Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection.

In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.

Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

Comparison of a new prototype immunochromatographic assay and a commercial enzyme-linked immunosorbent assay for the detection of serum antibodies against Toscana virus.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by Phlebotomus spp. Sandflies, which causes neurological diseases such as aseptic meningitis and meningoencephalitis. The commercial enzyme-linked immunosorbent assay (ELISA) is used widely to detect anti-TOSV IgG and IgM antibodies and to allow for rapid diagnosis of infection (Diesse Diagnostica Senese, Siena, Italy). Recently, an immunochromatographic assay (ICA) was developed for human anti-TOSV IgG or IgM detection by InBios International (Seattle, WA, USA). A comparison of the two diagnostic assays was performed on one hundred serum samples collected from patients hospitalized with suspected TOSV meningitis. Both assays were in excellent agreement, for both IgG and IgM detection. For IgM, 64/65 ELISA positive samples were positive by ICA. One serum, positive for specific IgM by ELISA but negative by ICA, was confirmed by direct diagnosis, with TOSV RNA detection in the patient's cerebrospinal fluid by PCR. For IgG, 64 samples were positive by ICA out of 71 ELISA positive samples. The discordant sera were positive by immunofluorescence and neutralization tests. Three out of these seven samples were also positive by IgM ICA. The sensitivity of these new assays compared to ELISA, which is used routinely, was 98.5% for IgM and 90.1% for IgG, while specificity was 100% in both cases. This data shows that ICA could be a reliable alternative test for serological diagnosis of TOSV infection in humans.

Comparison of two immunochromatographic assays and the indirect immunofluorescence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana.

Two rapid tests evaluated in dogs considered to be of high risk of infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays: Trypanosoma Detect for canine, InBios, Seattle, WA and CHAGAS STAT-PAK assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test. From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT. The serological survey using IFAT showed a prevalence of T. cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays, 13 and 11 animals were positive on rapid assay: Trypanosoma Detect for canine, InBios and CHAGAS STAT-PAK, Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T. cruzi infection. Early detection of T. cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk, clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana.

Lateral flow immunoassay for diagnosis of Trypanosoma cruzi infection with high correlation to the radioimmunoprecipitation assay.

The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) "gold standard" in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a specificity of 99.1%.

Evaluación de una prueba rápida para el diagnóstico de la infección por Trypanosoma cruzi en suero / Evaluation of a rapid diagnosis test for trypanosoma cruzi infection in serum sample

Con el fin de evaluar la utilidad práctica, y la sensibilidad y especificidad del test rápido Dipstick test Trypanosoma cruzi Detect, (Inbios, Seattle, WA) se estudiaron 284 sueros humanos de los cuales 145 correspondieron a casos probados de infección chagásica y 139 a individuos sanos de zonas no endémicas. Todos ellos analizados previamente con las Técnicas de ELISA y de Inmunofluorescencia Indirecta para la detección de anticuerpos IgG anti T. cruzi. Además se estudiaron 56 muestras serologicamente negativas para enfermedad de Chagas pero que presentan otras patologías parasitarias y no parasitarias. El "test" rápido de INBIOS demostró una especificidad de 99,3 por ciento al igual que la sensibilidad. Y una concordancia con La ELISA y la RIFI de de 98,2 por ciento. De acuerdo a estos resultados y a la facilidad de su ejecución, Dipstick test T. cruzi Detect (INBIOS) resulta ideal como tamiz para la vigilancia y los programas de intervención de la enfermedad de Chagas.

We tested a Rapid Diagnostic Test (RDT) to detect Trypanosoma cruzi infection using a total of 284 human sera, some of them from provedcases ofChagas disease (n = 145) and healthy individuals from non-endemic areas (n = 139). Another group included was non chagasic serum samples of individuals of other parasitic and non parasitic disease (n = 56), all of them with known serological test results. The RDT had a specificity of 99,3 percent and a sensitivity of 99,3 percent. The agreement with ELISA and IFAT was 98,2 percent. According to the results and the feasibility of the RDT should be an ideal tool for screening purposes in disease surveillance and intervention programs ofChagas disease.

Canine visceral leishmaniasis: performance of a rapid diagnostic test (Kalazar Detect) in dogs with and without signs of the disease.

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms.