RESUMEN
The objective of this study was to evaluate a novel automated random-access test, mariPOC CDI (ArcDia Ltd., Finland), for the detection of Clostridioides difficile glutamate dehydrogenase (GDH) and toxins A and B directly from fecal specimens. The mariPOC test was compared with both the GenomEra C. difficile PCR assay (Abacus Diagnostica Oy, Finland) and the TechLab C. diff Quik Chek Complete (Alere Inc.; now Abbot) membrane enzyme immunoassay (MEIA). Culture and the Xpert C. difficile assay (Cepheid Inc., USA) were used to resolve discrepant results. In total, 337 specimens were tested with the mariPOC CDI test and GenomEra PCR. Of these specimens, 157 were also tested with the TechLab MEIA. The sensitivity of the mariPOC test for GDH was slightly lower (95.2%) than that obtained with the TechLab assay (100.0%), but no toxin-positive cases were missed. The sensitivity of the mariPOC test for the detection of toxigenic C. difficile by analyzing toxin expression was better (81.6%) than that of the TechLab assay (71.1%). The analytical specificities for the mariPOC and the TechLab tests were 98.3% and 100.0% for GDH and 100.0% and 99.2% for toxin A/B, respectively. The analytical specificity of the GenomEra method was 100.0%. The mariPOC and TechLab GDH tests and GenomEra PCR had high negative predictive values of 99.3%, 98.3%, and 99.7%, respectively, in excluding infection with toxigenic C. difficile The mariPOC toxin A/B test and GenomEra PCR had an identical analytical positive predictive value of 100%, providing highly reliable information about toxin expression and the presence of toxin genes, respectively.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Infecciones por Clostridium , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Clostridioides , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Enterotoxinas/genética , Heces , Finlandia , Glutamato Deshidrogenasa/genética , Humanos , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
mariPOC is a novel point-of-care test system for rapid detection of respiratory tract infections. We compared the performance of the mariPOC test to that of bacterial culture for detecting group A streptococcus (GAS) in 219 pharyngitis patients (ages 1-64 years) and 109 healthy asymptomatic controls (ages 19-69 years). In addition, 42 patient samples were analyzed by quantitative PCR (qPCR). Of the 219 pharyngeal patient samples, 32 were positive in a GAS bacterial culture (prevalence 15%) and 65 (30%) in the mariPOC test. The amount of GAS in samples reported positive by the mariPOC test and negative by culture was, on average, 10-fold less than that of those positive in both methods. This indicated that the negative results in bacterial cultures were due to lower sensitivity. The qPCR results were positive and in line with the mariPOC results in 43% of the discordant samples studied. Two GAS culture-positive samples were negative by the mariPOC test. The prevalences of GAS in the control subjects were 2% and 6% by culture and mariPOC results, respectively. We conclude that the mariPOC antigen detection test is more sensitive than the conventional bacterial culture for the detection of GAS among symptomatic pharyngitis patients. The higher prevalence of GAS by the mariPOC test among symptomatic patients was probably not due to carriership, since among the control patients, the difference in the prevalence of GAS by the mariPOC test and culture was not nearly as high, 15% versus 4%, respectively. Clinical trials are needed to show the clinical importance of our findings.
