RESUMEN
BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
Asunto(s)
Interleucina-8 , Neoplasias de la Vejiga Urinaria , Humanos , Factor A de Crecimiento Endotelial Vascular , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/orina , Inmunoensayo , Urinálisis , Medición de RiesgoRESUMEN
Background: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. Methods: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusion: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment esponsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. Trial Registration: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
RESUMEN
In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.
Asunto(s)
Mpox , Orthopoxvirus , Humanos , Estudios Retrospectivos , Infecciones Asintomáticas , Bioensayo , Reacciones CruzadasRESUMEN
In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.
RESUMEN
BACKGROUND: The 2014 Zaire Ebola virus disease epidemic accelerated vaccine development for the virus. We aimed to assess the safety, reactogenicity, and immunogenicity of one dose of monovalent, recombinant, chimpanzee adenovirus type-3 vectored Zaire Ebola glycoprotein vaccine (ChAd3-EBO-Z) in adults. METHODS: This phase 2, randomised, observer-blind, controlled trial was done in study centres in Cameroon, Mali, Nigeria, and Senegal. Healthy adults (≥18 years) were randomly assigned with a web-based system (1:1; minimisation procedure accounting for age, gender, centre) to receive ChAd3-EBO-Z (day 0), or saline placebo (day 0) and ChAd3-EBO-Z (month 6). The study was observer-blind until planned interim day 30 analysis, single-blind until month 6, and open-label after month 6 vaccination. Primary outcomes assessed in the total vaccinated cohort, which comprised all participants with at least one study dose administration documented, were serious adverse events (up to study end, month 12); and for a subcohort were solicited local or general adverse events (7 days post-vaccination), unsolicited adverse events (30 days post-vaccination), haematological or biochemical abnormalities, and clinical symptoms of thrombocytopenia (day 0-6). Secondary endpoints (subcohort; per-protocol cohort) evaluated anti-glycoprotein Ebola virus antibody titres (ELISA) pre-vaccination and 30 days post-vaccination. This study is registered with ClinicalTrials.gov, NCT02485301. FINDINGS: Between July 22, 2015, and Dec 10, 2015, 3030 adults were randomly assigned; 3013 were included in the total vaccinated cohort (1509 [50·1%] in the ChAd3-EBO-Z group and 1504 [49·9%] in the placebo/ChAd3-EBO-Z group), 17 were excluded because no vaccine was administered. The most common solicited injection site symptom was pain (356 [48%] of 748 in the ChAd3-EBO-Z group vs 57 [8%] of 751 in the placebo/ChAd3-EBO-Z group); the most common solicited general adverse event was headache (345 [46%] in the ChAd3-EBO-Z group vs 136 [18%] in the placebo/ChAd3-EBO-Z group). Unsolicited adverse events were reported by 123 (16%) of 749 in the ChAd3-EBO-Z group and 119 (16%) of 751 in the placebo/ChAd3-EBO-Z group. Serious adverse events were reported for 11 (1%) of 1509 adults in the ChAd3-EBO-Z group, and 18 (1%) of 1504 in the placebo/ChAd3-EBO-Z group; none were considered vaccination-related. No clinically meaningful thrombocytopenia was reported. At day 30, anti-glycoprotein Ebola virus antibody geometric mean concentration was 900 (95% CI 824-983) in the ChAd3-EBO-Z group. There were no treatment-related deaths. INTERPRETATION: ChAd3-EBO-Z was immunogenic and well tolerated in adults. Our findings provide a strong basis for future development steps, which should concentrate on multivalent approaches (including Sudan and Marburg strains). Additionally, prime-boost approaches should be a focus with a ChAd3-based vaccine for priming and boosted by a modified vaccinia Ankara-based vaccine. FUNDING: EU's Horizon 2020 research and innovation programme and GlaxoSmithKline Biologicals SA.
