Perspective on Improving Environmental Monitoring of Biothreats.

For more than a decade, the United States has performed environmental monitoring by collecting and analyzing air samples for a handful of biological threat agents (BTAs) in order to detect a possible biological attack. This effort has faced numerous technical challenges including timeliness, sampling efficiency, sensitivity, specificity, and robustness. The cost of city-wide environmental monitoring using conventional technology has also been a challenge. A large group of scientists with expertise in bioterrorism defense met to assess the objectives and current efficacy of environmental monitoring and to identify operational and technological changes that could enhance its efficacy and cost-effectiveness, thus enhancing its value. The highest priority operational change that was identified was to abandon the current concept of city-wide environmental monitoring because the operational costs were too high and its value was compromised by low detection sensitivity and other environmental factors. Instead, it was suggested that the focus should primarily be on indoor monitoring and secondarily on special-event monitoring because objectives are tractable and these operational settings are aligned with likelihood and risk assessments. The highest priority technological change identified was the development of a reagent-less, real-time sensor that can identify a potential airborne release and trigger secondary tests of greater sensitivity and specificity for occasional samples of interest. This technological change could be transformative with the potential to greatly reduce operational costs and thereby create the opportunity to expand the scope and effectiveness of environmental monitoring.

Enhancing the Department of Defense's Capability to Identify Environmental Exposures Into the 21st Century.

Military service members come in contact with a wide range of hazardous substances especially during deployment. The identification of service member's with potential exposures to infectious diseases and environmental toxic substances has been a problem for the U.S. military almost since the formation of the services and continues to be an issue today. In June and July of 2013, the Armed Forces Health Surveillance Center sponsored two meetings to address the need by the Department of Defense to perform retrospective exposure analysis that would support military force health protection efforts. The first meeting included medical professionals who were familiar with health problems that followed potential environmental or infectious disease exposures but that the military health system was unprepared to address. The second group was composed of technical experts who were asked to suggest potential material and nonmaterial solutions to address the needs of the military public health community. This supplement to Military Medicine includes the outcome of these two meetings, descriptions of some of the Department of Defense biorepositories including the large serum repository housed at Armed Forces Health Surveillance Center and discussion of additional topics related to the establishment and use of biorepositories that would support public health practice in the 21st century.

Building a DoD Biorepository for the Future: Potential Benefits and Way Forward.

Significant advances have been made in the molecular analyses of the human physiological state. In general, these techniques have been termed "omics" because of their requirements for sophisticated analyses of large datasets. Application of these new omics technologies has led to advances in medical practice related to public health as well as a new field termed personalized medicine. The Department of Defense (DoD) consistently needs the ability to identify people who have been exposed to environmental hazards during deployments and in their day-to-day jobs. The department currently has a biorepository of sera collected from military service members and has used that repository to study potential environmental exposures (toxins and infectious diseases) since 1987. The DoD Serum Repository is also linked to service member health records, making it a very powerful tool for studies related to force health protection and public health practice. However, this repository does not contain a reliable source of nucleic acid. Accordingly, to take advantage of modern molecular omics technologies, the DoD should establish an enhanced biospecimen repository that can support future questions related to force health protection. This article briefly discusses the various omics techniques, and how they can be used for analyses to support medical practice and public health.

Description and utilization of the United States department of defense serum repository: a review of published studies, 1985-2012.

Specimens in the United States Department of Defense (DoD) Serum Repository have accumulated in frozen storage since 1985 when the DoD began universal screening for human immunodeficiency virus. Use of the stored serum for health research has been carefully controlled, but the resulting publications have never been systematically identified or described. The Armed Forces Health Surveillance Center (AFHSC) information systems and open (online) sites were used as data sources. Through 2012, the repository contained 54,542,658 serum specimens, of which 228,610 (0.42%) have been accessed for any purpose. Between 2001 (the first year that comprehensive, digital records were available) and 2012, 65.2% of all approved requests for serum were for healthcare or public health investigations, but greater than 99% of all shipped samples were for research. Using two different methods - a structure search of PubMed and an exhaustive online search based on records from AFHSC - we identified 76 articles published between October 1988 and March 2013 that covered a multitude of infectious diseases, injuries, environmental exposures and mental health conditions through analysis of antibodies, biological metabolic, signaling and regulatory substances, Vitamin D, organochlorines, dioxin, omega-3-fatty acid, and portions of human deoxyribonucleic acid. Despite its operational and scientific value, it appears that the DoD Serum Repository has been underutilized. Changes to policy and increased capacity for specimen processing could increase use of the repository without risking privacy or the availability of specimens for the healthcare of individual service members in the future.

