Importance of diagnostics in epidemic and pandemic preparedness.

Diagnostics are fundamental for successful outbreak containment. In this supplement, 'Diagnostic preparedness for WHO Blueprint pathogens', we describe specific diagnostic challenges presented by selected priority pathogens most likely to cause future epidemics. Some challenges to diagnostic preparedness are common to all outbreak situations, as highlighted by recent outbreaks of Ebola, Zika and yellow fever. In this article, we review these overarching challenges and explore potential solutions. Challenges include fragmented and unreliable funding pathways, limited access to specimens and reagents, inadequate diagnostic testing capacity at both national and community levels of healthcare and lack of incentives for companies to develop and manufacture diagnostics for priority pathogens during non-outbreak periods. Addressing these challenges in an efficient and effective way will require multiple stakeholders-public and private-coordinated in implementing a holistic approach to diagnostics preparedness. All require strengthening of healthcare system diagnostic capacity (including surveillance and education of healthcare workers), establishment of sustainable financing and market strategies and integration of diagnostics with existing mechanisms. Identifying overlaps in diagnostic development needs across different priority pathogens would allow more timely and cost-effective use of resources than a pathogen by pathogen approach; target product profiles for diagnostics should be refined accordingly. We recommend the establishment of a global forum to bring together representatives from all key stakeholders required for the response to develop a coordinated implementation plan. In addition, we should explore if and how existing mechanisms to address challenges to the vaccines sector, such as Coalition for Epidemic Preparedness Innovations and Gavi, could be expanded to cover diagnostics

Identification of B-cell epitopes on domain 4 of anthrax protective antigen

Protective Antigen (PA) is the receptor binding subunit common to both Lethal (LT) and Edema (ET) toxins, which contribute to the mortality associated with Bacillus anthracis infection. While recombinant PA (rPA) is likely to be an important constituent of second generation anthrax vaccines, evaluating the effectiveness of candidate vaccines is currently difficult, because the specific B cell epitopes involved in toxin neutralization have not been completely defined. The only well characterized antibody, 14B7, has been shown to disrupt the association of PA with the anthrax toxin receptors (ATR) by binding to domain 4 of PA. I hypothesized that other domain 4 epitopes play a critical role in eliciting a protective immune response to anthrax. To test this hypothesis I first identified a novel rPA immunogen capable of eliciting a protective immune response in goats and mice. I next established an LT challenge mouse model to evaluate the ability of antibodies to disrupt the intoxication pathway in vivo. To identify neutralizing epitopes on domain 4 of PA, I screened a collection of murine B cell hybridomas for monoclonal antibodies (MAbs) that reacted with the native PA protein as well as linear peptides within domain 4. Two IgG1 MAbs, 1-F1 and 2-B12, were identified that recognize distinct domain 4 linear epitopes. 1-F1 recognized residues 692-703, part of the ATR recognition region. 1-F1 blocked PA’s ability to associate with ATR in an in vitro solid phase binding assay, and neutralized LT in vitro. 2-B12 recognized residues 716-727, a region not previously known to be a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro , although it only partially inhibited PA binding to ATR. Mice passively administered 1-F1 or 2-B12 were protected against LT challenge. This data confirm that several epitopes on domain 4 of PA contribute to the protective immune response against anthrax intoxication. The identification of neutralizing MAbs recognizing linear peptides provides us with a powerful tool for identifying the specific epitopes involved in the protective immune response to anthrax and advances our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin

Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen▿

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines

A novel sputum transport solution eliminates cold chain and supports routine tuberculosis testing in Nepal

This preliminary study evaluated the transport reagent OMNIgene SPUTUM (OMS) in a real-world, resource-limited setting: a zonal hospital and national tuberculosis (TB) reference laboratory, Nepal. The objectives were to: (1) assess the performance of OMS for transporting sputum from peripheral sites without cold chain stabilization; and (2) compare with Nepal’s standard of care (SOC) for Mycobacterium tuberculosis smear and culture diagnostics. Sixty sputa were manually split into a SOC sample (airline-couriered to the laboratory, conventional processing) and an OMS sample (OMS added at collection, no cold chain transport or processing). Smear microscopy and solid culture were performed. Transport was 0–8 days. Forty-one samples (68%) were smear-positive using both methods. Of the OMS cultures, 37 (62%) were positive, 22 (36%) were negative, and one (2%) was contaminated. Corresponding SOC results were 32 (53%), 21 (35%), and seven (12%). OMS “rescued” six (i.e., missed using SOC) compared with one rescue using SOC. Of smear-positives, six SOC samples produced contaminated cultures whereas only one OMS sample was contaminated. OMS reduced culture contamination from 12% to 2%, and improved TB detection by 9%. The results suggest that OMS could perform well as a no cold chain, long-term transport solution for smear and culture testing. The findings provide a basis for larger feasibility studies

Laboratory-Confirmed Transmission of Vaccinia Virus Infection through Sexual Contact with a Military Vaccinee

A laboratory-confirmed, inadvertent transmission of vaccinia virus from an unusual source highlights the importance of epidemiologic tracing, proper biosafety practices in the clinical diagnostic laboratories, and educating clinicians and laboratorians to potential bioterrorism-initiated outbreaks as well as look-alike disease discrimination

Laboratory Confirmation of Generalized Vaccinia following Smallpox Vaccination

The reinitiation of smallpox vaccination has renewed interest in implementing modern diagnostic methods to assess orthopoxvirus infection and adverse events following vaccination. We report here the laboratory confirmation of vaccinia virus in pustular lesions of a healthy adult vaccinee by use of a two-tier algorithm incorporating TaqMan PCR and electron microscopy

Sylvatic Typhus Associated with Flying Squirrels (\u3cem\u3eGlaucomys volans\u3c/em\u3e) in New York State, United States

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases\u27 residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection