A case report of staphylococcal protein A immunoadsorption therapy for refractory myasthenia gravis / 中华神经科杂志

Staphylococcal protein A (SPA) has a high affinity for human immunoglobulin, and SPA immunoadsorption can specifically reduce the titer of autoantibodies and quickly relieve the clinical symptoms of myasthenia gravis (MG). Recent studies have suggested that immunoadsorption has better clinical efficacy and a lower incidence of adverse reactions than plasma exchange. A case of refractory MG with poor response to corticosteroids, intravenous immunoglobulins and immunosuppressive therapy was reported. The patient had low immune function and progressive pulmonary infection in the later stage of the disease. Respiratory muscle weakness was relieved quickly after four times of immunoadsorption therapy. The value of immunoadsorption in the treatment of refractory MG was explored with literature review.

Molecular types and related clinical features of methicillin-resistant Staphylococcus aureus in ;Jingzhou area / 中华临床感染病杂志

Objective To investigate the molecular types and related clinical features of methicillin-resistant Staphylococcus aureus (MRSA) in Jingzhou area, Hubei Province.Methods A total of 80 MRSA strains confirmed by mecA gene were isolated from inpatients in Jingzhou Central Hospital of Hubei province during January and December 2014. Vitek 2 Compact was used for antibiotic susceptibility test . Staphylococcus protein A (SPA) types and Staphylococcal cassette chromosome mec (SCCmec) genotypes were detected by multiplex polymerase chain reaction ( PCR ) and gene sequencing . Panton-valentine leucocidin ( pvl) gene of the strains was detected by PCR .Chi-square test and Wilcoxon test were used for data analysis .Results There were 16 spa types in 80 MRSA isolates , in which t030 and t437 were the most prevalent ones accounting for 50.0% ( 40 strains ) and 28.8% ( 23 strains ) of the total strains, respectively.There were 77 strains of SCCmec type Ⅰ-Ⅴ, in which SCCmecⅢ and SCCmecⅣ were the most prevalent ones accounting for 45.0% (36 strains) and 35.0% (28 strains), respectively.t030 was the main spa type in isolates of SCCmecⅢ(33/36, 91.7%), while t437 was the main spa type in isolates of SCCmecⅣ(20/28, 71.4%).Patients infected with t030/SCCmecⅢMRSAs were with higher ages than those infected with t437/SCCmecⅣMRSAs (T=446.500 and 607.500, P<0.01).Patients infected with t030/SCCmecⅢ MRSAs were mainly from surgical wards and intensive care unit ( ICU ) , while those infected with t437/SCCmecⅣ MRSAs were mainly from pediatrics wards , and there were significant differences in ward distribution between two groups (χ2 =33.724 and 29.768, P <0.01).Seventy percent and above strains of t030/SCCmec type Ⅲ were resistant to rifampin, erythromycin, clindamycin, tetracycline, levofloxacin, moxifloxacin, ciprofloxacin and gentamicin .Strains of t437/SCCmec type Ⅳwere resistant to erythromycin , clindamycin and tetracycline , but were sensitive to most non-β-lactam antimicrobial drugs (with resistance rates <20%).Virulence gene pvl was found in 11 strains (13.8%), in which 7 were strains of t437-SCCmec typeⅣ.Conclusions MRSAs in Jinzhou are of various genotypes , in which t030-SCCmecⅢand t437-SCCmecⅣare the most prevalent ones .Strains of t030-SCCmec typeⅢare usually multiple-drug resistant , mainly seen in elderly patients in surgical wards and ICU .Strains of t437-SCCmecⅣare sensitive to most non-β-lactam antimicrobial drugs , and its infection is mainly seen in children and young people .

Characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus isolated from patients in intensive care units / 中国感染控制杂志

Objective To study the characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus (S .aureus)in the intensive care units(ICUs)of a hospital.Methods Clinical isolates of S .aureus collected from ICUs between January and December 2014 were identified and performed antimicrobial susceptibility testing,then typed by staphylococcal protein A (spa)typing and multilocus sequence typing (MLST) methods.Results Of 160 isolates of S .aureus ,120 (75.00%)were methicillin-resistant S .aureus (MRSA). Resistance rates of MRSA to erythromycin,clindamycin,and levofloxacin were all > 80%;methicillin-sensitive S .aureus (MSSA)were sensitive to cefazolin,resistance rates to erythromycin,clindamycin,and levofloxacin were 62.50%,35.00%,and 10.00% respectively.spa typing and MLST results showed that the main types of 120 isolates of MRSA were ST239-t030,ST239-t037,and ST5-t2460,the major epidemic strains were ST239-t030 (n=105,87.50%),and were isolated from 8 ICUs;MSSA had more types,ST59-t437 were detected only from depart-ment of neurology(n =8)and department of digestive diseases(n =2),ST6-t701 ,ST398-t3625,ST398-t1793,and ST121-t2092 were isolated from departments of neurology(n=7),anesthesiology(n=5),neurosurgery(n=4),and cardiac surgery(n=4)respectively.Conclusion Isolation rate of MRSA in ICUs in this hospital is high,ST239-t030 is the main type,which prevailed in hospital;different types of MSSA have epidemic trends in various departments.