Amount of usage and involvement in explosions not associated with increased contamination of prehospital vehicles with multi-drug-resistant organisms.

INTRODUCTION: The role of explosions and patient transport vehicles as sources and vectors of Gram-negative, multidrug-resistant organisms (MDROs) that predominate infections following lengthy evacuations after disasters due to natural hazards and in current war-trauma patients is unknown. HYPOTHESIS/PROBLEM: Damaged or heavily-used vehicles could be sources of the MDROs subsequently linked to nosocomial infections. METHODS: From January through May 2008 in Iraq, inside surfaces of heavily-used, tactical vehicles (Experimental Group) were sampled with sterile, pre-moistened swabs. Swabs, along with positive and negative controls, were shipped to the reference laboratory in Washington, DC, where they underwent culture, identification and susceptibility testing, and pulsed-field gel electrophoresis. Multidrug-resistant organisms were defined according to the standard Centers for Disease Control and Prevention definitions. High risk organisms (HROs) were defined as susceptible E. coli, A. baumannii, P. aeruginosa, Enterobacter spp, or Klebsiella spp. Concurrently, new counterparts (Control Group) were similarly surveyed in a storage lot in Georgia, USA. Groups were compared using the Chi-squared test. RESULTS: One hundred thirty-nine consecutive vehicles including all available ambulances were sampled, yielding 153 swabs. Nineteen were lost or damaged during shipping. Seventy-nine swabs yielded growth of one or more Gram-negative bacteria. The amount and genotype of MDROs in heavily-used vehicles, including those involved in roadside bombings, were compared to control vehicles and to strains isolated from wounds and environmental surfaces at the base hospital. Predominant organisms included P. agglomerans (34%), S. flexneri (8%), E. vulneris (6%), Pseudomonas sp. (6%), and K. pneumonia (6%). No MDROs were isolated. Thirteen vehicles (eight of 94 experimental and five of 45 control) yielded HRO. There was no difference in contamination rates (P = .63). No HROs were isolated from ambulances. No clonal association existed between vehicle and hospital strains. CONCLUSION: Given the implications that this knowledge gap has on military and civilian prehospital reservoirs of infection, further study is warranted to confirm these findings and identify targets for preventive intervention throughout civilian disaster and military casualty evacuation chains.

Influenza and wound infections: laboratory support for deployed U.S. forces.

Since 1997, the absence of a global, DoD public health laboratory system has been identified as a vulnerability in the U.S. military's effort to identify and quickly respond to emerging infections. The AFHSC Division of GEIS Operations has attempted to mitigate this vulnerability by supporting initiatives such as the DoD Global Influenza Surveillance Program and the DoD Directory of Public Health Laboratory Services. AFHSC continues to be engaged in identifying and addressing diagnostics needed to protect deployed forces. The GASI and the enhanced capability for identification of MDROs and threatening influenza strains in deployed areas are recent examples of GEIS utilizing its financial resources and position as a DoD organization to coordinate the efforts of the military services and other U.S. government organizations to improve preparedness for EID agents. However, the absence of a defined, comprehensive public health system that contains surveillance systems, reference laboratories, and public health communication systems functioning in unison to provide reach back and reference laboratory support to the global MHS remains a significant gap.

Serosurvey of bacterial and viral respiratory pathogens among deployed U.S. service members.

BACKGROUND: Respiratory illnesses can cause substantial morbidity during military deployments. Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, adenovirus, parainfluenza, and respiratory syncytial virus (RSV) are hypothesized causes. PURPOSE: To determine pathogen-specific seroprevalence prior to and after deployment in support of Operation Enduring Freedom (OEF). METHODS: A retrospective cohort study of 1000 service members deployed between June 30, 2004, and June 30, 2007, was conducted from 2008 through 2009. Pre- and post-deployment sera were tested for the presence of antibody to each pathogen. RESULTS: Pre-deployment IgG seropositivity was high for adenovirus, RSV, and parainfluenza (98.7%, 97.8%, and 81.6%, respectively), whereas seropositivity for B. pertussis, M. pneumoniae, and C. pneumoniae was 14.2%, 21.9%, and 65.1%, respectively. As defined by seroconversion in 1000 subjects, the following were identified: 43 new parainfluenza infections (24% of susceptibles); 37 new pertussis infections (4% of susceptibles); 33 new C. pneumoniae infections (10% of susceptibles); and 29 new M. pneumoniae infections (4% of susceptibles). B. pertussis seroconversion was two to four times higher than reports for the general U.S. population. Overall, 14.2% of the service members seroconverted to at least one of these six pathogens; this increased to 30.1% seroconversion when influenza was included. However, serologic testing was not clearly associated with clinical illness in this report. CONCLUSIONS: Serologic evidence for respiratory infections was common among the 2004-2007 OEF-deployed military, sometimes at a higher rate than the general U.S. population. Awareness of this risk and implementation of preventive measures should be emphasized by leadership prior to and during deployment.

