RESUMO
Shiga toxin (stx)-producing Escherichia coli (STEC) causes potentially severe gastrointestinal infections. Due to its public health importance, control measures are required, and carriers may need to refrain from work or daycare when the risk of spread to vulnerable people is high. We evaluated the use of direct stool multiplex PCR compared to culture for primary STEC diagnostics and for follow-up in order to update the national guidelines for STEC monitoring. We analyzed primary and follow-up samples of 236 STEC PCR-positive cases at HUSLAB, Helsinki, Finland in 2016-2017, altogether 858 samples. All STEC PCR-positive samples were inoculated on non-selective chromogenic agar plates. Culture positivity was confirmed from culture sweeps by PCR. 211 (89%) of the cases were culture positive in their primary sample. Of all primary and follow-up samples, 499 were PCR positive and of these 450 (90%) were culture positive. PCR-negative follow-up samples were available from 125 cases. Of these, 88 cases were followed for at least three consecutive PCR-negative samples. Two cases (2%) had culture-positive sample(s) after two consecutive PCR-negative samples. The median time for STEC clearance was 22-23 days. The laboratory-developed multiplex PCR test used in this study is a reliable method for STEC diagnostics and follow-up in a clinical laboratory. When non-selective methodology is used, the majority of PCR-positive samples (90%) are also culture positive. Furthermore, only two cases (2%) in our material had two consecutive PCR-negative samples followed by positive samples. Consequently, to demonstrate the clearance from STEC infection, we consider two PCR-negative follow-up samples sufficient. The Finnish national guidelines for STEC monitoring have been updated accordingly.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Humanos , Escherichia coli Shiga Toxigênica/genética , Reação em Cadeia da Polimerase Multiplex , Seguimentos , Infecções por Escherichia coli/diagnóstico , Técnicas Bacteriológicas/métodos , Fezes , Proteínas de Escherichia coli/genéticaRESUMO
The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.
Assuntos
Antígenos Virais/análise , Teste Sorológico para COVID-19/métodos , Transferência Ressonante de Energia de Fluorescência , SARS-CoV-2/isolamento & purificação , Antígenos Virais/imunologia , COVID-19/diagnóstico , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Estudo de Prova de Conceito , Proteínas Recombinantes/imunologia , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de TempoRESUMO
BACKGROUND: Understanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR. METHODS: This population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR. RESULTS: All 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. CONCLUSIONS: The clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.
Assuntos
Teste de Ácido Nucleico para COVID-19/normas , Adulto , Idoso , Teste de Ácido Nucleico para COVID-19/métodos , Reações Falso-Negativas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição Aleatória , Kit de Reagentes para Diagnóstico/normasRESUMO
Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.
Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , COVID-19/epidemiologia , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
BACKGROUND: Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. OBJECTIVES: We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. RESULTS: In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8â¯hours earlier than that obtained with the large-scale PCR tests. CONCLUSIONS: While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.
Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pneumonia Viral/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus , COVID-19 , Teste para COVID-19 , Criança , Pré-Escolar , Técnicas de Laboratório Clínico , Serviço Hospitalar de Emergência/estatística & dados numéricos , Feminino , Finlândia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , SARS-CoV-2 , Sensibilidade e Especificidade , Atenção Terciária à Saúde/estatística & dados numéricos , Adulto JovemRESUMO
BackgroundTick-borne encephalitis (TBE) is a potentially severe neurological disease caused by TBE virus (TBEV). In Europe and Asia, TBEV infection has become a growing public health concern and requires fast and specific detection.AimIn this observational study, we evaluated a rapid TBE IgM test, ReaScan TBE, for usage in a clinical laboratory setting.MethodsPatient sera found negative or positive for TBEV by serological and/or molecular methods in diagnostic laboratories of five European countries endemic for TBEV (Estonia, Finland, Slovenia, the Netherlands and Sweden) were used to assess the sensitivity and specificity of the test. The patients' diagnoses were based on other commercial or quality assured in-house assays, i.e. each laboratory's conventional routine methods. For specificity analysis, serum samples from patients with infections known to cause problems in serology were employed. These samples tested positive for e.g. Epstein-Barr virus, cytomegalovirus and Anaplasma phagocytophilum, or for flaviviruses other than TBEV, i.e. dengue, Japanese encephalitis, West Nile and Zika viruses. Samples from individuals vaccinated against flaviviruses other than TBEV were also included. Altogether, 172 serum samples from patients with acute TBE and 306 TBE IgM negative samples were analysed.ResultsCompared with each laboratory's conventional methods, the tested assay had similar sensitivity and specificity (99.4% and 97.7%, respectively). Samples containing potentially interfering antibodies did not cause specificity problems.ConclusionRegarding diagnosis of acute TBEV infections, ReaScan TBE offers rapid and convenient complementary IgM detection. If used as a stand-alone, it can provide preliminary results in a laboratory or point of care setting.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/diagnóstico , Imunoglobulina M/sangue , Anticorpos Antivirais/sangue , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
Number of tick-borne encephalitis (TBE) cases has increased and new foci have emerged in Finland during the last decade. We evaluated risk for locally acquired TBE in the capital region inhabited by 1.2 million people. We screened ticks and small mammals from probable places of TBE virus (TBEV) transmission and places without reported circulation. The TBEV positive samples were sequenced and subjected to phylogenetic analysis. Within the study period 2007-2017, there was a clear increase of both all TBE cases and locally acquired cases in the Helsinki area. The surveillance of ticks and small mammals for TBEV confirmed four distinct TBEV foci in the Helsinki area. All detected TBEV strains were of the European subtype. TBEV genome sequences indicated that distinct TBEV lineages circulate in each focus. Molecular clock analysis suggested that the virus lineages were introduced to these foci decades ago. In conclusion, TBE has emerged in the mainland of Helsinki area during the last decade, with at least four distinct virus lineages independently introduced into the region previously. Although the overall annual TBE incidence is below the threshold for recommending general vaccinations, the situation requires further surveillance to detect and prevent possible further emergence of local TBE clusters.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/classificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/virologia , Variação Genética , Mamíferos/virologia , Carrapatos/virologia , Animais , Transmissão de Doença Infecciosa , Vírus da Encefalite Transmitidos por Carrapatos/genética , Finlândia/epidemiologia , Genótipo , Humanos , Incidência , Epidemiologia Molecular , Filogenia , Análise de Sequência de DNARESUMO
The generalist tick Ixodes ricinus is the most important vector for tick-borne pathogens (TBP), including Borrelia burgdorferi sensu lato, in Europe. However, the involvement of other sympatric Ixodes ticks, such as the specialist vole tick I. trianguliceps, in the enzootic circulations of TBP remains unclear. We studied the distribution of I. ricinus and I. trianguliceps in Central Finland and estimated the TBP infection likelihood in the most common rodent host in relation with the abundance of the two tick species. Ixodes trianguliceps was encountered in all 16 study sites whereas I. ricinus was frequently observed only at a quarter of the study sites. The abundance of I. ricinus was positively associated with open water coverage and human population density around the study sites. Borrelia burgdorferi s. l.-infected rodents were found only in sites where I. ricinus was abundant, whereas the occurrence of other TBP was independent of I. ricinus presence. These results suggest that I. trianguliceps is not sufficient, at least alone, in maintaining the circulation of B. burgdorferi s. l. in wild hosts. In addition, anthropogenic factors might affect the distribution of I. ricinus ticks and, hence, their pathogens, thus shaping the landscape of tick-borne disease risk for humans.
