Evaluación de tres pruebas serológicas para la detección de anticuerpos contra el virus linfotrópico T humano tipo 1 (HTLV-1)

Introducción: El Perú es endémico al virus linfotrópico T humano tipo 1 (HTLV-1), por esas razones es importante conocer la fiabilidad de las pruebas diagnósticas que se usan en el país, con la finalidad de continuar o no su uso. El objetivo fue evaluar el rendimiento de tres pruebas serológicas ELISA Murex, ELISA Wantai e IFI INS-Perú para la detección de anticuerpos anti HTLV-1 frente a muestras peruanas. El estudio. Las tres pruebas fueron evaluadas frente a 382 sueros: 215 positivos y 167 negativos a HTLV-1 (Gold Standar: inmunoblot). Hallazgos. IFI no presentó falsos positivos, Wantai tuvo más falsos negativos (siete) y Murex más falsos positivos (ocho). Las tres pruebas mostraron resultados superiores a 95% para los parámetros estimados de exactitud diagnóstica. Conclusiones. IFI INS-Perú y ELISA Murex tuvieron buen rendimiento diagnóstico para la detección de anticuerpos contra HTLV-1 y son buenos candidatos para continuar siendo usados en Perú.

Background: Peru is endemic to the human T-lymphotropic virus type 1 (HTLV-1), for these reasons it is important to know the reliability of the diagnostic tests used in the country, in order to continue their use or not. The objective was to evaluate the performance of three serological tests ELISA Murex, ELISA Wantai and IFI INS-Peru for the detection of anti-HTLV-1 antibodies against Peruvian samples. The study. The three tests were evaluated against 382 sera: 215 positive and 167 negative for HTLV-1 (Gold Standard: immunoblot). Findings. IFI had no false positives, Wantai had more false negatives (seven) and Murex more false positives (eight). The three tests showed results above 95% for the estimated parameters of diagnostic accuracy. Conclusions. IIF INS-Perú and ELISA Murex had good diagnostic performance for the detection of antibodies against HTLV-1 and are good candidates to continue being used in Peru.

Clinical and immunoserological features of 7 cases of anti-p200 pemphigoid / 中华皮肤科杂志

Objective:To analyze clinical and immunoserological features of patients with anti-p200 pemphigoid.Methods:Clinical data were collected from patients with confirmed anti-p200 pemphigoid in Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2015 to October 2021, and their clinical and immunoserological characteristics were retrospectively analyzed.Results:Seven patients with anti-p200 pemphigoid were included. Indirect immunofluorescence on salt-split skin (IIF-SSS) showed that serum IgG antibodies of the 7 patients were located in the dermis of the salt-split skin, and Western blot analysis with dermal extracts as substrates revealed a protein band with a relative molecular mass of 200 000. Four patients presented with classic bullous pemphigoid-like skin lesions, 2 initially presented with eczematous lesions, and 1 presented with linear IgA bullous dermatosis-like skin lesions. Circulating IgG antibodies could recognize the recombinant laminin γ1 C-terminal region in 6 cases. Four patients received different doses of systemic glucocorticoids, 1 of whom was resistant to high-dose systemic glucocorticoids (equivalent to 1.4 mg·kg -1·d -1 prednisone) ; 2 responded well to minocycline and dapsone; 1 was lost to follow-up. Four patients achieved complete remission and discontinued the treatment at a mean follow-up of 22.5 months; 2 received complete remissiona on minimal therapy at a mean follow-up of 8 months. Conclusion:Patients with anti-p200 pemphigoid presented with heterogeneous clinical manifestations, and the recombinant C-terminal fragment of laminin γ1 can serve as a reliable antigen substrate for the detection of autoantibodies in patients with anti-p200 pemphigoid; some patients can eventually achieve complete remission off treatment.

Optimization of indirect immunofluorescence on salt-split skin and its application in detection of bullous pemphigoid antibodies / 中华皮肤科杂志

Objective:To optimize indirect immunofluorescence on salt-split skin (IIF-SSS), and to evaluate its performance in detection of bullous pemphigoid (BP) antibodies.Methods:Normal human foreskin and non-foreskin skin tissues were used to prepare salt-split substrates under 3 different experimental conditions: traditional group rotated at 4 ℃ for 48 - 72 hours, low-temperature immersion group soaked at 4 ℃ for 48 - 72 hours, room-temperature immersion group soaked at 25 ℃ (range: 23 - 27 ℃) for 24 hours. Serum samples were obtained from 20 patients with bullous pemphigoid (BP) in Hospital of Dermatology, Chinese Academy of Medical Sciences between August 2019 and August 2020, and subjected to IIF on the intact skin or salt-split substrates by using a multiple dilution method. Paired-sample t test was used for comparisons of means between two paired samples. Results:No dermal-epidermal separation was observed in the substrates prepared in the low-temperature immersion group at 48 - 72 hours, while dermal-epidermal separation occurred in the lower lamina lucida of the foreskin and non-foreskin substrates in the room-temperature immersion group and the traditional group. For the 20 patients with BP, the reciprocal end-point titers ( M[ Q1, Q3]) detected with the salt-split non-foreskin skin and salt-split foreskin in the room-temperature immersion group, and with the salt-split non-foreskin skin in the traditional group were 5 120 (2 560, 17 920), 1 280 (640, 2 560), 1 280 (640, 2 560), respectively. Moreover, 19 (95%) patients with BP showed that the reciprocal end-point titers detected with the substrates in the room-temperature immersion group were 1 - 5 times those in the traditional group ( t = 8.04, P<0.001), suggesting that the performance of salt-split skin in the room-temperature immersion group was superior to that in the traditional group in the detection of BP antibodies; however, there was no significant difference in the reciprocal end-point titers of BP antibodies between the salt-split foreskin in the room-temperature immersion group and salt-split non-foreskin skin in the traditional group ( t<0.001, P>0.05). The reciprocal end-point titers in 20 BP sera detected by conventional IIF on the intact non-foreskin skin and foreskin were 320 (160, 640) and 480 (160, 1 120), respectively; the reciprocal end-point titers detected by IIF on the salt-split foreskin and non-foreskin skin in the room-temperature immersion group, as well as on the salt-split non-foreskin skin in the traditional group, were all consistent with or 1 - 7 times higher than those detected by conventional IIF ( t = 6.47, 14.83, 5.26, respectively, all P<0.001) . Conclusion:The soaking method at room temperature 25 ℃ (23 - 27 ℃) for preparing salt-split substrates has advantages of short duration and simple procedure, and the sensitivity of IIF-SSS using the substrates prepared by this method is equal or superior to the traditional salt-split method for detecting BP antibodies.