Antimicrobial resistance surveillance in the AFHSC-GEIS network.

International infectious disease surveillance has been conducted by the United States (U.S.) Department of Defense (DoD) for many years and has been consolidated within the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) since 1998. This includes activities that monitor the presence of antimicrobial resistance among pathogens. AFHSC-GEIS partners work within DoD military treatment facilities and collaborate with host-nation civilian and military clinics, hospitals and university systems. The goals of these activities are to foster military force health protection and medical diplomacy. Surveillance activities include both community-acquired and health care-associated infections and have promoted the development of surveillance networks, centers of excellence and referral laboratories. Information technology applications have been utilized increasingly to aid in DoD-wide global surveillance for diseases significant to force health protection and global public health. This section documents the accomplishments and activities of the network through AFHSC-GEIS partners in 2009.

Hepatitis E seroprevalence and seroconversion among US military service members deployed to Afghanistan.

BACKGROUND: Hepatitis E virus (HEV) has been recognized as a threat to military forces since its discovery. Although HEV seroprevalence in Afghanistan is not known, HEV infection is thought to be highly endemic in that country. This study determined the incidence of HEV seroconversion among United States (US) service members who were deployed to Afghanistan, as well as the prevalence of antibodies to HEV prior to the deployment. METHODS: A random sample of 1500 subjects was selected from the cohort of service members who were deployed to Afghanistan between 2002 and 2006. Predeployment and postdeployment serum samples from these subjects were tested by enzyme immunoassay for total antibodies to HEV. Results. The seroprevalence of antibodies to HEV in US service members prior to deployment was 1.1%. The seroconversion rate among service members deployed to Afghanistan was 0.13%. CONCLUSIONS: Although subpopulations may be at higher risk for HEV exposure during deployment, the risk among US service members deployed to Afghanistan in this study was low. Previously implemented and current preventive measures in theater appear to have been adequate. With future deployments to new areas or changes in military operations in areas of risk, continued surveillance for HEV infection in the military will be warranted.

Influenza and respiratory disease surveillance: the US military's global laboratory-based network.

The US Department of Defense influenza surveillance system now spans nearly 500 sites in 75 countries, including active duty US military and dependent populations as well as host-country civilian and military personnel. This system represents a major part of the US Government's contributions to the World Health Organization's Global Influenza Surveillance Network and addresses Presidential Directive NSTC-7 to expand global surveillance, training, research and response to emerging infectious disease threats. Since 2006, the system has expanded significantly in response to rising pandemic influenza concerns. The expanded system has played a critical role in the detection and monitoring of ongoing H5N1 outbreaks worldwide as well as in the initial detection of, and response to, the current (H1N1) 2009 influenza pandemic. This article describes the system, details its contributions and the critical gaps that it is filling, and discusses future plans.

Attenuation of defined Brucella melitensis wboA mutants.

Rough Brucella mutants have been sought as vaccine candidates because they do not induce seroconversion. In this study, two defined nonreverting rough mutants were derived from virulent Brucella melitensis strain 16M: a wboA deletion mutant designated WRR51 and a wboA purEK dual deletion mutant designated WRRP1. Strain WRRP1 exhibited reduced survival in human monocyte-derived macrophages (hMDMs) compared with parent strain WRR51 or with ΔpurEK strain WR201. Strain WRRP1 persisted for 1 week or less in BALB/c mice after intraperitoneal infection, while less severe attenuation was exhibited by the two single mutants in this model. Trans complementation of wboA restored the survival of WRR51 in hMDMs comparable to strain 16M and the survival of WRRP1 comparable to strain WR201.