Asunto(s)
Adenovirus de los Simios , Vacunas contra el Virus del Ébola/efectos adversos , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Adolescente , Adulto , Animales , Anticuerpos Antivirales/sangre , Femenino , Vectores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Pan troglodytes , Método Simple Ciego , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: During the large 2013-16 Ebola virus outbreak caused by the Zaire Ebola virus, about 20% of cases were reported in children. This study is the first, to our knowledge, to evaluate an Ebola vaccine in children younger than 6 years. We aimed to evaluate the safety, reactogenicity, and immunogenicity of a monovalent, recombinant, chimpanzee adenovirus type-3 vectored Zaire Ebola glycoprotein vaccine (ChAd3-EBO-Z) in a paediatric population. METHODS: This phase 2, randomised, observer-blind, controlled trial was done in a vaccine centre in Mali and a university hospital centre in Senegal. Healthy children were randomly assigned through a web-based system (1:1; stratified by age group, gender, and centre) to receive ChAd3-EBO-Z (day 0) and meningococcal serogroups A,C,W-135,Y tetanus toxoid conjugate vaccine (MenACWY-TT; month 6), or MenACWY-TT (day 0) and ChAd3-EBO-Z (month 6). The study was observer-blind from study start until interim day 30 analysis and became single-blind as of interim analysis. Primary outcomes assessed were serious adverse events (up to study end, month 12), solicited local or general adverse events (7 days post-vaccination), unsolicited adverse events (30 days post-vaccination), haematological or biochemical abnormalities, and clinical symptoms of thrombocytopenia (day 0-6). As secondary endpoints, we evaluated anti-glycoprotein Zaire Ebola virus antibody titres (ELISA) pre-vaccination and 30 days post-vaccination. This study is registered with ClinicalTrials.gov, NCT02548078. FINDINGS: From Nov 11, 2015, to May 9, 2016, of 776 children screened for eligibility, 600 were randomly assigned (200 [33%] in each age strata: 1-5, 6-12, 13-17 years), 300 (50%) to the ChAd3-EBO-Z/MenACWY-TT group and 300 (50%) to the MenACWY-TT/ChAd3-EBO-Z group; all were included in the total vaccinated cohort. Post-day 0 vaccination, the most common solicited injection site symptom was pain (127 [42%] of 300 in the ChAd3-EBO-Z/MenACWY-TT group vs 60 [20%] of 300 in the MenACWY-TT/ChAd3-EBO-Z group); the most common solicited general adverse event was fever (95 [32%] of 300 in the ChAd3-EBO-Z/MenACWY-TT group vs 28 [9%] of 300 in the MenACWY-TT/ChAd3-EBO-Z group). Unsolicited adverse events post-day 0 vaccination were reported by 41 (14%) of 300 participants in the ChAd3-EBO-Z/MenACWY-TT group and 24 (8%) of 300 MenACWY-TT/ChAd3-EBO-Z recipients. Serious adverse events were reported for two (1%) of 300 children in each group; none were considered vaccination related. No clinical symptoms of thrombocytopenia were reported. At day 30, anti-glycoprotein Ebola virus antibody geometric mean concentrations (GMC) in the ChAd3-EBO-Z/MenACWY-TT group were 1564 (95% CI 1340-1826) for those aged 13-17 years, 1395 (1175-1655) for 6-12 years, and 2406 (1942-2979) for 1-5 years. Anti-glycoprotein Ebola virus IgG antibody responses persisted up to 12 months post-vaccination, with a GMC of 716 (95% CI 619-828) for those aged 13-17 years, 752 (645-876) for 6-12 years, and 1424 (1119-1814) for 1-5 years. INTERPRETATION: ChAd3-EBO-Z was immunogenic and well tolerated in children aged 1-17 years. This study provides the first ChAd3-EBO-Z data in a paediatric population. Further development should focus on multivalent approaches including Sudan and Marburg strains, and heterologous prime-boost strategies, for instance using modified vaccinia Ankara-based vaccine to boost the immune response. FUNDING: EU's Horizon 2020 research and innovation programme and GlaxoSmithKline Biologicals SA.
