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Background: The circulation of human adenovirus type 21 (HAdV21) in the United States has been documented since the 1960s in association with outbreaks of febrile respiratory illness (FRI) in military boot camps and civilian cases of respiratory disease., Methods: To describe the molecular epidemiology of HAdV21 respiratory infections across the country, 150 clinical respiratory isolates obtained from continuous surveillance of military recruit FRI, and 23 respiratory isolates recovered from pediatric and adult civilian cases of acute respiratory infection were characterized to compile molecular typing data spanning 37 years (1978-2014)., Results: Restriction enzyme analysis and genomic sequencing identified 2 clusters of closely related genomic variants readily distinguishable from the prototype and designated 21a-like and 21b-like. A-like variants predominated until 1999. A shift to b-like variants was noticeable by 2007 after a 7-year period (2000-2006) of cocirculation of the 2 genome types. US strains are phylogenetically more closely related to European and Asian strains isolated over the last 4 decades than to the Saudi Arabian prototype strain AV-1645 isolated in 1956., Conclusions: Knowledge of circulating HAdV21 variants and their epidemic behavior will be of significant value to local and global FRI surveillance efforts., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense's (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization's (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

Two cases of febrile respiratory illness associated with untypeable influenza A virus were identified in Southern California in March 2009. One was initially detected as influenza virus using an experimental diagnostic device in a clinical trial, while the other was detected at a local reference lab using a diagnostic PCR assay. In both cases, analyses yielded negative results for strain-specific tests targeting circulating strains of influenza A virus (seasonal H1 and H3). These two samples became the first reported cases of the pandemic 2009/H1N1 influenza virus. The first reportable characterization was made from the second collected specimen on 15 April 2009 at the Centers for Disease Control and Prevention central lab using traditional culture and sequencing methods. The novel nature of the strain and its apparent zoonotic origins were initially characterized using the first collected specimen at the Naval Health Research Center in San Diego, CA, on 13 April using an experimental molecular analysis tool, PCR electro-spray ionization-mass spectrometry (PCR/ESI-MS), designed to amplify PCR products from any strain of influenza virus and to generate informative (phylogenetic) strain identifications through mass spectrometry of PCR amplicons. The ability of this high-throughput tool to correctly identify both well-characterized and novel influenza strains offers the possibility to integrate surveillance for emerging strains with on-site rapid diagnosis used for patient management, shortening the times between the emergence of new strains, their detection and identification, and appropriate public health response activities. Here we describe the initial characterization of the pandemic 2009/H1N1 influenza strain and discuss the possible roles of diagnostic tools with discovery potential.

Background: First isolated in the Netherlands in 1955 during an outbreak of acute respiratory disease (ARD) among military recruits, human adenovirus 14 (HAdV-14) has historically been considered rare. With no precedent of circulation in North America, HAdV-14 has been isolated from military and civilian cases of ARD of variable severity since 2003 in the United States., Methods: Ninety-nine isolates from military and civilian cases from different geographic locations and circulation periods were characterized by restriction enzyme analysis of viral DNA and select gene sequencing., Results: All examined viruses were found to be identical and to belong to a new genome type designated "HAdV-14p1" (formerly known as "14a"). Comparative alignments of E1A, hexon, and fiber gene sequences with other subspecies B2 HAdVs suggest that HAdV-14p1, like the closely related HAdV-11a, arose from recombination among similar HAdV-11 and HAdV-14 ancestral strains. A deletion of 2 amino acids in the knob region of the fiber protein is the only identified unique characteristic of HAdV-14p1., Conclusion: The current geographic distribution of HAdV-14p1 involves at least 15 states in the Unites States. The role of the fiber mutations in the recent emergence of HAdV-14p1 ARD in North America warrants further study.

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.

Background: Although several studies have shown a positive association between evidence of anti-adenovirus 36 (Ad-36) antibodies (Ad-36 exposure) and (1) obesity and (2) serum cholesterol in animals, there is limited research demonstrating this association in humans. There is also limited research on transmission, presentation and demographics of Ad-36 infection., Design: (1) Body mass (body mass index (BMI)), (2) fasting serum cholesterol and triglyceride levels and (3) demographic characteristics were compared between Ad-36 seropositive and seronegative groups. The majority of subjects were matched as cases versus controls on a number of demographic variables., Subjects: A total of 150 obese and 150 lean active-duty military personnel were studied., Measurements: Subjects completed a questionnaire regarding demographic and behavioral characteristics. Subject serum samples were tested by serum neutralization assay for the presence of anti-Ad-36 antibodies., Results: In all, 34% of obese and 39% of lean subjects had Ad-36 exposure, an insignificant difference. Serum cholesterol and triglyceride levels were significantly higher among the obese subjects than among the lean, but there were no associations between serum cholesterol and triglyceride levels and Ad-36 exposure. Positive associations were found between Ad-36 exposure and age, race and gender., Conclusion: The study stands in contrast to previous work that has shown a positive relationship between Ad-36 exposure and (1) obesity, and (2) levels of serum cholesterol and triglycerides. In this study there was no association in either case. Unanticipated relationships between Ad-36 exposure and age, race and gender were found, and this is the first time that such a link between Ad-36 exposure and demographics has been found.

We identified 179 cases of acute respiratory illness including 50 cases of radiographically confirmed pneumonia over the course of 4 months on a deployed U.S. Navy vessel. Laboratory tests showed Mycoplasma pneumoniae to be the etiological agent. This report represents the first published description of a shipboard outbreak of this pathogen.

This study reveals diverse-length polymorphisms in long mononucleotide repeats (microsatellites) in several serotypes of epidemic human respiratory adenovirus. The length of one of these microsatellites, a homopolymeric thymidine [poly(T)] repeat, is measured in 68 isolates of adenovirus serotype 14. These isolates were collected during a series of sudden and sometimes fatal outbreaks among both military recruits and civilians as the virus emerged for the first time in the United States in 2006 and 2007. The results demonstrate the usefulness of adenoviral microsatellites as high-resolution molecular strain markers. The described homopolymer is hypervariable in length, varying from 12 to 17 bp in the analyzed sample set. All intermediate lengths were identified in at least one isolate. Furthermore, the specific length of the marker is stable for significant periods of time (up to 7 months) at individual sites where the virus is in consistent circulation. The microsatellite also can maintain specific length identity through site-to-site transmission events, as determined by the analysis of isolates from three advanced training sites that appeared to be subject to pathogen transfer from one of the affected recruit training installations. Public database searches revealed that the polymorphic nature of the microsatellite extends to other species B serotypes, and that other polymorphic microsatellites can be identified readily in a variety of epidemic respiratory adenovirus clades. This study shows that microsatellites are a ubiquitous source of polymorphic markers for human adenoviruses and demonstrates their use through an epidemiological analysis of isolates from a recent North American epidemic.

Background: In 2007, a US Air Force training facility reported a cluster of severe respiratory illnesses associated with a rare human adenovirus (Ad) serotype, Ad14. We investigated this outbreak to better understand its epidemiology, clinical spectrum, and associated risk factors., Methods: Data were collected from ongoing febrile respiratory illness (FRI) surveillance and from a retrospective cohort investigation. Because an Ad7 vaccine is in development, Ad7 antibody titers in pretraining serum samples from trainees with mild and those with severe Ad14 illness were compared., Results: During 2007, an estimated 551 (48%) of 1147 trainees with FRI were infected with Ad14; 23 were hospitalized with pneumonia, 4 required admission to an intensive care unit, and 1 died. Among cohort members (n = 173), the Ad14 infection rate was high (50%). Of those infected, 40% experienced FRI. No cohort members were hospitalized. Male sex (risk ratio [RR], 4.7 [95% confidence interval {CI}, 2.2-10.1]) and an ill close contact (RR, 1.6 [95% CI, 1.2-2.2]) were associated with infection. Preexisting Ad7 neutralizing antibodies were found in 7 (37%) of 19 Ad14-positive trainees with mild illness but in 0 of 16 trainees with Ad14 pneumonia (P = .007)., Conclusions: Emergence of Ad14, a rare Ad serotype, caused a protracted outbreak of respiratory illness among military recruits. Most infected recruits experienced FRI or milder illnesses. Some required hospitalization, and 1 died. Natural Ad7 infection may protect against severe Ad14 illness.

