory-based testing of A. aegypti, A. aegypti (Trinidad strain), Culex pipiens, Culex quinquefasciatus, Anopheles stephensi, and Ochlerotatus taeniorhynchus individual adult mosquitoes and mixed pools (n = 10) demonstrated 100% concordance in both in vitro sensitivity (six of six samples) and specificity (10 of 10 samples). A single adult A. aegypti was identified in a pool of 100 non-A. aegypti mosquitoes. The limit of detection of A. aegypti egg pools was five individual eggs. Field testing was conducted in central Honduras. An A. aegypti and Culex spp. panel of individual and mixed pools (n = 30) of adult mosquitoes, pupae, and larvae demonstrated 100% concordance in sensitivity (22 of 22 samples) and 97% concordance in specificity (29 of 30 samples), with one false-positive result. Field testing of an A. aegypti and Culex spp. blind panel (n = 16) consisting of individual and mixed pools of adult mosquitoes, pupae, and larvae demonstrated 90% concordance in sensitivity (nine of 10 samples) and 88% concordance in specificity (14 of 16 samples).

Potential use of microarray technology for rapid identification of central nervous system pathogens.

Outbreaks of central nervous system (CNS) diseases result in significant productivity and financial losses, threatening peace and wartime readiness capabilities. To meet this threat, rapid clinical diagnostic tools for detecting and identifying CNS pathogens are needed. Current tools and techniques cannot efficiently deal with CNS pathogen diversity; they cannot provide real-time identification of pathogen serogroups and strains, and they require days, sometimes weeks, for examination of tissue culture. Rapid and precise CNS pathogen diagnostics are needed to provide the opportunity for tailored therapeutic regimens and focused preventive efforts to decrease morbidity and mortality. Such diagnostics are available through genetic and genomic technologies, which have the potential for reducing the time required in serogroup or strain identification from 500+ hours for some viral cultures to less than 3 hours for all pathogens. In the near future, microarray diagnostics and future derivations of these technologies will change the paradigm used for outbreak investigations and will improve health care for all.

Laboratory response to anthrax bioterrorism, New York City, 2001.

In October 2001, the greater New York City Metropolitan Area was the scene of a bioterrorism attack. The scale of the public response to this attack was not foreseen and threatened to overwhelm the Bioterrorism Response Laboratory's (BTRL) ability to process and test environmental samples. In a joint effort with the Centers for Disease Control and Prevention and the cooperation of the Department of Defense, a massive effort was launched to maintain and sustain the laboratory response and return test results in a timely fashion. This effort was largely successful. The development and expansion of the facility are described, as are the special needs of a BTRL. The establishment of a Laboratory Bioterrorism Command Center and protocols for sample intake, processing, reporting, security, testing, staffing, and and quality control are also described.

Hyperbaric oxygen exposure temporarily reduces Mac-1 mediated functions of human neutrophils.

Highly elevated partial pressures of oxygen achievable during hyperbaric oxygenation (HBO) have been shown to reduce leukocyte sequestration following ischemia/reperfusion injury suggesting a clinical role for HBO in treatment of various disease states characterized by transient ischemia. Previous studies have suggested that this effect may be due to inhibition of beta2-integrin function. In this study the effect of HBO on various CD11b/CD18 (Mac-1) mediated neutrophil functions was investigated in healthy human subjects. HBO 3.0 ATA, 23 m reduced adhesion 50% at 2 h with return to pre-HBO levels by 6 h. Homotypic aggregation, a Mac-1 dependent function, under fluid shear following stimulation with f-MLP was reduced from 20+/-2.6 to 3.4+/-1.0% 2 h after HBO. However, HBO did not inhibit adhesion to IL-1beta stimulated HUVEC. Mac-1 mediated oxidative burst induced by opsonized zymosan was reduced 38.2+/-10.6% (P<0.05) by HBO. However, oxidative burst induced by PMA or f-MLP was not affected. HBO did not alter the distribution of neutrophils displaying morphologies associated with stimulation (ruffled, bipolar, uropod) over a 24 h period after HBO nor did HBO change the percentages of mature versus immature cells. Taken together these findings demonstrate that HBO specifically inhibits Mac-1 mediated functions.