RESUMEN
Enterovirus 71 (EV71) has caused large hand, foot, and mouth disease (HFMD) epidemics among young children, and EV71 infection is the leading cause of severe HFMD cases and deaths. In mainland China, the prevalence and risk factors of non-C4 EV71 strains are still unclear. In this study, we monitored non-C4 strains over a 10-year HFMD epidemiological surveillance period in Xiamen. The 5'UTR and VP1 coding region of EV71 strains were amplified by RT-nested PCR and sequenced. Thirty-two non-C4 EV71 strains were identified during 2009-2018. This study provides important information about the prevalence of EV71 in China that will be applicable for development of vaccines and diagnostic reagents as well as establishment of policies for HFMD prevention and control.
Asunto(s)
Proteínas de la Cápside/genética , Enterovirus Humano A/clasificación , Enfermedad de Boca, Mano y Pie/epidemiología , Regiones no Traducidas 5' , Niño , China/epidemiología , Enterovirus Humano A/genética , Enterovirus Humano A/aislamiento & purificación , Enfermedad de Boca, Mano y Pie/virología , Humanos , Masculino , Filogenia , Prevalencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Coxsackievirus A10 (CVA10) recently has become one of the major pathogens of hand, foot, and mouth disease (HFMD) in children worldwide, but no cure or vaccine against CVA10 is available yet. Serological evaluation of herd immunity to CVA10 will promote the development of vaccine. The traditional neutralization assay based on inhibition of cytopathic effect (Nt-CPE) is a common method for measuring neutralizing antibody titer against CVA10, which is time-consuming and labor-intensive. In this study, an efficient neutralization test based on a monoclonal antibody (mAb) 3D1 against CVA10, called Elispot-based neutralization test (Nt-Elispot), was developed. In the Nt-Elispot, the mAb 3D1 labeled with horseradish peroxidase (HRP) was used to detect the CVA10-infected RD cells at a 1:4000 dilution and the optimal infectious dose of CVA10 was set at 105 TCID50/well when combined with a fixed incubation time of 14 h. Compared with the Nt-CPE, the Nt-Elispot method effectively shortened the detection period and presented a good correlativity with it. Using the Nt-Elispot, a total of 123 sera from healthy children were tested for neutralizing antibody against CVA10, demonstrating that the overall seroprevalence was 49.3% (54/123) and the geometric mean titer (GMT) had been calculated as 574.2. Furthermore, 2 anti-CVA10 neutralizing mAbs were obtained by screening via the Nt-Elispot. Overall, the established Nt-Elispot could be used as an efficient and high-throughput method for evaluating immunity to CVA10 and screening the neutralizing antibodies.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Enterovirus/inmunología , Enfermedad de Boca, Mano y Pie/inmunología , Pruebas de Neutralización/métodos , Preescolar , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Lactante , Estudios SeroepidemiológicosRESUMEN
Zika virus (ZIKV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) share the same mosquito vectors and have similar clinical manifestations early stage of infection. Therefore, simultaneously differentiating these viruses from each other is necessary. We developed a multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the differentiation of these four viruses in a single tube. The linear range was established by regression analysis, and the R2 value for each virus was ≥0.98, and the 95% lower limit of detection for each virus was as follows (copies/reaction): ZIKV-Asian, 9; ZIKV-Africa, 15; CHIKV, 11; DENV-1, 19; DENV-2, 13; DENV-3, 24; DENV-4, 36; and YFV, 17. Meanwhile, our multiplex real-time RT-PCR has a good consistency with the commercial singleplex assay. In summary, the developed assay can be effectively used for the diagnosis of ZIKV, DENV, CHIKV, and YFV infections.
