Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia.

BACKGROUND: Lateral flow immunoassays (LFIs) are point-of-care diagnostic assays that are designed for single use outside a formal laboratory, with in-home pregnancy tests the best-known example of these tests. Although the LFI has some limitations over more-complex immunoassay procedures, such as reduced sensitivity and the potential for false-positive results when using complex sample matrices, the assay has the benefits of a rapid time to result and ease of use. These benefits make it an attractive option for obtaining rapid results in an austere environment. In an outbreak of any magnitude, a field-based rapid diagnostic assay would allow proper patient transport and for safe burials to be conducted without the delay caused by transport of samples between remote villages and testing facilities. Use of such point-of-care instruments in the ongoing Ebola virus disease (EVD) outbreak in West Africa would have distinct advantages in control and prevention of local outbreaks, but proper understanding of the technology and interpretation of results are important. METHODS: In this study, a LFI, originally developed by the Naval Medical Research Center for Ebola virus environmental testing, was evaluated for its ability to detect the virus in clinical samples in Liberia. Clinical blood and plasma samples and post mortem oral swabs submitted to the Liberian Institute for Biomedical Research, the National Public Health Reference Laboratory for EVD testing, were tested and compared to results of real-time reverse transcription-polymerase chain reaction (rRT-PCR), using assays targeting Ebola virus glycoprotein and nucleoprotein. RESULTS: The LFI findings correlated well with those of the real-time RT-PCR assays used as benchmarks. CONCLUSIONS: Rapid antigen-detection tests such as LFIs are attractive alternatives to traditional immunoassays but have reduced sensitivity and specificity, resulting in increases in false-positive and false-negative results. An understanding of the strengths, weaknesses, and limitations of a particular assay lets the diagnostician choose the correct situation to use the correct assay and properly interpret the results.

Monitoring of Ebola Virus Makona Evolution through Establishment of Advanced Genomic Capability in Liberia.

To support Liberia's response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014-February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.

Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

The type 1 pili regulator gene fimX and pathogenicity island PAI-X as molecular markers of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) fall within a larger group of isolates producing extraintestinal disease. UPEC express type 1 pili as a critical virulence determinant mediating adherence to and invasion into urinary tract tissues. Type 1 pili expression is under regulation by a family of site-specific recombinases, including FimX, which is encoded from a genomic island called PAI-X for pathogenicity island of FimX. Using a new multiplex PCR, fimX and the additional PAI-X genes were found to be highly associated with UPEC (144/173 = 83.2 %), and more prevalent in UPEC of lower urinary tract origin (105/120 = 87.5 %) than upper urinary tract origin (39/53 = 74 %; P<0.05) or commensal isolates (28/78 = 36 %; P≤0.0001). The Fim-like recombinase gene fimX is the only family member that has a significant association with UPEC compared to commensal isolates. Our results indicate PAI-X genes, including the type 1 pili regulator gene fimX, are highly prevalent among UPEC isolates and have a strong positive correlation with genomic virulence factors, suggesting a potential role for PAI-X in the extraintestinal pathogenic E. coli lifestyle.

Artificial Blood Development Implications for Military Medicine.

Massive hemorrhaging remains the most common cause of preventable battlefield deaths. Blood used for trauma care requires a robust donation network, capacity for long-term storage, and extensive and accurate testing. Bioengineering technologies could offer a remedy to these constraints in the form of blood substitutes-fluids that could be transfused into patients to provide oxygen, carry away waste, and aid in coagulation-that would be used in prolonged casualty care and in far-forward settings, overcoming the obstacles of distance and time. The different molecular properties of red blood cells (RBCs), blood substitutes, and platelet replacements contribute to their respective utilities, and each type is currently represented in ongoing clinical trials. Hemoglobin oxygen carriers (HBOCs) are the most advanced RBC replacements, many of which are currently being evaluated in clinical trials in the United States and other countries. Despite recent advancements, challenges remaining in the development of blood alternatives include stability, oxygen capacity, and compatibility. The continued research and investment in new technologies has the potential to significantly benefit the treatment of life-threatening emergency injuries, both on the battlefield and in the civilian sector. In this review, we discuss military blood-management practices and military-specific uses of individual blood components, as well as describe and analyze several artificial blood products that could be options for future battlefield use.

Description of a COVID-19 Beta variant outbreak, Joint Base Lewis-McChord, WA, February-March 2021.

An outbreak of SARS CoV-2 infection occurred in an infantry battalion from Joint Base Lewis-McChord following participation in a field training exercise in the vicinity of Yakima, WA in February of 2021. Extreme weather during the exercise disrupted planned COVID-19 mitigation measures and caused 110 soldiers to be sheltered in a small aircraft hangar for several nights. The probable index case reported to sick call with symptoms compatible with COVID-19, but the soldier was not diagnosed with COVID-19, was returned to duty, and was allowed to remain in the enclosed hangar for 3 additional days. In total, 143 individuals with epidemiologic ties to the field training exercise tested positive for SARS-CoV-2 during the outbreak. Nine samples sent for sequencing were determined to be the SARS-CoV-2 Beta variant. This report illustrates important lessons learned whose implementation in the future will enable better protection of service members from COVID-19 and similar health risks associated with training.

