Zika virus surveillance in active duty U.S. military and dependents through the Naval Infectious Diseases Diagnostic Laboratory.

The Naval Infectious Diseases Diagnostic Laboratory (NIDDL) serves as a reference clinical laboratory that supports Department of Defense (DoD) military treatment facilities worldwide in the detection and identification of high-risk and emerging infectious diseases. Since the emergence of Zika virus (ZIKV) in the Western Hemisphere in 2016, the NIDDL has been a central hub for ZIKV testing for DoD personnel and beneficiaries. Samples collected during patients' clinical evaluations were screened for evidence of possible exposure to ZIKV using molecular and serological methods. An in-house ZIKV plaque reduction neutralization test was used to confirm the presence of ZIKV immunoglobulin M antibody. Of 1,420 individuals tested, ZIKV infection was confirmed by quantitative real-time polymerase chain reaction (PCR) assay in 11 (0.8%); an additional 26 recent flaviviral infections (possibly ZIKV) were identified based on serology (1.8%). These findings contribute to the understanding of the burden of ZIKV infections among DoD personnel and beneficiaries and highlight the role of the NIDDL in clinical diagnosis during emerging infectious disease outbreaks.

Clinical, Serological, and Molecular Observations from a Case Series Study during the Asian Lineage Zika Virus Outbreak in Grenada during 2016.

This paper describes the spatial and temporal distribution of cases, demographic characteristics of patients, and clinical manifestations of Zika virus (ZIKV) during the 2016 outbreak in Grenada. The first reported case was recorded in St. Andrew Parish in April, and the last reported case was seen in November, with peak transmission occurring in the last week of June, based on test results. Data were collected from a total of 514 patients, of whom 207 (40%) tested positive for ZIKV. No evidence was found that testing positive for ZIKV infection was related to age, gender, or pregnancy status. Clinical presentation with rash (OR = 2.4, 95% CI = 1.5 to 3.7) or with lymphadenopathy (OR = 1.7, 95% CI = 1.0 to 2.9) were the only reported symptoms consistent with testing positive for ZIKV infection. During the Zika outbreak, the infection rate was 20 clinical cases per 10,000 in the population compared to 41 cases per 10,000 during the chikungunya outbreak in Grenada in 2014 and 17 cases per 10,000 during the dengue outbreak in 2001-2002. Even though the country has employed vector control programs, with no apparent decrease in infection rates, it appears that new abatement approaches are needed to minimize morbidity in future arbovirus outbreaks.

Clinical and Serological Insights from the Asian Lineage Chikungunya Outbreak in Grenada, 2014: An Observational Study.

Chikungunya virus (CHIKV) spread rapidly throughout the Caribbean region in 2014, and the first serologically confirmed case was seen in Grenada in July. This study investigated the outbreak of CHIKV in Grenada to identify the distinguishing clinical manifestations and the symptoms that corresponded the closest with serological test results. Sera were tested by IgM enzyme-linked immunosorbent assay and polymerase chain reaction to distinguish between cases positive or negative for CHIKV. Of 493 cases, 426 (86%) tested positive for CHIKV. The diagnostic decision rule, "Define as CHIKV positive a patient presenting with joint pain and any combination of fever, body pain, or rash," produced the closest agreement (85%) with the serological test results (Cohen's kappa, k = 0.289, P value < 0.001). When laboratory facilities are not available for diagnostic confirmation, syndromic surveillance using these four symptoms could be useful to define cases during a CHIKV outbreak when CHIKV is the predominant circulating arbovirus.

Seroconversions for Coxiella and Rickettsial Pathogens among US Marines Deployed to Afghanistan, 2001-2010.

We assessed serum samples from 1,000 US Marines deployed to Afghanistan during 2001-2010 to find evidence of 4 rickettsial pathogens. Analysis of predeployment and postdeployment samples showed that 3.4% and 0.5% of the Marines seroconverted for the causative agents of Q fever and spotted fever group rickettsiosis, respectively.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

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.

Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell.

In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of approximately 100 genes. The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites. To estimate the number of soxboxes in the cell, we searched the E. coli genome for SoxS binding sites using as query sequence the previously determined optimal SoxS binding sequence. We found approximately 12,500 sequences that match the optimal binding sequence under the conditions of our search; this agrees with our previous estimate, based on information theory, that a random sequence the size of the E. coli genome contains approximately 13,000 soxboxes. Thus, fast-growing cells with 4-6 genomes per cell have approximately 65,000 soxboxes. This large number of potential SoxS binding sites per cell raises the interesting question of how SoxS distinguishes between the functional soxboxes located within the promoters of target genes and the plethora of equivalent but nonfunctional binding sites scattered throughout the chromosome. To address this question, we treated cells with paraquat and used Western blot analysis to determine the kinetics of SoxS accumulation per cell; we also determined the kinetics of SoxS-activated gene expression. The abundance of SoxS reached a maximum of 2,500 molecules per cell 20 min after induction and gradually declined to approximately 500 molecules per cell over the next 1.5 h. Given that activation of target gene expression began almost immediately and given the large disparity between the number of SoxS molecules per cell, 2,500, and the number of SoxS binding sites per cell, 65,000, we infer that SoxS is not likely to activate transcription by the usual "recruitment" pathway, as this mechanism would require a number of SoxS molecules similar to the number of soxboxes. Instead, we propose that SoxS first interacts in solution with RNA polymerase and then the binary complex scans the chromosome for promoters that contain a soxbox properly positioned and oriented for transcription activation. We name this new pathway "pre-recruitment."