Patrones diferenciales entre Leishmaniasis y chagas, empleando epimastigotes de Trypanosoma cruzi / Differential patterns between leishmaniasis and chagas disease using Trypanosoma cruzi epimastigotes

En diferentes regiones de Latinoamérica la infección por T. cruzi y Leishmania se superponen, por lo cual se reportan infecciones mixtas circulantes, debido a esto; deben realizarse pruebas diagnósticas específicas para evitar reacciones cruzadas entre estas dos patologías. OBJETIVO: determinar patrones de fluorescencia que permitan la diferenciación entre Leishmaniasis, enfermedad de Chagas e infección mixta empleando epimastigotes de T. cruzi. MÉTODOS: se empleó la técnica de Inmunofluorescencia Indirecta utilizando epimastigotes de T. cruzi (TcV autóctono) como antígeno figurado frente a un panel de muestras de suero codificados como A, B, C y D correspondientes a pacientes con infección por: Leishmaniasis (A), Infección mixta por Leishmania y Chagas(B), Enfermedad de Chagas (C) y sin ninguna de las dos infecciones (D). RESULTADOS: en los cuatro paneles de muestras se observaron diferentes patrones de intensidad de fluorescencia a nivel de membrana y núcleo de los epimastigotes de T. cruzi (TcV autóctono). CONCLUSIONES: la técnica de Inmunofluorescencia (IFI) con antígenos de epimastigotes de T. cruzi a demostrado utilidad en la diferenciación entre enfermedad de Chagas, Leishmaniasis y/o infecciones mixtas por ambos parásitos en aquellas zonas donde la coexistencia de ambas es habitual

In different regions of Latin America, infection by T. cruzi and Leishmania overlap, for which mixed circulating infections are reported, due to this; Specific diagnostic tests must be performed to avoid cross reactions between these two pathologies. OBJECTIVE: to determine fluorescence patterns that allow the differentiation between Leishmaniasis, Chagas disease and mixed infection using T. cruzi epimastigotes. METHODS: the Indirect Immunofluorescence technique was used using epimastigotes of T. cruzi (autochthonous TcV) as figurative antigen against a panel of serum samples coded as A, B, C and D corresponding to patients with infection by: Leishmaniasis (A) , Mixed infection by Leishmania and Chagas (B), Chagas disease (C) and without either of the two infections (D). RESULTS: in the four sample panels, different patterns of fluorescence intensity were observed at the membrane and nucleus level of the epimastigotes of T. cruzi (autochthonous TcV). CCONCLUSIONS: the Immunofluorescence technique (IFI) with T. cruzi epimastigote antigens has proven useful in differentiating between Chagas disease, Leishmaniasis and / or mixed infections by both parasites in areas where the coexistence of both is common.

Aislamiento y caracterización de una cepa temprana de SARS-CoV-2 durante la epidemia de 2020 en Medellín, Colombia / Isolation and characterization of an early SARS-CoV-2 isolate from the 2020 epidemic in Medellín, Colombia

Introducción. El nuevo coronavirus causante de un brote de enfermedad respiratoria aguda en China en diciembre de 2019 se identificó como SARS-CoV-2. La enfermedad, denominada COVID-19, fue declarada pandemia por la Organización Mundial de la Salud (OMS). El primer caso de COVID-19 en Colombia se reportó el 6 de marzo de 2020; en este estudio se caracterizó un aislamiento temprano del virus SARS-CoV-2 de una muestra recolectada en abril de 2020. Objetivos. Describir y caracterizar una cepa temprana a partir de un aislamiento de SARS-CoV-2 durante la pandemia en Colombia. Materiales y métodos. Se obtuvo una muestra de un paciente con COVID-19 confirmada por qRT-PCR; la muestra fue inoculada en diferentes líneas celulares hasta la aparición del efecto citopático. Para confirmar la presencia de SARS-CoV-2 en el cultivo, se utilizó la qRT-PCR a partir de los sobrenadantes, la inmunofluorescencia indirecta (IFI) en células Vero-E6, así como microscopía electrónica y secuenciación de nueva generación (next-generation sequencing). Resultados. Se confirmó el aislamiento de SARS-CoV-2 en células Vero-E6 por la aparición del efecto citopático tres días después de la infección, así como mediante la qRT-PCR y la IFI positiva con suero de paciente convaleciente positivo para SARS-CoV-2. Además, en las imágenes de microscopía electrónica de trasmisión y de barrido de células infectadas se observaron estructuras compatibles con viriones de SARS-CoV-2. Por último, se obtuvo la secuencia completa del genoma, lo que permitió clasificar el aislamiento como linaje B.1.5. Conclusiones. La evidencia presentada en este artículo permite confirmar el primer aislamiento de SARS-CoV-2 en Colombia. Además, muestra que esta cepa se comporta en cultivo celular de manera similar a lo reportado en la literatura para otros aislamientos y que su composición genética está acorde con la variante predominante en el mundo. Finalmente, se resalta la importancia que tiene el aislamiento viral para la detección de anticuerpos, para la caracterización genotípica y fenotípica de la cepa y para probar compuestos con potencial antiviral.

Introduction: SARS-CoV-2 has been identified as the new coronavirus causing an outbreak of acute respiratory disease in China in December, 2019. This disease, currently named COVID-19, has been declared as a pandemic by the World Health Organization (WHO). The first case of COVID-19 in Colombia was reported on March 6, 2020. Here we characterize an early SARS-CoV-2 isolate from the pandemic recovered in April, 2020. Objective: To describe the isolation and characterization of an early SARS-CoV-2 isolate from the epidemic in Colombia. Materials and methods: A nasopharyngeal specimen from a COVID-19 positive patient was inoculated on different cell lines. To confirm the presence of SARS-CoV-2 on cultures we used qRT-PCR, indirect immunofluorescence assay, transmission and scanning electron microscopy, and next-generation sequencing. Results: We determined the isolation of SARS-CoV-2 in Vero-E6 cells by the appearance of the cytopathic effect three days post-infection and confirmed it by the positive results in the qRT-PCR and the immunofluorescence with convalescent serum. Transmission and scanning electron microscopy images obtained from infected cells showed the presence of structures compatible with SARS-CoV-2. Finally, a complete genome sequence obtained by next-generation sequencing allowed classifying the isolate as B.1.5 lineage. Conclusion: The evidence presented in this article confirms the first isolation of SARS-CoV-2 in Colombia. In addition, it shows that this strain behaves in cell culture in a similar way to that reported in the literature for other isolates and that its genetic composition is consistent with the predominant variant in the world. Finally, points out the importance of viral isolation for the detection of neutralizing antibodies, for the genotypic and phenotypic characterization of the strain and for testing compounds with antiviral potential.