Asunto(s)
Adenovirus de los Simios , Vacunas contra el Virus del Ébola/efectos adversos , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Adolescente , Animales , Anticuerpos Antivirales/sangre , Niño , Preescolar , Femenino , Vectores Genéticos , Humanos , Lactante , Masculino , Pan troglodytes , Método Simple Ciego , Vacunas Sintéticas/inmunologíaRESUMEN
Herpes simplex virus type 2 (HSV-2) is a common human pathogen that can cause a variety of clinical manifestations in humans. In order to provide near-patient results to allow for faster counseling and treatment, a rapid point-of-care test that is accurate and simple to use is desirable. Here, we describe the development and evaluation of an HSV-2 immunoglobulin G (IgG)-specific antibody lateral-flow immunochromatographic assay (LFIA) based on colloidal gold nanoparticles. A total of 359 serum samples and 100 whole-blood samples were tested in the newly developed HSV-2 LFIA. Serum results were compared to those from the HerpeSelect HSV-2 enzyme-linked immunosorbent assay (ELISA), and whole-blood sample results were compared to those of both ELISA and HerpeSelect HSV-1 and -2 immunoblotting (IB). The sensitivity of the HSV-2 LFIA compared to that of the HerpeSelect ELISA was 100% (89/89), and the specificity was 97.3% (257/264). Cross-reactivity with HSV-1 IgG-positive serum samples was observed in 2.6% (5/196) of samples, 2.9% (1/34) for rubella virus, and 6.2% (1/16) for Epstein-Barr virus. No cross-reactivity in varicella-zoster virus or cytomegalovirus IgG-positive serum samples was observed. No interference was observed from bilirubin-, triglyceride-, albumin-, or hemoglobin-spiked samples. The concordance of the LFIA results between capillary whole blood, EDTA-treated venous whole blood, heparin-treated venous whole blood, and serum was 99% (99/100). In conclusion, the LFIA for HSV-2 IgG-specific antibodies demonstrated excellent sensitivity, specificity, and concordance for both serum and whole-blood samples compared to the sensitivity, specificity, and concordance of both HSV-2 ELISA and IB.
Asunto(s)
Anticuerpos Antivirales/sangre , Cromatografía/métodos , Herpes Genital/sangre , Herpesvirus Humano 2/aislamiento & purificación , Inmunoglobulina G/sangre , Sistemas de Atención de Punto , Adulto , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Oro Coloide/química , Herpes Genital/inmunología , Herpes Genital/virología , Herpesvirus Humano 2/inmunología , Humanos , Immunoblotting/métodos , Inmunoglobulina G/inmunología , Valor Predictivo de las Pruebas , Curva ROC , Sensibilidad y Especificidad , Suero/inmunología , Interferencia ViralRESUMEN
BACKGROUND: In South Africa, human immunodeficiency virus-1 (HIV-1) infection correlates with herpes simplex virus-2 (HSV-2; genital herpes) seropositivity in genitourinary disease clinic attendees. HSV-2 infection may be a marker for risk behavior and/or directly facilitate HIV-1 transmission. The rate of HSV-2 infection in HIV-infected South African and US blood donations was assessed, and whether the infections were correlated in donors screened and found negative for high-risk behavior by predonation interview was questioned. STUDY DESIGN AND METHODS: A total of 625 South African and 393 US HIV-1-infected repository samples previously characterized for longstanding or recent HIV-1 infection were tested with two commercially available HSV-2-specific assays. The prevalence of HSV-2 antibodies in South Africa was further assessed in 106 HIV-1-infected and 106 HIV-1-negative donors matched for sex, race, and donation history, as well as 200 random HIV-1-negative donors. RESULTS: A total of 52.2 percent of US and 69.3 percent of South African HIV-1-infected donations were HSV-2-seropositive. Age, race, and sex were independent risk factors for HSV-2 antibody prevalence in HIV-1-infected South African donors, who were more likely to be HSV-2 antibody-reactive than random HIV-1-negative donors (72.6% vs. 8.5%: odds ratio [OR], 28.6; 95% confidence interval [CI], 14.5-55) or matched donors (71.6% vs. 19.6%: OR, 10.3; 95% CI, 5.4-19.8). HIV-1 infection and HSV-2 seropositivity correlated in white and black populations when analyzed by age group. CONCLUSIONS: HIV-1 infection correlates strongly with HSV-2 seropositivity in US and South African blood donors. Our data describe the characteristics of HSV-2 antibody testing as a surrogate marker for HIV-1 infection and support a facilitating role for HSV-2 infection in HIV-1 transmission.
