A growing global network's role in outbreak response: AFHSC-GEIS 2008-2009.

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.

Initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread.

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.

Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection.

BACKGROUND: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. OBJECTIVES: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. STUDY DESIGN: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. RESULTS: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). CONCLUSIONS: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.

Local changes in rates of group A Streptococcus disease and antibiotic resistance are associated with geographically widespread strain turnover events.

This study addresses the effects of dynamic strain turnover and antibiotic prophylaxis on rates of group A Streptococcus (GAS) antibiotic resistance and disease. The authors analyzed the strain distributions, disease rates, and patterns of antibiotic resistance of 802 GAS isolates collected from 2002 through 2007. These samples were collected from patients with GAS infection symptoms at 10 military facilities. Macrolide resistance peaked at 25% during 2004, due to the geographically widespread dominance of a single resistant strain (M75). The resistant strain was not retained regardless of local patterns of macrolide use, and resistance rates decreased upon replacement of M75 with macrolide-susceptible strains. Disease rates were similarly correlated with dominance of specific M types. Statistical analysis revealed temporal correlations between strain distributions at multiple locations. Only the most common strains yielded enough data at multiple sites for statistically significant comparison of temporal fluctuations in dominance, but these (including M44, M3, M18, M118, and M6) all yielded highly significant temporal correlations of 90% or greater on yearly scales. As expected given the complexity and variability of strain distributions on shorter time scales, analysis on a monthly scale yielded lower degrees of positive correlation (31-62%), but in this case all significant correlations were still positive. Shifts in antibiotic resistance profiles and disease rates at specific sites appear to be associated with strain replacements happening on larger scales, independent of antibiotic use at individual sites.

Inference of antibiotic resistance and virulence among diverse group A Streptococcus strains using emm sequencing and multilocus genotyping methods.

BACKGROUND: Group A Streptococcus pyogenes (GAS) exhibits a high degree of clinically relevant phenotypic diversity. Strains vary widely in terms of antibiotic resistance (AbR), clinical severity, and transmission rate. Currently, strain identification is achieved by emm typing (direct sequencing of the genomic segment coding for the antigenic portion of the M protein) or by multilocus genotyping methods. Phenotype analysis, including critical AbR typing, is generally achieved by much slower and more laborious direct culture-based methods. METHODOLOGY/PRINCIPAL FINDINGS: We compare genotype identification (by emm typing and PCR/ESI-MS) with directly measured phenotypes (AbR and outbreak associations) for 802 clinical isolates of GAS collected from symptomatic patients over a period of 6 years at 10 military facilities in the United States. All independent strain characterization methods are highly correlated. This shows that recombination, horizontal transfer, and other forms of reassortment are rare in GAS insofar as housekeeping genes, primary virulence and antibiotic resistance determinants, and the emm gene are concerned. Therefore, genotyping methods offer an efficient way to predict emm type and the associated AbR and virulence phenotypes. CONCLUSIONS/SIGNIFICANCE: The data presented here, combined with much historical data, suggest that emm typing assays and faster molecular methods that infer emm type from genomic signatures could be used to efficiently infer critical phenotypic characteristics based on robust genotype: phenotype correlations. This, in turn, would enable faster and better-targeted responses during identified outbreaks of constitutively resistant or particularly virulent emm types.

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling.

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.