Assuntos
Arvicolinae/parasitologia , Vetores de Doenças , Ixodes/patogenicidade , Simpatria , Doenças Transmitidas por Carrapatos/veterinária , Animais , Finlândia/epidemiologia , Humanos , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/transmissãoRESUMO
In most locations except for Russia, tick-borne encephalitis is mainly caused by the European virus subtype. In 2015, fatal infections caused by European and Siberian tick-borne encephalitis virus subtypes in the same Ixodes ricinus tick focus in Finland raised concern over further spread of the Siberian subtype among widespread tick species.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/virologia , Adulto , Idoso , Animais , Evolução Fatal , Feminino , Finlândia/epidemiologia , Humanos , Masculino , RNA Viral/genética , RNA Viral/isolamento & purificação , Carrapatos/virologiaRESUMO
BACKGROUND: The tick species Ixodes ricinus and I. persulcatus are of exceptional medical importance in the western and eastern parts, respectively, of the Palaearctic region. In Russia and Finland the range of I. persulcatus has recently increased. In Finland the first records of I. persulcatus are from 2004. The apparent expansion of its range in Finland prompted us to investigate if I. persulcatus also occurs in Sweden. METHODS: Dog owners and hunters in the coastal areas of northern Sweden provided information about localities where ticks could be present. In May-August 2015 we used the cloth-dragging method in 36 localities potentially harbouring ticks in the Bothnian Bay area, province Norrbotten (NB) of northern Sweden. Further to the south in the provinces Västerbotten (VB) and Uppland (UP) eight localities were similarly investigated. RESULTS: Ixodes persulcatus was detected in 9 of 36 field localities in the Bothnian Bay area. Nymphs, adult males and adult females (n = 46 ticks) of I. persulcatus were present mainly in Alnus incana - Sorbus aucuparia - Picea abies - Pinus sylvestris vegetation communities on islands in the Bothnian Bay. Some of these I. persulcatus populations seem to be the most northerly populations so far recorded of this species. Dog owners asserted that their dogs became tick-infested on these islands for the first time 7-8 years ago. Moose (Alces alces), hares (Lepus timidus), domestic dogs (Canis lupus familiaris) and ground-feeding birds are the most likely carriers dispersing I. persulcatus in this area. All ticks (n = 124) from the more southern provinces of VB and UP were identified as I. ricinus. CONCLUSIONS: The geographical range of the taiga tick has recently expanded into northern Sweden. Increased information about prophylactic, anti-tick measures should be directed to people living in or visiting the coastal areas and islands of the Baltic Bay.
Assuntos
Ixodes/classificação , Infestações por Carrapato/epidemiologia , Animais , DNA Ribossômico/química , DNA Ribossômico/genética , Reservatórios de Doenças , Cães , Feminino , Geografia , Humanos , Ilhas , Ixodes/genética , Masculino , Ninfa , Análise de Sequência de DNA , Suécia/epidemiologia , Taiga , Infestações por Carrapato/parasitologiaRESUMO
The first tick-borne encephalitis (TBE) cases in Kotka, Finland appeared in 2010. Altogether ten human cases have been diagnosed by 2014. Four had long-lasting sequelae. We collected 195 Ixodes ricinus ticks, nine rodents, and eleven shrews from the archipelago of Kotka in 2011. Three Siberian subtype TBE virus (TBEV) strains were isolated from the ticks and three mammals were positive for TBEV antibodies. The archipelago of Kotka is a newly emerged TBE focus of Siberian subtype TBEV circulating notably in I. ricinus. The patients had on average longer hospitalization than reported for the European subtype infection.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/virologia , Ixodes/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Finlândia/epidemiologia , Humanos , Ilhas , Pessoa de Meia-Idade , RNA Viral , Adulto JovemRESUMO