Asunto(s)
Donantes de Sangre , Infecciones por VIH/sangre , Infecciones por VIH/virología , VIH-1/fisiología , Herpes Genital/sangre , Herpes Genital/virología , Herpesvirus Humano 2 , Adolescente , Adulto , Anticuerpos Antivirales/inmunología , Femenino , Herpesvirus Humano 2/inmunología , Humanos , Masculino , Sudáfrica , Factores de TiempoRESUMEN
Sera from 1,238 Vietnamese women in Hanoi were tested for herpes simplex virus type 2 (HSV-2). HSV-2 prevalence was 2.0%. The Kalon and Biokit assays showed significantly higher concordance to Western blotting data than did the Focus assay (P < 0.01). Screening by Focus and then retesting with Kalon/Biokit of positive samples can reduce falsely positive results significantly (P < 0.01).
Asunto(s)
Anticuerpos Antivirales/sangre , Herpes Simple/diagnóstico , Técnicas para Inmunoenzimas/métodos , Proteínas del Envoltorio Viral/inmunología , África Occidental , Reacciones Falso Positivas , Femenino , Herpes Simple/epidemiología , Herpes Simple/virología , Herpesvirus Humano 2/inmunología , Humanos , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Vietnam/epidemiologíaRESUMEN
In the United States most cases of hantavirus pulmonary syndrome (HPS) are caused by the Sin Nombre virus (SNV) and are typically identified by serology. The goal of this study was to assess the performance of our hantavirus serologic testing algorithm by reviewing results generated over five years. Sera were screened for pan-hantavirus immunoglobulin (Ig)G and IgM by enzyme-linked immunosorbent assay (ELISA). Screen IgG+ sera were then tested by immunoblot for SNV glycoprotein-specific IgG, and screen IgM+ sera were tested for SNV-specific IgM using an ELISA that measured differential reactivity to SNV and Seoul nucleocapsid proteins. Although only 13% of sera were positive in one or both screening assays, 85% of screen+sera lacked SNV antibodies. Nearly all (97%) screen IgM-IgG+ samples lacked SNV IgG, and 90% of screen IgM+IgG- samples lacked SNV IgM. However, SNV IgM testing of screen IgM+IgG- samples appears to be necessary, since this test identified nine of 37 patients with acute HPS (based on clinical feedback). A screen IgM+IgG+ result was a good predictor of SNV antibody detection and acute HPS. These findings were used to design a modified algorithm that identified all 37 patients with acute HPS, but reduced the number of specimens that required SNV antibody testing by 42%.
Asunto(s)
Algoritmos , Anticuerpos Antivirales/sangre , Síndrome Pulmonar por Hantavirus/diagnóstico , Virus Sin Nombre/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Orthohantavirus/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Pruebas SerológicasRESUMEN
In 2003, the Nebraska Public Health Laboratory tested more than 10,371 serum and 516 cerebral spinal fluid specimens. Results showed that without performing the interfering factors screen for specimens in the low positive index value range of >1.1 to Asunto(s)
Anticuerpos Antivirales/sangre
, Inmunoglobulina M
, Interferencia Viral/inmunología
, Fiebre del Nilo Occidental/diagnóstico
, Virus del Nilo Occidental/inmunología
, Nebraska
, Estudios Prospectivos
, Juego de Reactivos para Diagnóstico
, Estudios Retrospectivos
, Pruebas Serológicas
, Fiebre del Nilo Occidental/sangre
, Fiebre del Nilo Occidental/inmunología
RESUMEN
Southern tick-associated rash illness (STARI), also known as Masters disease, affects people predominantly in the Southeast and South Central United States. These patients exhibit skin lesions that resemble erythema migrans (EM), the characteristic skin lesion in early Lyme disease. The etiology of STARI remains unknown, and no serologic test is available to aid in its diagnosis. The C6 Lyme enzyme-linked immunosorbent assay was used to evaluate coded serum specimens from patients with STARI at two laboratory sites. The specimens tested at one site consisted of acute- and convalescent-phase samples that were obtained from nine STARI patients from Missouri and from one patient with documented Borrelia lonestari infection who acquired this infection in either North Carolina or Maryland. All of these samples were C6 negative. Seventy acute- or convalescent-phase specimens from 63 STARI patients from Missouri were C6 tested at the second site. All but one of these STARI specimens were also negative. In contrast, of nine acute- and nine convalescent-phase serum specimens obtained from culture-confirmed Lyme disease patients with EM from New York state, seven were C6 positive at the acute stage, and eight were positive at convalescence. The C6 test is negative in patients with STARI, providing further evidence that B. burgdorferi is not the etiologic agent of this disease.