Asunto(s)
Antígenos Bacterianos/análisis , Técnicas Microbiológicas/métodos , Faringitis/diagnóstico , Faringe/microbiología , Sistemas de Atención de Punto , Infecciones Estreptocócicas/diagnóstico , Streptococcus pyogenes/aislamiento & purificación , Adolescente , Adulto , Anciano , Antígenos Bacterianos/inmunología , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Faringitis/microbiología , Sensibilidad y Especificidad , Adulto JovenRESUMEN
During pregnancy, alterations take place in mother's immune system with the goal of maintaining a successful pregnancy, and delivering healthy offspring. Immune alterations include activation of the innate immune system and dampening of cell-mediated adaptive immunity. Due to these alterations, cell-mediated autoimmune diseases typically ameliorate during pregnancy. The objectives of this study were to evaluate whether C-reactive protein (CRP) concentration, a sensitive marker of systemic inflammation (1) is increased during MS pregnancy (2) predicts pregnancy-related co-morbidities associated with MS (3) predicts MS disease activity after delivery. CRP concentration was measured using a high sensitivity assay from seven prospectively collected serum samples of 41 MS patients and 19 controls during pregnancy and 6 months after delivery. Annualized relapse rates, EDSS, fatigue scores and obstetric details of the patients were recorded. Delivery-related CRP levels were significantly elevated both among MS patients and in controls. CRP levels were higher during pregnancy than during the postpartum period in both study groups. Delivery-related elevated CRP levels did not correlate with postpartum disease activity. MS patients with eventual gestational diabetes had a significantly higher median CRP in the beginning of pregnancy compared to non-diabetic MS patients (9.28 vs. 2.98 mg/l, p = 0.0025). MS patients reporting fatigue had a significantly higher CRP throughout pregnancy compared to patients without fatigue. Higher CRP values were associated with pregnancy-related co-morbidities but not with MS disease activity.
Asunto(s)
Proteína C-Reactiva/análisis , Esclerosis Múltiple/complicaciones , Complicaciones del Embarazo/sangre , Adulto , Comorbilidad , Complicaciones de la Diabetes/sangre , Fatiga/complicaciones , Femenino , Humanos , Esclerosis Múltiple/sangre , Periodo Posparto , Embarazo , Recurrencia , Adulto JovenRESUMEN
The aim of the study was to evaluate the performance of the new mariPOC(®) method against the direct fluorescent antibody assay (DFA) as the primary reference method for rapid virus detection from nasopharyngeal aspirates and swab samples. The study was an open prospective evaluation during the seasonal winter epidemics in the Mikkeli Central Hospital, Finland. Altogether, 283 samples were analyzed; 124 (43.8%) were from young children (<5 years old). Discrepant samples were resolved by PCR. With nasopharyngeal aspirate samples, the sensitivity and clinical specificity of the mariPOC(®) assay for influenza A virus and respiratory syncytial virus, were 85.7% (CI 69.7-95.2) and 90% (CI 52.0-80.5), and 100% and 99.5%, respectively. The mariPOC(®) performed less well with swab samples having sensitivities at 77.3% (CI 54.6-92.2) and 67.4% (CI 52-80.5), respectively. The specificities were as for nasopharyngeal aspirates. Importantly, similar performance was observed regardless of the cohort age group. In conclusion, the mariPOC(®) test system has a high potential and utility in duty units because it is fast, simple, and multianalyte. The importance of personnel training for proper sample collection should be emphasized.
Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Sistemas de Atención de Punto , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitiales Respiratorios/aislamiento & purificación , Virología/métodos , Preescolar , Finlandia , Humanos , Gripe Humana/virología , Nasofaringe/virología , Estudios Prospectivos , Infecciones por Virus Sincitial Respiratorio/virología , Sensibilidad y EspecificidadRESUMEN
COVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 min with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.
Asunto(s)
Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Adulto , Anciano , Antígenos Virales/análisis , COVID-19/inmunología , Reacciones Cruzadas/inmunología , Femenino , Finlandia/epidemiología , Humanos , Inmunoensayo/métodos , Masculino , Persona de Mediana Edad , Nasofaringe/virología , ARN Viral/genética , Reproducibilidad de los Resultados , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses. OBJECTIVE: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings. STUDY DESIGN: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens. RESULT: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR). CONCLUSION: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.
Asunto(s)
Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Infecciones del Sistema Respiratorio/diagnóstico , Rhinovirus/aislamiento & purificación , Humanos , Técnicas de Amplificación de Ácido Nucleico/normas , Sistemas de Atención de Punto , ARN Viral/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Diagnosis of human bocavirus 1 (HBoV1) has been based on qualitative PCRs detecting HBoV1 DNA or detection of HBoV1 mRNA. OBJECTIVE: This study aims to assess whether a rapid and automated HBoV1 antigen test is suitable for diagnosis of acute HBoV1 infection. STUDY DESIGN: HBoV1 antigen detection has been compared with quantitative HBoV1 DNA PCR and HBoV1 mRNA RT-PCR. RESULTS AND CONCLUSION: We conclude that HBoV1 antigen detection has higher clinical specificity and positive predictive value than HBoV1 DNA qualitative PCRs, yet a lower sensitivity than HBoV1 mRNA detection. Additionally, HBoV1 antigen detection is beneficial in its rapidity and availability as a point-of-care test.