BACKGROUND: In many European countries (including Finland, Estonia, Latvia and Russia) two subtypes of tick-borne encephalitis virus (TBEV) occur with overlapping geographic distribution yet with apparently different severity and persistence of symptoms. However, it has not usually been possible to distinguish these infections in the laboratory, as TBEV RNA or sequences have rarely been retrieved from patients seeking medical care in the second phase of infection when the neurological symptoms occur, and serological tests have so far not been able to discriminate between the subtype-specific responses. OBJECTIVES: The aim of this study was to assess the applicability of a µ-capture enzyme immunoassay (EIA) based on TBEV prME subviral particles produced in mammalian cells from Semliki-Forest virus replicons (SFV-prME EIA) to distinguish reactivity to European and Siberian strains of TBEV. STUDY DESIGN: Altogether 54 TBEV IgM positive acute human serum samples and 6 positive cerebrospinal fluid (CSF) samples from different regions of Finland were tested in EIA with subtype-specific antigens and TBEV-IgM subtype-specific index ratios were determined. RESULTS: All 30 samples from patients whose transmission had occurred in foci where only Siberian subtype of TBEV is occurring had an index ratio of more than 1.8, whereas all 30 acute TBE samples from an area where only European subtype circulates had an index ratio below 1.5. CONCLUSIONS: We conclude that the assay is a useful tool to distinguish between acute infections of European and Siberian strains of TBEV, and should help in further studies of the clinical outcome of these two subtypes.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/classificação , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/imunologia , Diagnóstico Diferencial , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Europa (Continente) , Humanos , Imunoensaio , Imunoglobulina M/sangue , Imunoglobulina M/líquido cefalorraquidiano , Cadeias mu de Imunoglobulina/sangue , Cadeias mu de Imunoglobulina/líquido cefalorraquidiano , SibériaRESUMO
Tick-borne encephalitis virus (TBEV) is a member of the family Flaviviridae. It is transmitted by Ixodes spp. ticks in a cycle involving rodents and small mammals. TBEV has three subtypes: European, Siberian and Far Eastern. The virus causes thousands of cases of meningoencephalitis in Europe annually, with an increasing trend. The increase may be attributed to a complex network of elements, including climatic, environmental and socio-economic factors. In an attempt to understand the evolutionary history and dispersal of TBEV, to existing genetic data we add two novel complete ORF sequences of TBEV strains from northern Europe and the completion of the genome of four others. Moreover, we provide a unique measure for the natural rate of evolution of TBEV by studying two isolations from the same forest on an island in Åland archipelago 44 years apart. For all isolates, we analysed the phylogeny, rate of evolution and probable time of radiation of the different TBEV strains. The results show that the two lineages of TBEV in different Ixodes species have evolved independently for approximately 3300 years. Notably, rapid radiation of TBEV-Eur occurred approximately 300 years ago, without the large-scale geographical clustering observed previously for the Siberian subtype. The measurements from the natural rate of evolution correlated with the estimates done by phylogenetic programs, demonstrating their robustness.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/virologia , Evolução Molecular , Animais , Sequência de Bases , Encefalite Transmitida por Carrapatos/epidemiologia , Estônia/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Finlândia/epidemiologia , Variação Genética/genética , Humanos , Ixodes/virologia , Masculino , Camundongos , Epidemiologia Molecular , Dados de Sequência Molecular , FilogeniaAssuntos
Vetores Artrópodes/virologia , Doenças Transmissíveis Emergentes/epidemiologia , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/epidemiologia , Ixodes/virologia , Animais , Arvicolinae/virologia , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/transmissão , Encefalite Transmitida por Carrapatos/virologia , Finlândia/epidemiologia , Humanos , Camundongos , Filogenia , Prevalência , RNA Viral/sangue , Doenças dos Roedores/epidemiologia , Doenças dos Roedores/transmissão , Doenças dos Roedores/virologiaRESUMO
Rodents might maintain tick-borne encephalitis virus (TBEV) in nature through latent persistent infections. During 2 subsequent winters, 2008 and 2009, in Finland, we detected RNA of European and Siberian subtypes of TBEV in Microtus agrestis and Myodes glareolus voles, respectively. Persistence in rodent reservoirs may contribute to virus overwintering.