Asunto(s)
Borrelia burgdorferi/inmunología , Eritema Crónico Migrans/inmunología , Enfermedad de Lyme/diagnóstico , Anticuerpos Antibacterianos/sangre , Ensayo de Inmunoadsorción Enzimática , Eritema Crónico Migrans/diagnóstico , Eritema Crónico Migrans/microbiología , Humanos , Enfermedad de Lyme/sangre , Enfermedad de Lyme/inmunología , MissouriRESUMEN
OBJECTIVE: The objective of this study was to define the positive predictive value (PPV) of the Focus herpes simplex virus type 2 (HSV-2) enzyme-linked immunosorbent assay (ELISA) in a low HSV-2 prevalence population and to develop a new test interpretation algorithm. METHODS: HSV-2 Western blots were performed on sera from male sexually transmitted disease clinic patients testing HSV-2 ELISA-positive and used to define a new class of indeterminate HSV-2 ELISA result. HSV-2 Western blots were then prospectively performed on sequential sera with indeterminate HSV-2 ELISAs. RESULTS: Ninety-one (84%) of 108 HSV-2 ELISA-positive sera tested HSV-2 Western blot-positive. Western blot positivity was more common in men without herpes simplex virus type 1 (HSV-1) antibody than in those with HSV-1 antibody (93% vs 76%, P = 0.02) and in men with a history or clinical evidence of genital lesions (88% vs 80%, P = 0.30). Selectively raising the ELISA index value defining HSV-2 positivity from >1.1 to >or=3.0 either among HSV-1-positive men or among those without a history or clinical evidence of genital lesions increased the PPV to >or=93%. Prospective evaluation of an algorithm incorporating HSV-1 serostatus found that 11 of 70 persons with indeterminate HSV-2 ELISAs were Western blot-positive. CONCLUSIONS: Clinicians should consider selectively using a higher index value to define Focus ELISA HSV-2 positivity based on either HSV-1 serostatus or clinical circumstances.
Asunto(s)
Algoritmos , Anticuerpos Antivirales/sangre , Herpes Genital/diagnóstico , Herpes Genital/epidemiología , Herpesvirus Humano 2/inmunología , Adolescente , Adulto , Anciano , Instituciones de Atención Ambulatoria , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Prevalencia , Sensibilidad y Especificidad , Enfermedades de Transmisión Sexual/prevención & controlRESUMEN
Vaccines for infectious diseases have in the past, and will into the future, relied on a variety of surrogate markers to monitor vaccine efficacy. The primary surrogate markers have been either the antibody titer to vaccine antigens or the measurement of antibody function such as anti-viral neutralizing activity. In recent years, the measurement of T-cell function in conjunction with or independent of antibody measurements have been used to assess vaccine efficacy. ELISPOT, flow cytometry and intra-cellular staining methods are used to determine the impact of vaccines on immune mediators such as interleukins, interferons, MHC expression and pro-inflammatory mediators. The relevant B-cell and T-cell surrogate markers for vaccine efficacy is dependent on the vaccine being used, so that no universal set of surrogate markers can be applied to all vaccines. The use of T-cell surrogate markers can be complicated by the lack of sensitivity to accurately measure intra-cellular mediators. Although typically this is not a problem for infectious disease vaccines, it is a major problem for cancer vaccines.
Asunto(s)
Biomarcadores/metabolismo , Vacunas/uso terapéutico , Animales , Linfocitos B/metabolismo , Vacunas contra el Cáncer , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Citometría de Flujo , Humanos , Inmunoglobulina G/química , Modelos Biológicos , Linfocitos T/metabolismoRESUMEN
Focus Technologies developed an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a mu-capture IgM ELISA for the detection of West Nile virus (WNV)-specific antibodies based on a WNV preM/E protein recombinant antigen. Normal and disease state serum panels were used to assess the performance characteristics of the two WNV ELISA kits. Totals of 807 and 1,423 sera were used to assess the IgG ELISA and IgM ELISA kits, respectively. The Focus Technologies IgG ELISA had a sensitivity of 97.6% and a specificity of 92.1% (excluding non-WNV flavivirus sera). The comparative method for WNV IgG may lack sensitivity in detecting IgG in early WNV infection, so the specificity of the Focus IgG ELISA may be higher than 92.1%. When sera from patients either infected with or vaccinated against other flaviviruses were tested on the WNV IgG assay, 35% of the sera reacted as positive for WNV IgG. Yellow fever and Japanese encephalitis vaccinees were less reactive in the IgG ELISA than St. Louis and dengue fever patients. The Focus Technologies IgM ELISA had a sensitivity and a specificity of 99.3% (excluding the non-WNV flavivirus sera). The overall cross-reactivity for the IgM ELISA to flavivirus sera was 12%, with 31% of St. Louis encephalitis patients found to be WNV IgM positive and no yellow fever vaccinees found to be WNV IgM positive. In a selected population of 706 sera, 15 false-positive WNV IgM sera were identified. The use of a background subtraction method for the IgM ELISA eliminated all 15 false-positive results, giving a specificity of 100% for the Focus IgM ELISA.
