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Stabel J.R.,U.S. Department of Agriculture | Waters W.R.,U.S. Department of Agriculture | Bannantine J.P.,U.S. Department of Agriculture | Lyashchenko K.,Chembio Diagnostic Systems, Inc.
Clinical and Vaccine Immunology | Year: 2011

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis. Copyright © 2011, American Society for Microbiology. All Rights Reserved. Source

Ayove T.,Lihir Medical Center International | Houniei W.,Disease Control Branch | Wangnapi R.,Papua New Guinea Institute of Medical Research | Bieb S.V.,Disease Control Branch | And 12 more authors.
The Lancet Global Health | Year: 2014

Background: To eradicate yaws, national control programmes use the Morges strategy (initial mass treatment and biannual resurveys). The resurvey component is designed to actively detect and treat remaining yaws cases and is initiated on the basis of laboratory-supported reactive non-treponemal serology (using the rapid plasma reagin [RPR] test). Unfortunately, the RPR test is available rarely in yaws-endemic areas. We sought to assess a new point-of-care assay-the Dual Path Platform (DPP) syphilis assay, which is based on simultaneous detection of antibodies to treponemal and non-treponemal antigens-for guiding use of antibiotics for yaws eradication. A secondary goal was to ascertain at what timepoint the DPP assay line reverted to negative after treatment. Methods: 703 children (aged 1-18 years) with suspected clinical yaws living in two remote, yaws-endemic villages in Papua New Guinea were enrolled. Clinical suspicion of yaws was established according to a WHO pictorial guide. We obtained blood samples from all patients. We calculated the sensitivity and specificity of the DPP assay for detection of antibodies to treponemal (T1) and non-treponemal (T2) antigens and compared values against those obtained with standard laboratory tests (the Treponema pallidum haemagglutination assay [TPHA] and the RPR test). We followed up a subsample of children with dually positive serology (T1 and T2) to monitor changes in DPP optical density (using an automatic reader) at 3 and 6 months. This trial is registered with ClinicalTrials.gov, number NCT01841203. Findings: Of 703 participants, 389 (55%) were reactive for TPHA, 305 (43%) for the RPR test, and 287 (41%) for both TPHA and the RPR test. The DPP T1 (treponemal) assay had a sensitivity of 88·4% (95% CI 84·8-91·4) and specificity of 95·2% (92·2-97·3). The DPP T2 (non-treponemal) assay had a sensitivity of 87·9% (83·7-91·3) and specificity of 92·5% (89·4-94·9). In subgroup analyses, sensitivities and specificities did not differ according to type of specimen (plasma vs whole blood). For specimens with an RPR titre of 1:8 or greater, the sensitivity of the DPP T2 assay was 94·1% (95% CI 89·9-96·9). Serological cure (including seroreversion or a fourfold reduction in optical density value) was attained at 6 months in 173 (95%) of 182 children with dual-positive serology. Interpretation: The DPP assay is accurate for identification of antibodies to treponemal and non-treponemal antigens in patients with yaws and avoids the need for laboratory support. A change of diagnostic procedure from the currently implemented RPR test to the simpler DPP assay could ease the implementation of yaws eradication activities. Funding: Chembio Diagnostic Systems, Newcrest Mining, and the Papua New Guinea National Department of Health. © 2014 Ayove et al. Source

Lyashchenko K.P.,Chembio Diagnostic Systems, Inc.
Clinical and vaccine immunology : CVI | Year: 2013

Bovine tuberculosis (TB) in cervids remains a significant problem affecting farmed herds and wild populations. Traditional skin testing has serious limitations in certain species, whereas emerging serological assays showed promising diagnostic performance. The recently developed immunochromatographic dual-path platform (DPP) VetTB assay has two antigen bands, T1 (MPB83 protein) and T2 (CFP10/ESAT-6 fusion protein), for antibody detection. We evaluated the diagnostic accuracy of this test by using serum samples collected from groups of white-tailed deer experimentally inoculated with Mycobacterium bovis, M. avium subsp. paratuberculosis, or M. bovis BCG Pasteur. In addition, we used serum samples from farmed white-tailed deer in herds with no history of TB, as well as from free-ranging white-tailed deer culled during field surveillance studies performed in Michigan known to have bovine TB in the wild deer population. The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks postinoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%. The higher seroreactivity found in deer with naturally acquired M. bovis infection was associated with an increased frequency of antibody responses to the ESAT-6 and CFP10 proteins, resulting in a greater contribution of these antigens, in addition to MPB83, to the detection of seropositive animals, compared with experimental M. bovis infection. Deer experimentally inoculated with either M. avium subsp. paratuberculosis or M. bovis BCG Pasteur did not produce cross-reactive antibodies that could be detected by the DPP VetTB assay. The present findings demonstrate the relatively high diagnostic accuracy of the DPP VetTB test for white-tailed deer, especially in the detection of naturally infected animals. Source

Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.65K | Year: 2015

Bovine tuberculosis (TB), a zoonotic disease caused by Mycobacterium bovis, continues to plague the cattle industry in the United States (US) and worldwide. Current diagnostic methods are inadequate; therefore, improved tests are urgently needed for disease control/eradication. The goal of the proposed research is to develop a simple, rapid, accurate, and cost-effective blood test for bovine TB in cattle. The assay will use proprietary Dual Path Platform (DPP) technology and rationally selected antigens of M. bovis to detect specific antibodies animal-side within 15-20 minutes under field conditions.Phase I has two technical objectives: 1) identify key antigens, and 2) develop a DPP test prototype. Novel M. bovis antigens will be screened out of a large panel of recombinant candidates using serum samples from M. bovis-inoculated cattle. Novel polyepitope fusions will be designed for highly sensitive multi-antigen cocktails providing >90% serodiagnostic accuracy. DPP test prototype will be evaluated with extended panels of well-characterized serum samples. Phase II studies will focus on product development, validation, and field evaluation in preparation for regulatory approval.The proposed research will be translated into significant commercial applications. The Chembio manufacturing facility in Medford, NY, is registered with FDA and licensed by USDA. The company has an extensive track record of new diagnostic product commercialization and an established network of international distributors. A new blood test with superior performance and practical features will improve bovine TB control/eradication programs. Thus, the proposed research will meet the goals of the USDA Program Priorities and Societal Challenge Areas related to animal production and protection, and it will satisfy the public interest to increase food security and promote exports of agricultural biotechnology.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 2.43M | Year: 2011

DESCRIPTION (provided by applicant): Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis with an estimated two billion people currently infected worldwide and over nine million new cases detected annually worldwide. This is the largest killer of patients with HIV/AIDS. Current methods of diagnosis are complex, time-consuming, unreliable or unaffordable for most countries where the disease presents a significant public health threat. New diagnostics are critically needed to address the global tuberculosis burden and improve control programs. Our Phase I studies have clearly demonstrated the feasibility of developing a rapid and accurate test for tuberculosis. In collaboration with the Infectious Disease Research Institute, scientists at Chembio Diagnostic Systems, Inc. have identified a set of novel seroreactive antigens, designed several highly performing polyepitope fusion proteins, and applied the selected antigens in the Chembio's innovative immunoassay format, the Dual Path Platform (DPP(R)), with proven high accuracy of rapid serodiagnosis. We found that a DPP test prototype had a sensitivity of 81% and specificity of 95% in evaluations with serum samples obtained from culture-confirmed patients with active pulmonary tuberculosis from endemic regions in Brazil and Indonesia. In this Phase II application we propose to complete development and evaluation of the rapid diagnostic test for tuberculosis which will have required performance and operational characteristics. The specific aims are to: 1) optimize assay design, 2) determine diagnostic test performance, and 3) validate test production protocols in preparation for regulatory approval. We expect that the proposed DPP assay will demonstrate improved sensitivity and specificity over the existing rapid tests and will be applicable for expedited diagnosis of tuberculosis worldwide. PUBLIC HEALTH RELEVANCE: Tuberculosis remains one of the leading causes of mortality due to infectious disease worldwide. The current methods for diagnosis of tuberculosis are time-consuming, complex and laborious, and/or too expensive for routine use in resource-limited settings where most of patients live. The delayed or missed diagnosis leads to spreading of infection, progression of disease,and increased mortality. The research proposed here aims at developing a simple and rapid (20 minutes) point-of-care diagnostic test that would improve control programs by helping accurately detect new cases of tuberculosis, enabling timely antibiotic therapy, and preventing disease transmission. In addition to the visual reading, there will be an option for automated readout of the test result. The proposed assay will be fully validated and evaluated with serum samples from six countries representing various geographical areas and epidemiological setting associated with tuberculosis. These studies will demonstrate superior sensitivity and specificity as well as improved operational characteristics over the existing technologies.

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