Comparison between enzyme-linked immunosorbent assay and indirect immunofluorescence for detection of antineutrophil cytoplasmic antibodies / Comparação entre os métodos de ensaio imunoenzimático e imunoflurescência indireta para a pesquisa de anticorpos anticitoplasma de neutrófilos

ABSTRACT Objective To evaluate the performance of enzyme-linked immunosorbent assay and indirect immunofluorescence methods for the detection of antineutrophil cytoplasmic antibodies in a routine clinical laboratory setting. Methods A total of 227 samples were tested by indirect immunofluorescence and enzyme-linked immunosorbent assay with antigen specificity for antiproteinase 3 and antimyeloperoxidase. The proportions of positive samples were compared by McNemar hypotheses and agreement was described by Cohen's Kappa coefficient. Results The agreement of the tests was 96.5%, and the Kappa coefficient obtained was 0.70 (95%CI: 0.50-0.90; p<0.001). Considering indirect immunofluorescence as the gold standard, the sensitivity of the enzyme-linked immunosorbent assay was 0.62 and the specificity was 0.99, with diagnostic accuracy in 96% of cases. Some samples were negative in enzyme-linked immunosorbent assay and positive in indirect immunofluorescence. This situation occurred in all immunofluorescence patterns, but particularly in atypical patterns. Two samples with antiproteinase 3 positivity were considered negative in indirect immunofluorescence. Conclusion The enzyme-linked immunosorbent assay had high specificity but lower sensitivity. The performance of indirect immunofluorescence increases diagnostic sensitivity, while the search for antiproteinase 3 by enzyme-linked immunosorbent assay may also add diagnostic power.

RESUMO Objetivo Avaliar o desempenho das metodologias de ensaio imunoenzimático e imunofluorescência indireta para a detecção de anticorpos anticitoplasma de neutrófilos em um contexto de laboratório clínico de rotina. Métodos Foram testadas 227 amostras pelas metodologias de imunofluorescência indireta e ensaio imunoenzimático com especificidades para anticorpos antiproteinase-3 e antimieloperoxidase. As proporções de amostras positivas foram comparadas por hipóteses de McNemar, e a concordância foi descrita pelo coeficiente Kappa de Cohen. Resultados A concordância dos testes foi 96,5%, e o coeficiente Kappa obtido foi 0,70 (IC95%: 0,50-0,90; p<0,001). Utilizando a imunofluorescência indireta como padrão-ouro, a sensibilidade do ensaio imunoenzimático foi de 0,62 e a especificidade, 0,99, com acurácia diagnóstica em 96% dos casos. Algumas amostras apresentaram resultados negativos por ensaio imunoenzimático e positivos por imunofluorescência. Isso ocorreu em amostras com vários padrões de fluorescência, mas particularmente nos casos com padrões atípicos. Duas amostras com positividade antiproteinase 3 foram consideradas negativas por imunofluorescência. Conclusão Os métodos de ensaio imunoenzimático tiveram alta especificidade, mas sensibilidade inferior. A realização da imunofluorescência indireta aumenta a sensibilidade diagnóstica, ao mesmo tempo que a pesquisa de antiproteinase 3 por ensaio imunoenzimático também pode agregar poder diagnóstico.

Infectious endocarditis by Bartonella species: report of two cases / Endocarditis infecciosa por Bartonella: descripción de dos casos en Chile

ABSTRACT Infectious endocarditis (IE) by Bartonella species is an emerging problem worldwide. We report two cases of native valve Bartonella-associated IE events, both affecting adult male patients with a history of alcohol abuse and a low socioeconomic status. Admissions were due to pancytopenia and bleeding in one case and embolic stroke in the other. Blood cultures were negative and IgG indirect immunofluorescence assays (IFA) were positive for B. henselae/B. quintana in high titers (1/16,384-1/16,384, and 1/32,768 -1/16,384, respectively). Cases were classified as definitive IE events according to modified Duke criteria due to the presence of valve vegetations with at least three minor criteria. One patient required aortic mechanical valve replacement and survived, and the other died after a massive hemorrhagic transformation of his stroke. PCR amplification and sequencing of the 16S ribosomal bacterial DNA from a valve tissue sample obtained at surgery in the patient who survived, confirmed B. quintana as the etiological agent. Bartonella-associated IE is an emerging problem in Chile, present in disadvantaged populations. It should be suspected in patients with culture-negative IE. IFA does not discriminate between B. henselae and B. quintana infection, but high titers suggest IE. Complementary PCR techniques may help to elucidate the final causative agent.

La endocarditis infecciosa(EI) asociada a Bartonella es un problema emergente a nivel mundial. Publicamos los 2 primeros casos de EI en válvula nativa asociados a Bartonella en Chile, los que afectaron a pacientes masculinos con historia de consumo de alcohol y bajos ingresos. La hospitalización fue provocada por pancitopenia y hemorragias en un caso y por un evento cerebrovascular en el otro. Se solicitó serología para Bartonella por inmunofluorescencia indirecta (IFI) para ampliar el estudio ante hemocultivos negativos y en ambos casos se reportaron resultados intensamente positivos para B. henselae y B. quintana1/16.384-1/16.384 y 1/32.768 -1/16.384, respectivamente). Los casos se clasificaron como eventos definitivos de EI según los criterios modificados de Duke debido a la presencia de vegetaciones valvulares con al menos 3 criterios menores. Un paciente requirió reemplazo valvular aórtico y sobrevivió, y el otro falleció tras una transformación hemorrágica masiva del infarto cerebral. La amplificación del ADN ribosomal 16S por RCP y posterior secuenciación de una muestra de tejido valvular confirmó la presencia de B. quintana. La EI por Bartonella sp. es un problema emergente en Chile, probablemente asociada a poblaciones desfavorecidas, la que debe ser sospechada en pacientes con cultivos negativos. La IFI no permite discriminar infecciones por B. henselae o B. quintana pero los títulos altos sugieren EI. Técnicas complementarias por RCP pueden ayudar a dilucidar el diagnóstico.

Infección y enfermedad por Rickettsia spp. del grupo de las fiebres manchadas en pacientes febriles del Urabá antioqueño, Colombia / Rickettsia spp. infection of the group of spotted fevers in febrile patients of the Urabá Antioquia, Colombia

RESUMEN Objetivo: determinar la frecuencia de la infección y enfermedad por Rickettsia spp. del grupo de las fiebres manchadas en pacientes febriles del Urabá antioqueño, que asistieron a centros hospitalarios de la región. Métodos: se incluyeron en el estudio pacientes febriles provenientes de 9 instituciones de salud de la región del Urabá, los cuales fueron encuestados para determinar sus variables clínicas y demográficas. De estos pacientes se obtuvieron muestras de suero durante las fases aguda y convaleciente de la enfermedad. Para cada muestra se determinó la seropositividad (título ≥ 64) y su título de anticuerpos seriados dobles mediante inmunofluorescencia indirecta para IgG contra el antígeno de Rickettsia rickettsii. Resultados: se analizaron 89 pacientes febriles con 89 muestras de fase aguda y 60 en fase convaleciente. Los síntomas más comunes de los pacientes fueron cefalea, ictericia, mialgias, náuseas, dolor abdominal, trombocitopenia y vómito. El 55,1 % de los pacientes provenía de áreas rurales. Se obtuvo seropositividad del 40,4 % con títulos entre 64-512, infección previa en un 33,7 % y rickettsiosis en 6 pacientes (6,7 %). Los pacientes con seroconversión o serorefuerzo provenían de los municipios de Apartadó (n = 2), Chigorodó (n = 1), Necoclí (n = 2) y Turbo (n = 1); el hallazgo clínico más destacado fue la trombocitopenia. Conclusiones: se demostró que la infección y la enfermedad rickettsial continúan siendo activas en la zona del Urabá. Este hallazgo permite alertar a las autoridades de salud de la región para que se brinde tratamiento con antibióticos a los casos sospechosos de manera temprana y de esta forma evitar las muertes o secuelas derivadas de este tipo de infecciones.

SUMMARY Objective: Determine the frequency of infection and disease by Rickettsia spp. of the spotted fever group in febrile patients from Urabá Antioquia attended by hospital centers of the region. Methods: Patients from nine health institutions of the Urabá region were included in the study. These patients received a survey with questions about their clinical and socio-demographic variables. Eighty-nine acutephase serum samples, and 60 convalescent serum samples, were obtained from these patients, and each sample was tested (IgG) by Indirect Immunofluoerscence Assay (IIFA) using a dilution of 1:64 against R. rickettsii. Furtherly, positive sera were tittered by two-fold serial dilutions using the same antigen. Results: Patients showed symptoms such as fever, headache, jaundice, myalgias, nausea, abdominal pain, petechiae, thrombocytopenia and vomiting. Most of these patients came from rural areas (55,1 %). Seropositivity was obtained in 40,4 % patients with titers between 64-512, a 33,7 % with previous infection and the disease was found in 6 patients (6,7 %). Patients with seroconversion, or a fourlfold rise antibody titer between acute and convalescent samples, came from the municipalities of Apartadó (n = 2), Chigorodó (n = 1), Necoclí (n = 2) and Turbo (n = 1), and the most relevant clinical finding was thrombocytopenia in four of the patients. Conclusions: This study demonstrated that infection and rickettsial disease continues being active in the Urabá region. This situation represents a warning for the health authorities of the region and suggests them to provide appropriate treatment to avoid deaths or sequelae derived from this type of infections.

Evaluation of the value of indirect immunofluorescence on different substrates in the diagnosis of autoimmune subepidermal bullous diseases / 中华皮肤科杂志

Objective To evaluate the value of indirect immunofluorescence (IIF) on three different substrates including normal human skin (NS),monkey esophagus (ME) and salt-split human skin (SS) in the diagnosis of autoimmune subepidermal bullous diseases.Methods A total of 56 patients with autoimmune subepidermal bullous diseases,including 47 with bullous pemphigoid (BP),6 with epidermolysis bullosa acquisita (EBA),2 with linear IgA bullous dermatosis,and 1 with anti-P200 pemphigoid,were diagnosed in and enrolled from Department of Dermatology,Institute of Dermatology,Chinese Academy of Medical Sciences between January 2015 and December 2016.Seventy patients with pemphigus,15 patients with chronic eczema and 15 healthy adults served as controls.Blood samples collected from these patients and controls were subjected to IIF on three different substrates including NS,ME and SS,and the fluorescence deposition was observed.The sensitivities and specificities of IIF in the diagnosis of different subepidermal bullous diseases were compared.Statistical analysis was carried out with SPSS 13.0 software by using chi-square test for the comparison of enumeration data.Results IIF on NS or ME in the serum of patients with BP showed linear deposition of fluorescent material along the basement membrane zone.IIF on SS showed linear deposition of fluorescent material in the epidermis in the patients with BP,but in the dermis in the patients with EBA and anti-P200 pemphigoid.The sensitivities of IIF on NS,ME or SS in the diagnosis of subepidermal bullous diseases were 73.2％,60.7％ and 94.6％ respectively,and the specificities were 98.0％,100％ and 97.1％ respectively.There were significant differences among the sensitivities (x2 =18.2,P ＜ 0.05),but no significant difference was observed among the specificities (P ＞ 0.05).The diagnostic sensitivity of IIF on SS was significantly higher than that of IIF on NS or ME(x2 =8.0,16.7,both P ＜ 0.05).Conclusion In the diagnosis of autoimmune subepidermal bullous diseases,IIF on SS is superior to IIF on ME or NS.