Asunto(s)
Antígenos Virales/metabolismo , Bocavirus Humano/genética , Bocavirus Humano/inmunología , Infecciones por Parvoviridae/diagnóstico , ARN Mensajero/genética , Infecciones del Sistema Respiratorio/virología , Automatización , Niño , ADN Viral/genética , Femenino , Genotipo , Humanos , Masculino , Infecciones por Parvoviridae/inmunología , Fenotipo , Sistemas de Atención de Punto , ARN Viral/genética , Infecciones del Sistema Respiratorio/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Carga ViralRESUMEN
Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Técnicas de Tipificación Bacteriana , Recuento de Colonia Microbiana , Relación Dosis-Respuesta a Droga , Humanos , Inmunoensayo/métodos , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Oxacilina/farmacología , Sensibilidad y Especificidad , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Two-photon excitation fluorometry (TPX) is a separation-free bioaffinity assay technique which enables accurate diagnostic testing in microvolumes. The technology is currently commercially applied in an automated mariPOC® test system for rapid phenotypic multi-microbe detection of pathogen antigens. The first TPX applications for diagnostics were intended for respiratory infection testing from nasopharyngeal and oropharyngeal samples. Feces and urine are more complex sample matrices and contain substances that may interfere with immunoassay binding or fluorescence detection. Our objective was to study the suitability of these complex matrices in the TPX technique. As expected, feces and urine elevated fluorescence levels but the methodology has the unique property of compensating for matrix effects. Compensation allows reliable separation of specific fluorescence from the fluorescence caused by the matrix. The studied clinical samples did not contain immunoassay inhibitors. The results suggest that the methodology is robust and may provide reliable testing of feces and urine samples with high accuracy.
Asunto(s)
Antígenos Bacterianos/análisis , Fluorometría , Nasofaringe/microbiología , Heces/microbiología , Fluorometría/instrumentación , Fluorometría/métodos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Orina/microbiologíaRESUMEN
A new technique for separation-free detection of antigen-specific antibodies is presented. The new technique employs antibody bridging assay principle and the recently developed ArcDia TPX fluorescence detection technology. According to the assay scheme, antibody molecules from the sample bind with one arm to an antigen on polymer microspheres and with the other arm to a fluorescently labeled secondary antigen reagent. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique, an assay for anti-adenovirus antibodies was constructed. The function of the assay method was tested both with monoclonal anti-adenovirus antibody preparation (standard analyte), and with positive serum samples. Standard class-specific ELISA was used as a reference method. The new assay method provides comparable sensitivity and precision, and wider dynamic range for IgG antibodies than the ELISA method. The standard curve showed linear response (R(2)=0.999) with a dynamic range of three orders of magnitude, detection limit (mean+3S.D.) of 8 pM, and intra-assay signal precision of 5%. Applicability of the new method for clinical serodiagnostics is discussed.
Asunto(s)
Anticuerpos/sangre , Fluorometría/métodos , Inmunoensayo/métodos , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Antígenos , Antígenos Virales , Proteínas de la Cápside/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Isotipos de Inmunoglobulinas/sangre , Indicadores y Reactivos , Cinética , Ratones , MicroesferasRESUMEN
A new lab-on-a-chip compatible binding assay platform is introduced. The platform combines dry-chemistry bioaffinity reagents and the recently introduced ArcDia TPX binding assay technique. The technique employs polymer microspheres as a solid phase reaction carrier, fluorescently labeled antibody conjugates, and detection of fluorescence emission from the surface of individual microspheres by two-photon excitation fluorescence. Signal response of the technique is independent of the reaction volume, thus the technique is particularly well suited for detection of bioaffinity reactions from miniature volumes. Performance of the new assay platform is studied by means of an immunometric assay of human alpha-fetoprotein (hAFP) in 384-plate format, and the results are compared to those of a corresponding wet-chemistry assay method. The results show that the ArcDia TPX detection technique can be combined with dry-chemistry reagents without compromises in assay performance. The microchip field has so far been characterized with a lack of microchip-compatible detection platforms which would allow cost-effective microchip design and sensitive bioaffinity detection. The presented detection technique is expected to provide a solution for this shortage.