Asunto(s)
Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/inmunología , Virus del Nilo Occidental/inmunología , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Antígenos Virales/genética , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Flavivirus/inmunología , Infecciones por Flavivirus/diagnóstico , Infecciones por Flavivirus/virología , Humanos , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/genética , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/virologíaRESUMEN
Two hundred forty-eight human immunodeficiency virus (HIV)-positive and 496 HIV-negative subjects in Uganda were tested by HerpeSelect herpes simplex virus type 2 enzyme-linked immunosorbent assay (ELISA) and Western blotting to optimize the ELISA for use in this population. A higher index cutoff value was required for optimal sensitivity and specificity, and overall performance of the assay was not affected by HIV status.
Asunto(s)
Anticuerpos Antivirales/sangre , Herpes Genital/diagnóstico , Herpesvirus Humano 2/inmunología , Juego de Reactivos para Diagnóstico , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Seronegatividad para VIH , Seropositividad para VIH/complicaciones , Humanos , Sensibilidad y Especificidad , UgandaRESUMEN
Focus Technologies has developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) kit that utilizes recombinant West Nile virus (WNV) antigens to detect WNV IgM in serum. We evaluate here the utility of the kit for detecting WNV IgM in cerebrospinal fluid (CSF). The sensitivity was evaluated by using 52 CSF specimens from the 2002 WNV season that were positive in both the Public Health Service Laboratories WNV IgM ELISA and an in-house WNV IgM ELISA with native WNV antigen. The specificity was evaluated with two groups of specimens: (i). 73 CSF specimens submitted for in-house WNV IgM ELISA testing from February through April 2003 and yielding a negative WNV IgM result and (ii). 60 CSF specimens determined to be positive for another virus by PCR testing. Using these 185 CSF specimens at a screening dilution of 1:2, the kit was determined to be 100% sensitive and 100% specific. Endpoint titers were determined for 20 IgM-positive CSF specimens by testing serial twofold dilutions and ranged from 1:8 to 1:512. Index values (specimen absorbance value/calibrator absorbance value) for the screening dilution (1:2) showed no correlation with IgM titers, whereas index values for higher dilutions showed significant correlation with IgM titers. CSF screening dilutions of greater than 1:2 are not recommended, however, due to the risk of obtaining false-negative results. These findings show that the Focus Technologies WNV IgM capture ELISA, when utilized as recommended, offers accurate qualitative detection of WNV IgM in CSF specimens.
Asunto(s)
Anticuerpos Antivirales/líquido cefalorraquídeo , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina M/líquido cefalorraquídeo , Virus del Nilo Occidental/inmunología , Humanos , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
Between 1 June and 31 December 2002, 30,677 serum samples and 4,554 cerebrospinal fluid (CSF) samples were tested for West Nile virus (WNV)-specific immunoglobulin M (IgM) by an in-house enzyme-linked immunosorbent assay (ELISA); 1,481 serum samples (4.8%) and 345 CSF samples (7.6%) were positive for WNV IgM. Positive samples were forwarded to public health service laboratories (PHSLs) for further testing. PHSLs supplied results from their WNV IgM ELISAs for 654 samples; 633 (97%) were positive. PHSLs supplied WNV plaque reduction neutralization test results for 128 samples; 123 (96%) were positive. WNV IgM seroconversion and seroreversion trends were evaluated for 749 patients who each provided two serum samples that were tested during the study period. Of 574 patients whose first serum sample was IgM negative, 41 (7%) seroconverted (the second serum sample was IgM positive); of 175 patients whose first serum sample was IgM positive, 22 (13%) seroreverted (the second serum sample was IgM negative). The seroreversion rate was directly proportional to the time between serum sample collection; whereas only 1% of patients whose sera were collected <20 days apart showed seroreversion, 54% of patients whose sera were collected >60 days apart showed seroreversion. Conversion and reversion trends for CSF were evaluated for 68 patients. Of 54 patients whose first CSF specimen was IgM negative, 9 (17%) converted; none of 14 patients whose first CSF specimen was IgM positive reverted. Concomitant detection of WNV IgM in serum and CSF was assessed for 1,188 patients for whom paired serum and CSF specimens were available; for all 130 patients for whom IgM was detectable in CSF, IgM was also detectable in serum. These findings show that an in-house WNV IgM ELISA accurately identifies patients with WNV infection, document WNV IgM conversion and reversion trends, and demonstrate that WNV IgM detection in CSF is accompanied by WNV IgM detection in serum.