Assuntos
Animais Selvagens/virologia , Arvicolinae/virologia , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/veterinária , Doenças dos Roedores/epidemiologia , Animais , Reservatórios de Doenças/virologia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/virologia , Finlândia/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças dos Roedores/virologia , Estações do AnoRESUMO
Tick-borne encephalitis (TBE) is a central nervous system infection caused by a flavivirus [tick-borne encephalitis virus (TBEV)], transmitted by Ixodes ticks and endemic in a large region in Eurasia. We collected 2411 ticks from Finland and Russia in 2003-2008, screened them for TBEV by RT-PCR and isolated and analysed eight strains belonging to all three TBEV subtypes; in addition, we obtained two European-subtype strains from human serum samples. TBEV RNA prevalence in unengorged ticks was approximately 1â% both in the northernmost TBE-endemic areas of Europe in Finland and Russian Karelia, and in Siberia in Buryatia. In Finland, both Ixodes ricinus and Ixodes persulcatus ticks were found from distinct areas and, in Russian Karelia, were overlapping in the same study site. TBEV E and NS3 gene sequences obtained showed a variability of 0-4â% within European-subtype strains, 2-9â% for Siberian-subtype strains and 3-13â% for Far Eastern-subtype strains.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/virologia , Ixodes/virologia , Animais , Análise por Conglomerados , Finlândia , Genótipo , Humanos , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Federação Russa , Análise de Sequência de DNA , Soro/virologia , Proteínas não Estruturais Virais/genéticaRESUMO
Recently, we presented a simple method for generating biological functional protein-based nanoparticles that are ready for use as label agents in bioaffinity assays (Jääskeläinen et al., 2007 Small 3:1362-1367). In this process, the particle shell (ferritin protein) and binding molecules are conjugated via genetic fusion, and particles with binding capacity are produced in a single bacterial cultivation. Production is combined with simple, non-chromatographic purification during which Europium ions are introduced into particles to serve as marker agents. Denaturation-refolding has previously performed by means of pH changes. Here, we test urea as an alternative agent for denaturation, and examine techniques to improve refolding of the functional particles. Three different types of binding molecules were employed in our experiments: biotin carboxyl carrier protein (a small protein with 87 amino acids), single chain antibody fragment (a complex binding protein) and calmodulin-binding peptide (27 amino acids). Urea was successfully utilized to generate functional particles with inherent binding activity and label function. Additionally, particle yield was effectively optimized by analyzing various refolding and bacterial production conditions. Our results clearly demonstrate that this simple biological method of producing functional ferritin-based particles is flexible, and different types of binding moieties can be applied by adjusting the production conditions.
Assuntos
Biotecnologia/métodos , Ferritinas/metabolismo , Nanopartículas , Desnaturação Proteica , Dobramento de Proteína , Renaturação Proteica , Ureia/metabolismoRESUMO
Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles.
Assuntos
Nanopartículas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Cátions/química , Eletroforese em Gel de Poliacrilamida , Európio/química , Ferritinas/química , Ferritinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Imunoensaio/métodos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Proteínas Recombinantes/imunologia , Tireotropina/química , Tireotropina/imunologia , Tireotropina/metabolismoAssuntos
Apoferritinas/química , Apoferritinas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Compostos Inorgânicos/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Proteínas de Bactérias/genética , Cristalização/métodos , Dimerização , Fermentação , Teste de Materiais , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Nanotecnologia/métodos , Tamanho da Partícula , Engenharia de Proteínas/métodos , Propriedades de SuperfícieRESUMO
We isolated 11 Siberian subtype tickborne encephalitis virus (TBEV) strains from Ixodes persulcatus ticks from a TBEV-endemic focus in the Kokkola Archipelago, western Finland. Thus I. persulcatus and the Siberian TBEV are reported in a focus considerably northwest of their previously known range in eastern Europe and Siberia.