Genome sequence of the deep-rooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium.

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

Survey of sexually transmitted disease laboratory methods in US Army laboratories.

BACKGROUND: Sexually transmitted diseases, in particular Chlamydia trachomatis and Neisseria gonorrhoeae, are ranked as the top 2 most commonly notified disease in the US Army. Although surveillance programs are in place to capture event data, no routine STD surveillance program captures laboratory test information. METHODS: To evaluate laboratory testing practices/methodologies in US Army laboratories in 2007, a questionnaire was distributed to all 38 US Army laboratories. The results of the survey were compared across Army installations to US civilian public health laboratories. RESULTS: Of 38 survey recipients, 35 (92.1%) completed the survey. Overall, 78.6% of C. trachomatis and 77.2% of N. gonorrhoeae specimens were tested by nucleic acid amplification tests. In addition, 48.6% used culture as a method of N. gonorrhoeae testing. Testing for genital herpes, trichomonas, bacterial vaginosis, syphilis, human papillomavirus, and/or premalignant/malignant cervical cells was performed by 33 of the 35 laboratories. CONCLUSIONS: A high proportion of US Army laboratories are using NAAT technology for C. trachomatis and N. gonorrhoeae testing. A more comprehensive questionnaire may be needed to accurately describe the type and volume of other STD tests. Despite the difference in survey data acquisition between the US civilian public health laboratory survey and the US Army laboratory survey, broad comparisons such as test types were able to be made. Future surveys should be extended to other US military services and should include both civilian and military laboratories.

A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes.

Francisella tularensis Schu S4, LVS and U112 have become model organisms for the study of Francisella pathogenesis, and represent a cross section of the different F. tularensis subspecies. Both Schu S4 and LVS are fastidious organisms, requiring medium fortified with supplements and nutrients for enhanced growth. Chamberlains defined medium, Tryptone Soy Broth supplemented with cysteine (TSBc), and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2% IsoVitaleX are typically used in the cultivation of these bacteria. In this report, we describe a simple brain heart infusion broth formulation that can be used to obtain superior growth characteristics in all of these model organisms, and can support bacterial growth from low inoculum. Surprisingly, CAMHB, which is favored in the literature for culturing Schu S4 and LVS, induced the worst growth characteristics of the four formulations studied. To expand on these observations, an additional seven strains of F. tularensis, representing types A.I, A.II, and B were selected from the Department of Defense United Culture Collection (UCC) and a comparative analysis of their growth characteristics performed in the four broth formulations. Results demonstrate differences in the growth characteristics of Francisella species that are significantly influenced by both strain type and the choice of growth medium. Though four of the five additional Type A strains displayed superior growth characteristics in Chamberlain's defined medium, growth characteristics of all three model organisms, as well the Type B strains, were enhanced by the new BHI-based broth formulation. We conclude that this medium represents the optimal choice for cultivation of the three model organisms used for Francisella research.

Draft genome sequences of Yersinia pestis isolates from natural foci of endemic plague in China.

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague.

Typing methods for the plague pathogen, Yersinia pestis.

Phenotypic and genotypic methodologies have been used to differentiate the etiological agent of plague, Yersinia pestis. Historically, phenotypic methods were used to place isolates into one of three biovars based on nitrate reduction and glycerol fermentation. Classification of Y. pestis into genetic subtypes is problematic due to the relative monomorphic nature of the pathogen. Resolution into groups is dependent on the number and types of loci used in the analysis. The last 5-10 years of research and analysis in the field of Y. pestis genotyping have resulted in a recognition by Western scientists that two basic types of Y. pestis exist. One type, considered to be classic strains that are able to cause human plague transmitted by the normal flea vector, is termed epidemic strains. The other type does not typically cause human infections by normal routes of infection, but is virulent for rodents and is termed endemic strains. Previous classification schemes used outside the Western hemisphere referred to these latter strains as Pestoides varieties of Y. pestis. Recent molecular analysis has definitely shown that both endemic and epidemic strains arose independently from a common Yersinia pseudotuberculosis ancestor. Currently, 11 major groups of Y. pestis are defined globally.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells.

BACKGROUND: Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. METHODS: To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. RESULTS: We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. CONCLUSION: Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.

Pestoides F, an atypical Yersinia pestis strain from the former Soviet Union.

Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lack of the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout a comprehensive genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla(-) strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains reveals differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single approximately 7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet sequenced.