Asunto(s)
Fiebre Chikungunya/diagnóstico , Dengue/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fiebre Amarilla/diagnóstico , Infección por el Virus Zika/diagnóstico , África , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Virus Chikungunya/aislamiento & purificación , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular/métodos , Factores de Tiempo , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/aislamiento & purificación , Virus Zika/genética , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/virologíaRESUMEN
OBJECTIVE: To investigate the prevalence of occult HBV infection in the normal population in Xiamen. METHODS: 4 437 registered permanent residents, aged 1-59 years old, were selected in Xiamen using stratified random sampling method from September to October in 2006. Serum samples were obtained, the basic characteristics, inoculation of HBV vaccine, and liver disease were surveyed. The serum samples were tested five HBV seroimmunological markers. The HBsAg-negative specimens were subjected to HBV-DNA detection by nested PCR targeting for multiple gene segments. The amplified products were sequenced and the sequence was used for determination of HBV genotype and mutation analysis of amino acids located in HBsAg "a" epitope. Subjects with serum detectable HBV-DNA and negative result of HBsAg were considered as occult HBV infection. RESULTS: Among the 4 437 subjects, 482 individuals were observed HBsAg positive and 3 944 were observed negative. Of the 3 955 HBsAg- negative specimens, 27 occult HBV infections were determined with the positive rate of 0.68% (27/3 955). There were 16 samples with genotype B and 11 with genotype C. 3 types of amino acid (AA) mutation (M133T, T140I, G145R) that influence "a" epitope conformation were observed in 9 subjects with occult HBV infection. S region was successfully sequenced in 312 of the 482 HBsAg positive samples. In subjects with occult HBV infection, the infection rate of genotype C HBV (40.74%, 11/27), inoculation rate of HBV vaccine (62.96%, 17/27), positive rate of HBsAb (51.85%, 14/27), and mutation rate of critical amino acid of "a" epitope (33.33%, 9/27) were higher than HBsAg positive individuals (22.76% (71/312), 13.78% (43/312),0.32% (1/312),0.99% (31/312), respectively), and all the difference were significant (χ(2) = 4.29, 41.26, 156.00, 13.07, respectively, and P value = 0.038, <0.001, <0.001, <0.001, respectively). While the average age in subjects with occult HBV infection (18.3 ± 16.2) were lower than that in HBsAg positive infection (34.4 ± 11.6), and the difference was significant (t = 6.67, P < 0.001). The reactive rate of HBeAb (11.11%, 3/27) and HBcAb (62.96%, 17/27) in subjects with occult HBV infection were lower than that in HBsAg positive infection (74.36% (232/312), 98.40% (307/312)), and the difference were significant (χ(2) = 46.74, 73.78, respectively, and P value <0.001, <0.001, respectively). CONCLUSION: In normal population in Xiamen, the infection rate of genotype C, the positive rate of HBsAb, the HBV vaccination rate, and the key AA mutation rate in "a" epitope are significantly higher in occult HBV infection than in HBsAg positive infection, and the age, the positive rate of HBeAb and HBcAb are significantly lower.
Asunto(s)
Análisis Mutacional de ADN , Genotipo , Hepatitis B/diagnóstico , Adolescente , Adulto , Niño , Preescolar , Anticuerpos contra la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Virus de la Hepatitis B , Humanos , Lactante , Persona de Mediana Edad , Mutación , Prevalencia , VacunaciónRESUMEN
Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-106 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings.
Asunto(s)
Infecciones por Enterovirus , Enterovirus , Nanopartículas , Humanos , Nanopartículas/química , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/diagnóstico , Enterovirus/genética , Enterovirus/clasificación , Enterovirus/aislamiento & purificación , Código de Barras del ADN Taxonómico/métodos , Sensibilidad y Especificidad , Técnicas de Genotipaje/métodos , Genotipo , ARN Viral/genéticaRESUMEN
SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.
Asunto(s)
Técnicas Biosensibles , COVID-19 , COVID-19/diagnóstico , Humanos , Microesferas , Reacción en Cadena de la Polimerasa Multiplex , Mutación , ARN Viral/genética , SARS-CoV-2/genéticaRESUMEN
OBJECTIVE: To identify the etiology of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city from 11 to 17 May, 2011. METHODS: A total of ten patients' throat swabs, anal swabs and cerebrospinal fluid were collected and detected by RT-PCR for pan-enterovirus. The samples containing detectable pan-enterovirus were tested by PCR with genotype-specific general primers located in VP1 region of enterovirus genotype A, B and C (HEV-A, B and C). The PCR products of VP1 segment were purified and sequenced, and phylogenetic analysis was performed. Meanwhile, the pathogens in those samples were isolated in Vero cell culture. Homologous analysis of VP1 sequences were carried out for the cultured virus samples and the original clinical samples to identify the outbreak etiology. RESULTS: Among the ten cases, seven cases were positive for pan-enterovirus nucleic acid. When tested by genotype-specific PCR, the throat and anal swab samples from those 7 patients were positive with HEV-B VP1 primers. Meanwhile, the HEV-B VP1 segments were sequenced and phylogenetic analyzed, which indicated the seven cases were all infected by enterovirus Echo 30. The sequences from those samples had homology of 95.3% - 97.1% with the epidemic strains in Zhejiang, 2004. Out of the seven cases, the sequences of XM2, XM3, XM4, XM8 throat swab samples and XM3, XM6 throat samples showed 99.4% - 100.0% homology which were different from the sequence of XM1, and the homology was 92.8% - 93.4%. Furthermore, the viruses were isolated using Vero cells from XM1, XM2, XM3, XM4 and XM8 throat swab samples, and the VP1 sequence showed more than 99.9% homology with the original specimens. CONCLUSION: The local outbreak of aseptic encephalitis was caused by Echo 30 of enterovirus genotype B, and the epidemic strains may have different genetic background.