Evaluation of the performance of a multiplex reverse transcription polymerase chain reaction kit as a potential diagnostic and surveillance kit for rotavirus in Kenya.

BACKGROUND: Diarrhea is a serious concern worldwide, especially in developing countries. Rotavirus is implicated in approximately 400,000 infant deaths annually. It is highly contagious elevating the risk of outbreaks especially in enclosed settings such as daycare centers, hospitals, and boarding schools. Reliable testing methods are critical for early detection of infections, better clinical management, pathogen surveillance and evaluation of interventions such as vaccines. Enzyme immunoassays have proved to be reliable and practical in most settings; however, newer multiplex reverse transcription polymerase assays have been introduced in the Kenya market but have not been evaluated locally. METHODS: Stool samples collected from an ongoing Surveillance of Enteric Pathogens Causing diarrheal illness in Kenya (EPS) study were used to compare an established enzyme immunoassay, Premier™ Rotaclone® (Meridian Bioscience, Cincinnati, Ohio, U.S.A.), that can only detect group A rotavirus against a novel multiplex reverse transcription polymerase chain reaction kit, Seeplex® Diarrhea-V ACE Detection (Seegene, Seoul, Republic of Korea), that can detect rotavirus, astrovirus, adenovirus, and norovirus genogroups I and II. Detection frequency, sensitivity, specificity, turnaround time, and cost were compared to determine the suitability of each assay for clinical work in austere settings versus public health work in well-funded institutes in Kenya. RESULTS: The Premier™ Rotaclone® kit had a detection frequency of 11.2%, sensitivity of 77.8%, specificity of 100%, turnaround time of 93 min and an average cost per sample of 13.33 United States dollars (USD). The Seeplex® Diarrhea-V ACE Detection kit had a detection frequency of 16.0%, sensitivity of 100%, specificity of 98.1%, turnaround time of 359 min and an average cost per samples 32.74 United States dollars respectively. The detection frequency sensitivity and specificity of the Seeplex® Diarrhea-V ACE Detection kit mentioned above are for rotavirus only. CONCLUSIONS: The higher sensitivity and multiplex nature of the Seeplex® Diarrhea-V ACE Detection kit make it suitable for surveillance of enteric viruses circulating in Kenya. However, its higher cost, longer turnaround time and complexity favor well-resourced clinical labs and research applications. The Premier™ Rotaclone®, on the other hand, had a higher specificity, shorter turnaround time, and lower cost making it more attractive for clinical work in low complexity labs in austere regions of the country. It is important to continuously evaluate assay platforms' performance, operational cost, turnaround time, and usability in different settings so as to ensure quality results that are useful to the patients and public health practitioners.

Intracellular Macrophage Infections with E. coli under Nitrosative Stress.

Escherichia coli (E. coli) produces disseminated infections of the urinary tract, blood, and central nervous system where it encounters professional phagocytes such as macrophages, which utilize reactive nitrogen intermediates (RNI) to arrest bacteria. In vitro, extraintestinal pathogenic E. coli (ExPEC) can survive within bone marrow-derived macrophages for greater than 24 h post-infection within a LAMP1+ vesicular compartment, and ExPEC strains, in particular, are better adapted to intracellular macrophage survival than commensal strains (Bokil et al., 2011). This protocol details an intracellular murine macrophage-like cell infection, including modulation of the host nitrosative stress response, to model this host-pathogen interaction in vitro. To accomplish this, RAW 264.7 murine macrophage-like cells are pre-incubated with either L-arginine, an NO precursor, or IFNγ to yield a high nitric oxide (NO) physiological state, or L-NAME, an inducible NO synthase (iNOS)-specific inhibitor, to yield a low NO physiological state. This protocol has been successfully utilized to assess the contribution of a novel ExPEC regulator to intracellular survival and the nitrosative stress response during macrophage infections (Bateman and Seed, 2012), but can be adapted for use with a variety of E. coli strains or isogenic deletions.

Promoter Orientation of Prokaryotic Phase-variable Genes by PCR.

One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic Escherichia coli (ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.

Procession to pediatric bacteremia and sepsis: covert operations and failures in diplomacy.

Despite advances in diagnosis and treatment, bacterial sepsis remains a major cause of pediatric morbidity and mortality, particularly among neonates, the critically ill, and the growing immunocompromised patient population. Sepsis is the end point of a complex and dynamic series of events in which both host and microbial factors drive high morbidity and potentially lethal physiologic alterations. In this article we provide a succinct overview of the events that lead to pediatric bloodstream infections (BSIs) and sepsis, with a focus on the molecular mechanisms used by bacteria to subvert host barriers and local immunity to gain access to and persist within the systemic circulation. In the events preceding and during BSI and sepsis, Gram-positive and Gram-negative pathogens use a battery of factors for translocation, inhibition of immunity, molecular mimicry, intracellular survival, and nutrient scavenging. Gaps in understanding the molecular pathogenesis of bacterial BSIs and sepsis are highlighted as opportunities to identify and develop new therapeutics.