Indirect immunofluorescence on salt-split skin and bullous pemphigoid 180 NC16a enzyme-linked immunosorbent assay in the diagnosis of bullous pemphigoid / 中华皮肤科杂志

Objective To evaluate the value of indirect immunofluorescence on salt-split skin (IIF-SSS) and bullous pemphigoid 180 N C 16a enzyme-linked immunosorbent assay (BP 180 N C 16a-ELISA) in the diagnosis of bullous pemphigoid (BP).Methods Serum samples were collected from 174 BP patients and 129 controls,who were enrolled from Institute of Dermatology of Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017,and subjected to IIF-SSS and BP180 NC16a-ELISA.Direct immunofluorescence (DIF) test was performed in 25 cases of BP,and its sensitivity for the diagnosis of BP was compared with that of IIF-SSS and BP180 NC16a-ELISA.Results The sensitivities for IIF-SSS and BP180 NC16a-ELISA were 93.67％ and 96.55％ respectively,and the specificities for IIF-SSS and BP180 NC16a-ELISA were 100％ and 96.12％ respectively.IIF-SSS was weakly correlated with BP180 NC16a-ELISA with a correlation coefficient of 0.147.There was no significant difference in the sensitivity between the serological diagnostic methods (IIF-SSS and BP180 NC 16a-ELISA) and DIF.Conclusion Serological diagnostic methods show high specificity and sensitivity in the diagnosis of BP,and are worthy of clinical promotion and application.

Rendimiento diagnóstico de la prueba de inmunofluorescencia indirecta para la detección de anticuerpos contra HTLV-1 / Diagnostic performance of the indirect immunofluorescence test for the detection of antibodies against HTLV-1

RESUMEN El objetivo del estudio fue determinar el rendimiento diagnóstico de la prueba de inmunofluorescencia indirecta (IFI) para la detección de anticuerpos contra HTLV-1. Se realizó un estudio de evaluación de prueba diagnóstica. Se usaron cultivos celulares MT2 infectados con HTLV-1 y K-562 sin infección, luego fueron sembrados, fijados en láminas para inmunofluorescencia y enfrentados a sueros. Se usaron 155 sueros (80 positivos para HTLV-1 y 75 positivos para otras enfermedades) procedentes de la seroteca del Instituto Nacional de Salud del Perú. Adicionalmente, se evaluó la repetibilidad (en el laboratorio) y reproducibilidad (en laboratorios de costa, sierra y selva) de la prueba. La prueba IFI para la detección de anticuerpos contra HTLV-1 tuvo una sensibilidad de 98,75% (IC 95%: 95,69-100%), una especificidad de 98,67% (IC 95%: 95,40-100%) y el índice de kappa de 0,975. No hubo falsos positivos ni falsos negativos; sin embargo, sí se obtuvo un resultado indeterminado y uno inespecífico. La prueba mostró 100% de concordancia en la repetibilidad y reproductibilidad. Concluimos que los resultados obtenidos son comparables a la prueba de referencia. La prueba de IFI presenta un buen rendimiento diagnóstico y sería de utilidad para la confirmación de HTLV-1.

ABSTRACT The objective of the study was to determine the diagnostic yield of the indirect immunofluorescence (IFI) test for the detection of antibodies against HTLV-1. A diagnostic test evaluation study was performed. HTLV-1-infected MT2 cells and HTLV-1-uninfected K-562 cells were cultured; then these cells were impregnated and fixed in sheets for immunofluorescence and faced to Peruvian sera. A total of 155 sera (80 HTLV-1-positive sera and 75 sera positive for other diseases) from the Peruvian Instituto Nacional de Salud were used. In addition, the parameters of repeatability (intra-laboratory) and reproducibility (in laboratories of the Peruvian coast, mountains and jungle) of the test were evaluated. The IFI test detected the presence of antibodies against HTLV-1 reaching a sensitivity of 98.75% (95% CI: 95.69 - 100.00%), a specificity of 98.67% (95% CI: 95.40 - 100.00%) and the Kappa index was 0.975. There were no false positives or false negatives; however, one undetermined result and one non-specific result were obtained. The test showed 100% qualitative agreement when performing the repeatability and reproducibility. The results obtained are comparable to the reference test. Therefore, the IFI test had a good diagnostic performance and would be useful for the confirmation of HTLV-1.

Analysis of common viral infections found in patients with chronic obstructive pulmonary disease with acute exacerbations / 中华实验和临床病毒学杂志

Objective@#To analyze characteristics of virus infections in patients with acute exacerbations of chronic obstructive pulmonary disease (COPD), and to analyze the role of virus infection in acute exacerbations of COPD.@*Methods@#Totally 193 patients of COPD with acute exacerbations admitted to the Hospital of Sichuan Provincial Armed Police Force from April 2015 to March 2016 were enrolled as group A, and 50 patients of COPD in stable stage in the same period were enrolled as group B. IgM antibodies to respiratory syncytial virus, adenovirus, parainfluenza virus, influenza A virus, and influenza B virus in two groups were detected. General conditions and total virus infection rate were analyzed. Infection rates of each virus in group A were calculated. Proportion of single virus infection, two virus infections and several virus infections in group A with virus infection were calculated. Virus infection rates in different seasons in group A were also analyzed.@*Results@#There was no significant difference in age, sex, and course of disease between the two groups(P>0.05). The virus infection rate of group A was 13.47%, which was higher than that of group B(χ2=5.29, P<0.05). The most common virus found in group A was influenza B virus(infection rate 12.44%), followed by influenza A virus(infection rate 7.78%), parainfluenza virus(infection rate 7.25%), adenovirus(infection rate 2.63%), respiratory syncytial virus(infection rate 1.32%). Single virus infection accounted for 34.6% of acute exacerbation COPD patients with virus infection, and two virus infections accounted for 15.4%, and three or more virus infections accounted for 50%. Virus infection rate in summer and autumn were higher than that in winter and spring, but the differences were not statistically significant(P>0.05).@*Conclusions@#This study showed that 14.37% of patients with acute exacerbations of COPD had viral infections, and most of them were infected with two or more viruses. Virus infection may play an important role in the acute exacerbation of COPD, and we need to pay attention to the prevention and treatment of virus infection in patients with COPD.