Background: Adenoviruses (Ads) cause continuous outbreaks of acute respiratory disease (ARD) in US military training facilities. In 1996, the loss of vaccines targeting the dominant recruit-associated serotypes precipitated the reemergence of Ads in these populations. Between 1999 and 2002, serotype 4 accounted for >95% of Ads isolated from recruits and for >50% of ARD cases in training facilities (15,000 cases/year)., Methods: Ads (n=1867) collected between 2002 and 2006 from recruits with ARD at 8 military training facilities in the United States were serotyped by serum neutralization and polymerase chain reaction., Results: The dominance of Ad4 continued through 2005, followed by a simultaneous emergence of diverse species B serotypes at the majority of sites. This included the subspecies B1 serotypes 3, 7, and 21 and the subspecies B2 serotype 14. Ad14 was the most prevalent species B serotype, appearing in high numbers at 3 sites and becoming dominant at 1., Conclusions: Subspecies B2 Ads have rarely been associated with ARD, and only in Eurasia. This survey represents the first report of AdB2-associated ARD in the Western Hemisphere. The simultaneous emergence of several species B Ads suggests a common external source (the civilian population) and a decrease in preexisting immunity to species B Ads.

Background: Military recruits are at a higher risk of respiratory infection than their civilian counterparts. Continuous outbreaks of adenovirus (Ad)-associated acute respiratory disease were documented among US trainees before the implementation of serotype 4 (Ad4) and serotype 7 vaccines in 1971. The discontinuation of Ad vaccination programs in 1999 precipitated the reemergence of Ad in training sites, with Ad4 accounting for 98% of all diagnosed cases., Methods: A total of 724 Ad4 strains isolated from recruits presenting with febrile respiratory illness at 8 training sites nationwide between 1997 and 2003 were genome typed by restriction enzyme analysis., Results: Seven genome types were identified, all of which were distinct from the prototype Ad4p and the vaccine type 4p1. Results showed very different, and often stable, genome type distributions at different geographic sites, despite the homogeneity of the recruit source population., Conclusions: The data support the hypothesis that reservoirs for Ad outbreaks are within recruit training sites or in their immediate environments, not in the incoming recruit population. Molecular characterization beyond serotype is critical to understanding the transmission dynamics of Ad infection in these unique susceptible populations and to the implementation of effective prevention approaches.

Despite the success of the adenovirus vaccine administered to US military trainees, acute respiratory disease (ARD) surveillance still detected breakthrough infections (respiratory illnesses associated with the adenovirus serotypes specifically targeted by the vaccine). To explore the role of adenoviral co-infection (simultaneous infection by multiple pathogenic adenovirus species) in breakthrough disease, we examined specimens from patients with ARD by using 3 methods to detect multiple adenoviral species: a DNA microarray, a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay, and a multiplex PCR assay. Analysis of 52 samples (21 vaccinated, 31 unvaccinated) collected from 1996 to 2000 showed that all vaccinated samples had co-infections. Most of these co-infections were community-acquired serotypes of species B1 and E. Unvaccinated samples primarily contained only 1 species (species E) associated with adult respiratory illness. This study highlights the rarely reported phenomenon of adenoviral co-infections in a clinically relevant environment suitable for the generation of new recombinational variants.