Asunto(s)
Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Encefalitis/epidemiología , Encefalitis/virología , Preescolar , China/epidemiología , Infección Hospitalaria/virología , Enterovirus/genética , Enterovirus Humano B/genética , Femenino , Genotipo , Humanos , Masculino , Datos de Secuencia MolecularRESUMEN
As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.
Asunto(s)
COVID-19 , Microfluídica , Humanos , Patología Molecular , Reacción en Cadena de la Polimerasa , SARS-CoV-2 , TemperaturaRESUMEN
Convenient and accurate nucleic acid quantification (NAQ) is crucial to clinical diagnosis, forensic medicine, veterinary medicine and food analysis. However, traditional NAQ relies on the preparation of a laborious, time-consuming and expensive calibration curve, which would also propagate pipette errors through serially dilutions. Besides, traditional NAQ is run in different tubes, which introduces bias from random tube-to-tube variations and is unable to detect inhibitors from biological samples. To solve these problems, a single-tube quantitative PCR (stqPCR) technique is proposed which enables accurate quantification without the need for a calibration curve. In this method, an internal quantitative standard DNA (IQS-DNA) for quantification was screened out by co-amplification with the target DNA. Then the difference between the quantification cycle value (ΔCq) of the IQS-DNA and the target DNA was used for NAQ. The method permitted high accuracy quantification with reliable data for concentrations in plasmid, serum standard, and clinical samples being confirmed (R2 values of 0.9951, 0.9889, and 0.9727, slope values of 1.011, 1.028, and 0.9327, and intercept values of -0.06037, -0.1486, and 0.3325, respectively). Accurate NAQ could also be achieved by stqPCR even though inhibitors were present in a sample; however, in the case of using a commercial assay kit, satisfactory performance was only attained after the same sample was diluted some 32-fold. Moreover, integration of the present method into a microfluidic system could achieve super-fast NAQ in less than 30 min and achieve super-fast "sample in, quantitative answer out" testing in less than 40 min. Thus, the stqPCR method present here would promote the development of NAQ in the laboratory and on site.
Asunto(s)
ADN , Análisis de los Alimentos , Calibración , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In a study of 315 HBV specimens obtained from southern China, 240 (76.9%) were assigned to genotype B, 72 (22.9%) were genotype C, two (0.6%) were genotype A and one (0.3%) was genotype D. Statistical analysis revealed that variables such as age, gender, HBV vaccination rate, hepatitis anamnesis rate, anti-HBs and HBeAg prevalence and virus load were insignificant between genotype B (n = 240) and genotype C cases (n = 72) (P > 0.05). However, the frequency of amino acid (aa) substitutions in the major hydrophilic region (MHR; aa 99-169) and the putative HLA class I-restricted cytotoxic T lymphocyte (CTL) epitope region of the S gene, as well as the overlapping polymerase/RT region (aa 32-212), were significantly higher in genotype C group than genotype B (P < 0.001). These results suggest that the higher variability within genotype C carriers may account for the pathogenic potential.
Asunto(s)
Variación Genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/virología , Adolescente , Adulto , Sustitución de Aminoácidos , Niño , Preescolar , China , ADN Polimerasa Dirigida por ADN/genética , Epítopos de Linfocito T/genética , Femenino , Genotipo , Virus de la Hepatitis B/clasificación , Humanos , Lactante , Masculino , Persona de Mediana Edad , Filogenia , Análisis de Secuencia de ADN , Proteínas Virales/genética , Adulto JovenRESUMEN
Enterovirus (EV)-A71 and Coxsackievirus (CV)-A16 have historically been the major pathogens of hand, foot, and mouth disease (HMFD) in China; however, CV-A6ï¼ which had previously received little attention, became the predominant pathogen in 2013, and has remained one of the common pathogens since then. In this work, we conducted a molecular epidemiology study of CV-A6-associated HFMD in Xiamen from 2009 to 2015. The data showed CV-A6 pandemics had a certain periodicity rather than occurring randomly. Evolution analysis based on near-complete VP1 nucleotide sequences showed subgenotype D5 lineage 4 strains account for the persistent outbreak of CV-A6-associated HFMD in China since 2013. Alignment analysis revealed eight candidate amino acid substitutions in VP1, which may provide useful information for the research of CV-A6 virulence enhancement. This study contributed to elucidating the circulation patterns and genetic characteristics of CV-A6 in China; however, further surveillance and intervention in CV-A6 epidemics is recommended.