Asunto(s)
Inmunoensayo/métodos , Microquímica/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , alfa-Fetoproteínas/análisis , Humanos , Inmunoensayo/instrumentación , Microquímica/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Integración de Sistemas , alfa-Fetoproteínas/inmunologíaRESUMEN
Exposure of the skin to UV radiation induces local inflammation. We hypothesized that inflammation induced by erythemal UV-B irradiation could elevate levels of serum C-reactive protein (CRP) and that suberythemal repeating doses of solar-simulating UV radiation (SSR) would produce photoadaptation to such inflammation. Separation-free high-sensitivity assays of CRP show an increase by 42% (P = 0.046) in CRP concentrations in healthy human subjects 24 h after a 3 minimal erythemal dose (MED) dose of UV-B delivered onto a 100 cm2 skin area. Preceding daily suberythemal doses of whole-body SSR for 10 or 30 consecutive days completely prevented the CRP increase. UV-B-induced skin erythema was partially attenuated by 30 preceding days of SSR only (P = 0.00066). After 10 daily SSR doses, the mean baseline CRP concentrations (0.24 +/- 0.21 mg/L) declined by 35% (P = 0.018). Using high-sensitivity analysis of serum CRP as the endpoint marker for cutaneous inflammation, we show that acute exposure of even a relatively small skin area to erythemal UV-B induces skin inflammation detectable also at the systemic level and that photoadaptation by preceding repeating suberythemal doses of SSR reduces signs of inflammation. Our data complement the view given by previous studies in that local photoadaptation also has systemic manifestations.
Asunto(s)
Adaptación Fisiológica/efectos de la radiación , Proteína C-Reactiva/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta , Proteína C-Reactiva/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Eritema/metabolismo , Eritema/prevención & control , Humanos , Inflamación/complicaciones , Piel/metabolismo , Factores de TiempoRESUMEN
A novel methodology is introduced for rapid serological diagnosis. This methodology combines the antibody bridging assay principle with the measurement of antibody avidity. The combination allows the determination of the infection phase with a single dilution of a single sample of serum. This is a significant improvement on current serological techniques which often require either paired-sample testing (IgG/IgM serology) or testing of the sample in several dilutions (IgG avidity testing). Assay methods were developed on two immunoassay platforms; the heterogeneous time-resolved fluoroimmunoassay and the separation-free two-photon excitation fluorometry. The new methods were compared to conventional class-specific IgG/IgM and IgG avidity techniques. The major findings were that the avidity results of the new methodology were independent of the sample dilution (specific antibody concentration in serum) and consistent between immunoassay platforms. This new methodology is simple, rapid, and quick to perform. It provides the possibility of running serodiagnostic tests at point-of-care with bench-top random-access analyzers.
Asunto(s)
Infecciones por Adenoviridae/diagnóstico , Anticuerpos Antivirales/sangre , Afinidad de Anticuerpos , Sistemas de Atención de Punto , Preescolar , Fluoroinmunoensayo/métodos , Fluorometría/métodos , Humanos , Pruebas Serológicas/métodos , Factores de TiempoRESUMEN
New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.
Asunto(s)
Antígenos Virales/análisis , Fluorometría/métodos , Inmunoensayo/métodos , Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Humanos , Indicadores y Reactivos , Nasofaringe/virología , Sistemas de Atención de Punto , Sensibilidad y EspecificidadRESUMEN
We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.