Asunto(s)
Inmunoglobulina M/sangre , Inmunoglobulina M/líquido cefalorraquídeo , Laboratorios/normas , Fiebre del Nilo Occidental/diagnóstico , Virus del Nilo Occidental/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Salud Pública/normas , Reproducibilidad de los Resultados , Estaciones del Año , Pruebas Serológicas , Factores de Tiempo , Estados UnidosRESUMEN
Clinical, epidemiological, and laboratory diagnostic issues of human monocytotropic ehrlichiosis (HME) were investigated in a retrospective case study conducted at a national reference laboratory (Focus Technologies, formerly MRL Reference Laboratory), and at the University of Texas Medical Branch at Galveston, Texas, during 1997 and 1998. Standard questionnaires were sent to physicians for each laboratory-diagnosed patient 2 days to 2 weeks after immunofluorescent antibody assay results were available. Among the 41 cases for which data were obtained, 32 (78%) were definite cases of HME, and 9 (22%) were probable cases of HME. Tick bite or exposure to ticks was recorded in more than 97% of cases. The most prominent clinical findings were fever, abdominal tenderness, and regional lymphadenopathy. There was an association between age and severity of illness. The main laboratory findings included leukopenia, thrombocytopenia, and elevated aspartate aminotransferase and alanine aminotransferase. Clinical and laboratory findings were nonspecific and were not good predictors of the severity of illness. The 90% of patients who received doxycycline treatment underwent rapid clinical improvement with a favorable outcome. The usual duration of effective treatment with doxycycline was 7 to 10 days. This retrospective study is unique because it was based in a commercial reference laboratory setting that receives specimens from different geographic locations. The clinical and laboratory information from 41 patients provides insight into the epidemiological, clinical, and laboratory characteristics of HME.
Asunto(s)
Ehrlichiosis/diagnóstico , Ehrlichiosis/epidemiología , Ehrlichiosis/fisiopatología , Adolescente , Adulto , Niño , Preescolar , Ehrlichia chaffeensis , Femenino , Humanos , Inmunoensayo , Lactante , Laboratorios , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Encuestas y Cuestionarios , Enfermedades por Picaduras de GarrapatasRESUMEN
During the 2001 U. S. West Nile virus (WNV) season, 163 specimens were reactive in an in-house WNV-specific immunoglobulin M (IgM) screening enzyme-linked immunosorbent assay (ELISA) and were referred to either the Centers for Disease Control and Prevention or the appropriate state public health laboratory (CDC/SPHL) for additional testing. CDC/SPHL supplied results for 124 specimens that could be further evaluated in-house: 70 specimens were nonreactive in the CDC/SPHL WNV-specific IgM screening assay, and 54 specimens were reactive. These specimens were used to evaluate a modified in-house WNV-specific IgM ELISA that incorporated background subtraction to identify nonspecific reactivity and thus improve assay specificity. Of the 70 CDC/SPHL nonreactive samples, 49 (70%) were nonreactive in the modified ELISA; of the 54 CDC/SPHL reactive samples, 51 (94%) were reactive in the modified ELISA. Confirmatory studies performed by CDC/SPHL indicated that 38 CDC/SPHL screen-reactive specimens represented true WNV infection; all 38 specimens were reactive in the modified in-house WNV-specific IgM ELISA. These findings demonstrate that an in-house ELISA system for WNV-specific IgM effectively identifies patients with WNV infection.