Asunto(s)
Brotes de Enfermedades , Enterovirus/clasificación , Enterovirus/genética , Genotipo , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/virología , China/epidemiología , Enterovirus/aislamiento & purificación , Epidemiología Molecular , Análisis de Secuencia de ADN , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: Morbidity and mortality from influenza A (Flu A) have increased in recent years. Timely diagnosis and management are critical for disease control. Therefore, the development of a rapid, accurate, and portable analytical method for on-site analysis is imperative. OBJECTIVES: The aim of this work was to develop a rapid, on-site, automated assay for the detection of Flu A and to evaluate the assay. METHODS: A handheld instrument (TD-01) based on capillary convective polymerase chain reaction (PCR) was developed for rapid on-site detection of Flu A. Since a previous version of the instrument, an automated motion mechanism has been introduced to TD-01 to achieve RNA automated testing. The primers and probe used for Flu A detection were designed according to the Flu A gene sequence of matrix proteins. Finally, we evaluated the detection spectra, sensitivity, specificity, and diagnostic performance of the assay. RESULTS: The TD-01 was able to successfully automatically detect Flu A RNA within 30 min. Results for serially diluted viruses indicated that the lower limit of detection for Flu A was 0.1 TCID50/ml (50% tissue culture infective dose). After evaluating known virus stocks, including 15 strains of Flu A, four strains of Flu B, and two strains of respiratory syncytial virus (RSV), the assay had a favorable detection spectrum and no obvious cross-reactivity. Method verification based on 554 clinical samples indicated that the sensitivity and specificity of TD-01 were 98.30% (231/235) and 98.75% (315/319), respectively. CONCLUSIONS: The results indicate that Flu A detection by TD-01 is particularly suitable for on-site testing and has the potential for application in point-of-care testing.
Asunto(s)
Convección , Virus de la Influenza A/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN Viral/análisis , Humanos , Gripe Humana/diagnóstico , Gripe Humana/virología , Límite de Detección , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Human enteroviruses (EVs) are the most common causative agents infecting human, causing many harmful diseases, such as hand, foot, and mouth disease (HFMD), herpangina (HA), myocarditis, encephalitis, and aseptic meningitis. EV-related diseases pose a serious worldwide threat to public health. To gain comprehensive insight into the seroepidemiology of major prevalent EVs in humans, we firstly performed a serological survey for neutralizing antibodies (nAbs) against Enterovirus A71 (EV-A71), Coxsackie virus A16 (CV-A16), Coxsackie virus A6 (CV-A6), Coxsackie virus A10 (CV-A10), Coxsackie virus B3 (CV-B3), Coxsackie virus B5 (CV-B5), Echovirus 25 (ECHO25), and Echovirus 30 (ECHO30) among the healthy population in Xiamen City in 2016, using micro-neutralization assay. A total of 515 subjects aged 5 months to 83 years were recruited by stratified random sampling. Most major human EVs are widely circulated in Xiamen City and usually infect infants and children. The overall seroprevalence of these eight EVs were ranged from 14.4% to 42.7%, and most of them increased with age and subsequently reached a plateau. The co-existence of nAbs against various EVs are common among people ≥ 7 years of age, due to the alternate infections or co-infections with different serotypes of EVs, while most children were negative for nAb against EVs, especially those < 1 year of age. This is the first report detailing the seroepidemiology of eight prevalent EVs in the same population, which provides scientific data supporting further studies on the improvement of EV-related disease prevention and control.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Infecciones por Enterovirus/inmunología , Enterovirus/inmunología , Vigilancia Inmunológica , Estudios Seroepidemiológicos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Niño , Preescolar , Enterovirus Humano A/inmunología , Enterovirus Humano B/inmunología , Femenino , Voluntarios Sanos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Coxsackievirus A10 (CV-A10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD) and usually causes mild cases. During 2009-2014, no severe cases caused by CV-A10 was reported in Xiamen, China, however, an increase in cases was seen in 2015. OBJECTIVES: We aimed to perform a retrospective molecular epidemiological analysis of HFMD associated with CV-A10 infections in Xiamen. STUDY DESIGN: CV-A10 VP1 (n=41) capsid and full-length or near full-length genomes (n=14) were sequenced. Phylogenetic trees were constructed based on these sequences and other reference sequences and nucleotide and amino acid changes were characterized. RESULTS: From 2009-2014, no laboratory-confirmed CV-A10 infections associated with severe cases were identified, however, in 2015, 39% (7/18) of severe HFMD cases were CV-A10 infections. Sequence analysis of severe and non-severe CV-A10 HFMD cases determined that severe cases predominantly clustered with an emerging clade E lineage A strain which contained 4 nucleotide changes in 5' UTR and 5 amino acid substitutions in structural and non-structural proteins. CONCLUSIONS: The results indicate CV-A10 infection may be emerging as a new and major cause of severe HFMD and CV-A10 surveillance should be increased and considered in HFMD prevention and control strategies.