Expression of serum antinuclear antibodies in lymphoma patients and their clinical significances / 白血病·淋巴瘤

Objective To study the expression characteristics of serum antinuclear antibodies (ANA) in lymphoma patients as well as their target antigens in cells, and to explore the possible relationship between lymphoma and ANA. Methods Indirect immunofluorescence was used to detect the ANA expression level in 100 cases of lymphoma and 200 population-based controls. Then the relationship between lymphoma and ANA was investigated by means of 1:2 matched with logistic regression models. Results The positive rate of ANA in lymphoma patients was higher than that in the control group [28 % (28/100) vs. 7 % (14/200)], with a statistical difference (OR= 13.66, 95 %CI 4.10-45.57, P< 0.01). The positive rate of ANA in females was higher than that in males, and the positive rate of ANA became higher with age. Lymphoma group had more complex fluorescence pattern and wider target antigen spectrum compared with the control group. Conclusions Detection of ANA in lymphoma may help in the early diagnosis,prognosis and treatment. ANA target antigen spectra of lymphoma patients are different from those in healthy people as well as patients with autoimmune disease. Further efforts should be made to identify the target antigens as well as their biological roles and clinical significances.

Podocitúria na doença de Fabry / Podocyturia in Fabry disease

Resumo Introdução: A doença de Fabry (DF) é uma desordem lisossômica ligada ao cromossomo X ocasionada por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução ou ausência da atividade dessa enzima leva ao acúmulo progressivo de gb3. A doença renal é uma importante consequência clínica da acumulação de Gb3. Podócito é o tipo celular mais afetado na doença renal, que mostra apenas uma resposta parcial à Terapia de Reposição Enzimática. Além disso, a disfunção podocitária é a principal contribuinte para a perda progressiva da função renal e pode ser encontrada alterada mesmo antes do início da microalbuminúria. Assim, a podocitúria na DF pode ser uma ferramenta importante para prever a doença renal. Objetivo: O objetivo deste estudo foi quantificar a excreção urinária de podócitos em pacientes com DF (V269M, n = 14) e controles saudáveis (n = 40), e relacioná-las com as variáveis sexo, idade, tempo de terapia e a razão albumina: creatinina (AUC). Métodos: Podócitos urinários foram identificados utilizando imunofluorescência para podocalixina e DAPI. O número de células podocalixina positivo foi contado e o número médio foi utilizado (faixa normal 0-0.6 podócitos/mL). Resultados: O número médio de podócitos na urina de pacientes com DF foi significativamente maior do que os controles saudáveis (p < 0.0001). Observou-se uma correlação positiva entre podocitúria e AUC (p = 0.004; r2 = 0.6417). Conclusão: A podocitúria pode ser uma ferramenta adicional para avaliar a progressão da doença renal em pacientes que se espera que tenha um fenótipo mais agressivo.

Abstract Introduction: Fabry disease is a lysosomal storage disorder due to abnormalities in the GLA gene (Xq22). Such changes result in the reduction/absence of activity of the lysosome enzyme α-GAL, whose function is to metabolize globotriaosylceramide (Gb3). Renal disease is a major clinical outcome of the accumulation of Gb3. Podocyte injury is thought to be a major contributor to the progressive loss of the renal function and may be found altered even before the onset of microalbuminuria. Objective: The aim of this study was to quantify the urinary excretion of podocytes in Fabry disease patients (V269M, n = 14) and healthy controls (n = 40), and to correlate podocyturia with the variables gender, age, time of therapy and albumin: creatinine ratio (ACR). Methods: Urinary podocytes were stained using immunofluorescence to podocalyxin and DAPi. The number of podocalyxin-positive cells was quantified and the average number was taken (normal range 0-0.6 podocytes/mL). Results: The average number of podocytes in the urine of Fabry disease patients was significantly higher than in healthy controls (p < 0.0001). We observed a positive correlation between podocyturia and ACR (p = 0.004; (r2 = 0.6417). We found no correlation between podocyturia and gender, age or duration of therapy. Conclusion: Podocyturia is an important parameter in the assessment of renal disease in general, and it may serve as an additional early tool for monitoring Fabry disease nephropathy even before changes in ACR are seen. This may prove to be a useful tool to assess disease progression in patients expected to have a more aggressive phenotype.

Diagnostic value of serum anti-phospholipase A2 receptor antibodies (PLA2R) in idiopathic membranous nephropathy / 中华检验医学杂志

Objective To investigate the diagnostic value and monitoring application of anti-phospholipase A2 receptor antibodies ( PLA2R ) in patients with idiopathic membranous nephropathy ( IMN) .Methods A retrospective study was used .48 patients were diagnosed as idiopathic membranous nephropathy by puncturing kidney from January of 2013 to June of 2014 in the Hospital of Liaoning Traditional Chinese Medical University and Shengjing Hospital of China Medical University were included. 43 patients were diagnosed as non-idiopathic membranous nephropathy ( NIMN ).35 healthy volunteers were selected as control groups.Serum PLA2R were detected by indirect immunofluorescence.The intensity of antibodies fluorescence and the level of urine protein in 24 hours(24 h PRO) ,serum UREA ,serum CYSC, serum UA,GFR were compared in each groups.Measurement data was shown as mean ±standard deviation;Count data was shown as frequency or composition, t test andχ2 test were used as statistical method.Results (1) Among 48 cases with IMN,37 cases showed positive PLA2R (positive rate 77.08%).All of cases were negative in NIMN group.The sensitivity and the specificity of serum PLA2R were 77.08%and 100%.(2) Compared with control groups, the levels of UREA, CYSC and UA were found statistically significant differences in patients with IMN ( P 0.05 ) . ( 3 ) Positive correlation were found between 24 h PRO(r=0.877,P=0.00),serum UA (r=0.766,P=0.00 ) with the level of PLA2R antibodies fluorescence intensity.( 4 ) There were no correlation between serum PLA2R antibody with IMN pathology aging (r=0.087,0.194,0.182;P=0.598,0.399,0.667).PLA2R positive rate(Ⅰstage:37.50%,Ⅱstage:84.85%, Ⅲstage 85.71%) may increases with the increasing of illness severity.Conclusion Serum PLA2R antibody would be a new laboratory diagnosis standard in patients with IMN.(Chin J Lab Med, 2015, 38:595-599 ) Objective To investigate the diagnostic value and monitoring application of anti-phospholipase A2 receptor antibodies ( PLA2R ) in patients with idiopathic membranous nephropathy ( IMN) .Methods A retrospective study was used .48 patients were diagnosed as idiopathic membranous nephropathy by puncturing kidney from January of 2013 to June of 2014 in the Hospital of Liaoning Traditional Chinese Medical University and Shengjing Hospital of China Medical University were included. 43 patients were diagnosed as non-idiopathic membranous nephropathy ( NIMN ).35 healthy volunteers were selected as control groups.Serum PLA2R were detected by indirect immunofluorescence.The intensity of antibodies fluorescence and the level of urine protein in 24 hours(24 h PRO) ,serum UREA ,serum CYSC, serum UA,GFR were compared in each groups.Measurement data was shown as mean ±standard deviation;Count data was shown as frequency or composition, t test andχ2 test were used as statistical method.Results (1) Among 48 cases with IMN,37 cases showed positive PLA2R (positive rate 77.08%).All of cases were negative in NIMN group.The sensitivity and the specificity of serum PLA2R were 77.08%and 100%.(2) Compared with control groups, the levels of UREA, CYSC and UA were found statistically significant differences in patients with IMN ( P 0.05 ) . ( 3 ) Positive correlation were found between 24 h PRO(r=0.877,P=0.00),serum UA (r=0.766,P=0.00 ) with the level of PLA2R antibodies fluorescence intensity.( 4 ) There were no correlation between serum PLA2R antibody with IMN pathology aging (r=0.087,0.194,0.182;P=0.598,0.399,0.667).PLA2R positive rate(Ⅰstage:37.50%,Ⅱstage:84.85%, Ⅲstage 85.71%) may increases with the increasing of illness severity.Conclusion Serum PLA2R antibody would be a new laboratory diagnosis standard in patients with IMN.

Clinical significance of antinuclear antibodies associated with the nuclear dense fine speckled immunofluorescence pattern in autoimmune disease / 中华检验医学杂志

Objective To explore the prevalence of the nuclear dense fine speckled ( DFS ) immunofluorescence pattern in routine antinuclear antibodies(ANA) testing and its significance in patients with autoimmune diseases( AID) .Methods The ANA in 13 728 specimens were measured by indirect immunofluorescence( IIF) using HEp-2 cell slides from department of clinical laboratory, wenling hospital from 2011 to 2014.The frequencies, clinical manifestations and laboratory features of DFS positivity were restrospectively analyzed in patients with AID,usingχ2 test.Results ANA was positive in 20.56%(2 822/13 728) of the total patients, and the frequency of DFS pattern was observed in 1.81%(248/13 728) of the total patients and in 8.79%(248/2 822) of the patients with ANA positivity.In different age groups (≤20 years old, 21-49 years old and≥50 years old) , there were statistical significance of DFS pattern positive rate (male:χ2 =18.17,P<0.01; female: χ2 =1 500.00,P<0.01).And the highest frequency of ANA positivity was observed in patients from department of rheumatology(30.07%).The frequency of DFS pattern was higher in the departments of infection ( 32.58%) , dermatology ( 21.76%) , neurology ( 18.58%) and nephrology(6.73%) among the patients with ANA positivity(χ2 =123.00,P<0.01).Amony the 248 cases with DFS pattern positivity.41 cases were AID ( 16.53%) and 207 cases were non-autoimmune diseases ( 83.47%) . In AID group 13 cases were autoimmune thyroiditis ( 31.71%) , 12 cases were rheumatoid arthritis ( 29.27%) , 4 cases were autoimmune liver disease ( 9.76%) , 4 cases were undifferentiated connective tissue disease (9.76%), 3 cases were ankylosing spondylitis(7.32%), 2 cases were Sj?gren′s disease ( 4.88%) , 2 cases were inflammatory bowel disease ( 4.88%) and 1 case was systemic lupus erythematosus(2.44%).The titers of DFS in patients with AID were predominantly above 1∶320 and less than 1∶100 in non-AID.AID patients with DFS pattern positivity have different clinical manifestations and laboratory features.Howerer, antinuclear antibodies ( ANAs ) in 15 specific auto-antibodies were all negative.Conclusions The DFS pattern is a common pattern in ANA positivity patients and it mainly exists in non-AID patients.Further more, it is suggested that patients with DFS pattern identified by IIF should then be tested for anti-DFS70 antibodies with a specific immunoassay.

Diagnostic value of serum and cerebral spinal fluid aquaporin 4-IgG detected by indirect immunofluorescence assays using different base in neuromyelitis optica / 中华神经科杂志

Objective To explore the diagnostic value of serum and cerebral spinal fluid (CSF) aquaporin 4(AQP4)-IgG detected by indirect immunofluorescence assay (ⅡFA) using monkey optical nerve and AQP4 transfected cell as base in neuromyelitis optica (NMO).Methods Serum and CSF AQP4-IgG in 32 NMO patients,41 multiple sclerosis (MS) patients,33 non-inflammatory neurological disease (NIND) patients and serum AQP4-IgG in 20 healthy controls (HC) were detected by monkey optical nerve/AQP4 transfected cell-based IIFA.Results (1) In both optical nerve and AQP4 transfected cell based IIFA,compared with MS,NIND and HC,the patients with NMO had significantly higher positive rate of AQP4-IgG in both serum (optical nerve-based IIFA:NMO 46.9％ (15/32),MS 7.3％ (3/41),NIND 3.0％ (1/33),HC 0 (0/20),P ＜ 0.01 ; cell-based IIFA:NMO 84.4％ (27/32),MS 2.4％ (1/41),NIND 3.0％ (1/33),HC 0 (0/20),P ＜0.01) and CSF (optical nerve-based ⅡFA:NMO 21.9％ (7/32),MS 0 (0/41),NIND 0 (0/33),P ＜ 0.01 ; cell-based IIFA:NMO 56.3％ (18/32),MS 0 (0/41),NIND 0(0/33),P＜0.01); sensitivity of serum AQP4-IgG (optical nerve-based IIFA 46.9％; cell-based IIFA 84.4％) was significantly higher than that of CSF AQP4-IgG (optical nerve-based IIFA 21.9％,P ＜0.01 ; cell-based IIFA 56.3 ％,P ＜ 0.05),while no significant difference was found in specificity between serum and CSF AQP4-IgG in diagnosing NMO; using combination of serum and CSF AQP4-IgG applied,the sensitivity increased (optical nerve-based IlFA 46.9％ vs 50.0％ ; cell-based IIFA 84.4％ vs 87.5％) while specificity remained no change.(2) Compared with optical nerve-based IIFA (serum 46.9％,CSF 21.9％),cell-based IIFA had higher sensitivity in diagnosing NMO (serum 84.4％ ; CSF 56.3％,P ＜ 0.01),while no significant difference of specificity between these two methods.Conclusion It has better clinical value to detect serum and CSF AQP4-IgG at the same time by AQP4 transfected cell based IIFA in diagnosing NMO.