Asunto(s)
Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/virología , Proteínas de la Cápside/genética , China/epidemiología , Genotipo , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/patología , Humanos , FilogeniaRESUMEN
Two colloidal gold immunochromatographic assays (CGIAs) for detection of EV71- and CA16- immunoglobulin M (IgM) were developed and evaluated. A total of 1465 sera collected from children with hand, foot, and mouth disease (HFMD), non-HFMD patients and healthy children. The sensitivity of IgM CGIA tests for EV71 and CA16 were 97.6% (330/338) and 91.6% (296/323) respectively, compared to those who were viral RNA positive by PCR. Their performances were comparable to those of commercial ELISA kits, with agreement of 98.1% for EV71-IgM and 97.3% for CA16-IgM. In addition, for EV71- and CA16-IgM CGIAs, the results of whole blood samples were 99.6% (248/249) and 100% (191/191) concordant to those with serum samples, respectively. As rapid point-of-care (POC) tests, the two CGIAs were suitable to be used in community clinic units, especially in resource-poor areas and will facilitate the control of HFMD.
Asunto(s)
Anticuerpos Antivirales/sangre , Cromatografía de Afinidad/métodos , Enterovirus Humano A/inmunología , Enterovirus/inmunología , Enfermedad de Boca, Mano y Pie/diagnóstico , Inmunoglobulina M/sangre , Sistemas de Atención de Punto , Pruebas Diagnósticas de Rutina/métodos , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preS1 (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/prevención & control , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Sitios de Unión/inmunología , Western Blotting , Epítopos/genética , Epítopos/inmunología , Femenino , Regulación Viral de la Expresión Génica , Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/genética , Antígenos e de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virión/inmunologíaRESUMEN
Interferon-gamma release assays (IGRAs) have been demonstrated to be useful in the diagnosis of Mycobacterium tuberculosis (MTB) infection. However, IGRAs have not been recommended for clinical usage in most low-income countries due to the shortage of clinical data available resulting from their high test cost. Recently, a cheaper domestic TB-IGRA was approved in China. In this study, we compared TB-IGRA with QuantiFERON-TB Gold In-Tube (QFT-GIT) for MTB infection diagnosis in 253 active TB patients, 48 non-TB lung disease patients, 115 healthcare workers and 216 healthy individuals. The proportion of positive TB-IGRA results in active TB patients, patients with non-TB lung disease, healthcare workers and healthy individuals was 88.3%, 27.1%, 40.9% and 17.6%, respectively, which was similar to the results of QFT-GIT, with an overall agreement of 95% (κ = 0.89) and a high correlation between their responses (r = 0.85, p < 0.001) being observed. In conclusion, the TB-IGRA has comparable clinical performance with QFT-GIT.
Asunto(s)
Ensayos de Liberación de Interferón gamma , Interferón gamma/sangre , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/diagnóstico , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , China , Femenino , Interacciones Huésped-Patógeno , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Tuberculosis/sangre , Tuberculosis/inmunología , Tuberculosis/microbiología , Adulto JovenRESUMEN
Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5'-Untranslated Regions (5'-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25.
Asunto(s)
ADN Complementario/genética , ADN Viral/genética , Enterovirus Humano B/genética , Infecciones por Enterovirus/virología , Anticuerpos Antivirales/inmunología , China , ADN Complementario/metabolismo , ADN Viral/metabolismo , Enterovirus Humano B/clasificación , Enterovirus Humano B/inmunología , Enterovirus Humano B/fisiología , Enfermedad de Boca, Mano y Pie/inmunología , Enfermedad de Boca, Mano y Pie/virología , Humanos , Datos de Secuencia Molecular , FilogeniaRESUMEN
The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.