A study on clinical values of antineutrophil cytoplasmic antibody detection in diagnosis of rheumatic diseases / 国际检验医学杂志

Objective To investigate the clinical values of antineutrophil cytoplasmic antibody (ANCA) detection in the diagnosis of rheumatic diseases .Methods 965 patients with rheumatic diseases were taken as rheumatic group ,including 256 cases of SLE , 124 of mixed connective tissue disease(MCTD) ,336 of rheumatoid arthritis(RA) ,166 of Sjogren′s Syndrome(SS) ,45 of systemic sclerosis(SSc) and 38 of dermatomyositis .400 healthy people were served as the control group .Indirect immunofluorescence (IIF) was employed to detect the cytoplasmic ANCA (c-ANCA ) ,perinuclear ANCA (p-ANCA ) .Enzyme-linked immunosorbent assay (ELISA) was utilized to assay the anti-myeloperoxidase(MPO) antibody among patients with positive ANCA .Results Difference of positive rates of serum ANCA of subjects in rheumatic group and control group showed statistical significance (P<0 .01) ,and the differences of positive rates of serum p-ANCA and c-ANCA of subjects with positive ANCA between the two group were also sta-tistically significant(P<0 .01) .The positive rate of serum p-ANCA of patients with SLE was markedly higher than those with MCTD ,RA ,SS ,SSc ,dermatomyositis and healthy people in control group (P< 0 .05) ,while that of patients with MCTD was obviously higher than those with RA ,SS ,SSc ,dermatomyositis and healthy people in control group (P<0 .05) .The positive rate of anti-MPO antibody of 165 patients with positive ANCA in rheumatic group was 58 .4% ,which was significantly higher than that in control group(3% )(P<0 .01) .The positive rate of patients with positive ANCA and impaired renal function in rheumatic group was 55 .4% ,which was obviously higher than those of patients with negative ANCA in rheumatic group (21 .8% ) and control group (6 .25% )(P<0 .01) .Conclusion Serum ANCA detection is of important significance for prevention of nephrotoxic damage in pa-tients with rheumatic diseases .

Obstrução tubária em mulheres com imunofluorescência indireta positiva para clamídia / Tubal occlusion in women with indirect positive immunofluorescence for chlamydia

Objetivos: Avaliar o desempenho da imunofluorescência indireta para Chlamydia trachomatis em rastrear obstrução tubária. Métodos: Este é um estudo retrospectivo com 204 pacientes atendidas em um centro universitário e particular de infertilidade na cidade de Goiânia no período de 2006 a 2009. Para avaliar o risco de obstrução tubária as pacientes foram divididas em dois grupos: pacientes “expostas” à clamídia (imunofluorescênciaindireta ≥1:16) e “não expostas” (imunofluorescência indireta <1:16). Verificou-se, então, as pacientes que tiveram a “doença” (obstrução tubária) e “controles” (sem obstrução tubária) na histerossalpingografia. Para os cálculos foram utilizados os testes Qui-quadrado (χ2) e Exato de Fisher. O nível de p escolhido foi 0,05. Resultados: Das 72 pacientes com titulação significativa, 34 (47,2%) apresentaram a ocorrência de obstrução tubária. Em relação às 132 pacientes com titulação não significativa, somente 18 (13,7%) apresentaram obstrução tubária (p<0,001). Foi observado também um aumento progressivo entre os níveis de anticorpos e a probabilidade de obstrução tubária (p<0,001). Conclusões: Os resultados deste estudorevelaram que a sorologia para Chlamydia trachomatis é válida para rastreamento de lesão tubária, portanto, pode facilitar decisões naquelas mulheres que devem prosseguir com novas investigações.

Purpose: To evaluate the ability of indirect immunofluorescence for Chlamydia trachomatis to screening tubal occlusion. Methods: This is a retrospective study with 204 electronic records of patients attended at a university and private infertility center in the city of Goiania, in the period of 2006 to 2009. To evaluate the risk of tubal occlusion the patients were divided into two groups: patients “exposed” to chlamydia (IFI≥1:16) e “unexposed” (IFI<1:16). It was verified patients who had the “disease” (tubal occlusion) and “control” (without tubal occlusion) in the hysterosalpingography. For the calculations the Chi-square (χ2) and Fisher Exact Test were used. The p chosen level was 0,05. Results: Of the 72 patients with significant titers, 34 (47,2%) showed the occurrence of tubal occlusion. Concerning the 132 patients with no significant titers, only 18 (13,7%) had tubal occlusion(p<0,001). We also observed a progressive increase in the levels of antibodies and the likelihood of tubal occlusion (p<0,001). Conclusions: The results indicate that serology for Chlamydia trachomatis is valid for screening of tubal damage and may facilitate decisions on which women should proceed with further investigations.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector / 中华骨科杂志

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector. Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase chain reaction (PCR). hBMP2 gene was inserted into pTA2-T-easy and pSELECT-GFPzeo-MCS eukaryotic expression vector, and then transferred into competence DHSα cells. After screening, pSELEC-GFPzeo-hBMP2 was obtained and identified by sequence analysis. The recombinant vector pSELECT-GFP zeo-rhBMP2 was transfected into CHO cells. The successful trasfection was verified by fluorescence microscope in 48-72 hours. The RT-PCR and immunofluorescence was used to confirm the hBMP2 expression. Western Blotting was used to detect the secretion of hBMP2.Results A 1216 bp fragment was obtained by PCR, the same as expectant fragment. The recombined pSE-LECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis. The results were identical with that of reported hBMP2 sequence (Genebank NM-001200). The successful transfection was verified by fluorescence microscope in 48-72 hours. The stable expression in eukaryotic cells was confirmed by immunofluorescence and RT-PCR which showed an obvious band between 1000-2000 bp. Western Blotting identified the immunogenicity of recombinant human BMP2 with the molecular weight of about 17×103. Conclusion The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.