Effect of staphylococcal protein A on pro-inflammatory cytokines expression in mononuclear macrophage / 重庆医学

Objective To study the expression and regulation of pro‐inflammatory cytokinesTNF‐α,IL‐1 ,IL‐6 in mononucle‐ar macrophages stimulated with staphylococcal protein A (SpA) .Methods THP‐1 was incubated with PMA and induced into mononuclear macrophages .Then the macrophages were incubated with varying concentrations of SpA under different time points . The effect of SpA on macrophage proliferation was measured by MTT method .The levels of inflammatory cytokines ,TNF‐α,IL‐1 and IL‐6 from the cultured cell media were measured by ELISA respectively .The levels of mRNA expression corresponding to TNF‐α,IL‐1 and IL‐6 were detected by RT‐PCR from the macrophages stimulated with SpA .All statistical analyses were performed by SPSS17 .0 software .Results The MTT result indicated that SpA had a positive effect on the proliferation of THP‐1 cells in a dosage depended manner .The addition of SpA could enhance the mRNA expression of TNF‐α,IL‐1 and IL‐6 in the stimulated mac‐rophages .It also showed a specific dose‐effect and time‐effect correlation .The macrophages secreted inflammatory cytokines and its corresponding mRNA reached its peak levels at 12 h post stimulation .Compared with the control group ,the expression and release of TNF‐α,IL‐1 and IL‐6 in macrophages from the experimental group was increased with statistical significance(P<0 .01) .Conclu‐sion SpA can promote the secretion and expression of early pro‐inflammatory cytokines ,such as TNF‐α,IL‐1 and IL‐6 in macro‐phages .Therefore ,SpA plays a very important role in the initiation and development of the staphylococcus aureus sepsis .

A study of rapid genotyping method for methicillin-resistant Staphylococcus aureus / 国际检验医学杂志

Objective To establish a rapid genotyping method of for methicillin-resistant Staphylococcus aureus(MRSA) based on polymerase chain reaction(PCR)-high resolution melting (HRM ) curve analysis and staphylococcal protein A (SPA ) classifica-tion .Methods 71 strains of MRSA clinically isolated were collected as test strains .Gene sequencing and HRM curve analysis were employed to conduct SPA gene typing .Results According to gene sequencing method ,SPA gene of 71 strains of MRSA was divided into four types ,namely t570 ,t030 ,t002 and t588 .The most predominant type was t570 (74 .65% ) ,followed by t030 and t002(both 7 cases) .The result of SPA gene typing by HRM analysis were basically consistent with that by gene sequencing .Con-clusion PCR-HRM analysis is expected to become a fast ,efficient genotyping for MRSA SPA gene ,providing the basis for hospital infection control .

Use of Staphylococcal Protein-A and Streptococcal Protein-G for Detection of Red Blood Cells (RBC) Antibodies and Comparison with Other Techniques.

Background: Detection of red blood cells antibodies is important for the diagnosis of autoimmune hemolytic anemia, hemolytic disease of newborn, pre-transfusion testing and other problems. The aim of this study was to use Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) as reagents in immunological tests for detecting red blood cells (RBC) antibodies and to compare the method with other techniques. Study Design & Methods: Sera from 60 patients, comprising forty-four anti-D positive sera from pregnant women and 16 from healthy controls were, used for the study. The anti-globulin gel test and the standard Coombs’ test were used to determine RBC antibodies in these sera and the result were compared with that of protein A and protein G tests. Results: With various degree of agglutination all 4 techniques detected the presence of RBC antibodies (anti-D) in the sera from 44 pregnant women, and tested negative for the remaining 16 sera (from healthy controls). The sensitivity and the specificity of the 4 techniques was 100%. Conclusions: This preliminary study demonstrates that both SpA and SpG tests can be used for the detection of RBC antibodies and therefore requires more study and testing before they can become useful standard tests in transfusion medicine.

Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species / Comparación de las técnicas de detección de la reactividad de la proteína de unión de la inmunoglobulina frente a la inmunoglobulina producida por diferentes especies aviarias y mamíferas

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.

Drug-resistance and genotyping of methicillin-resistant Staphylococcus aureus isolated from intensive care unit / 中华临床感染病杂志

Objective To investigate drug resistance and genotypes of methicillin-resistant Staphylococcus aureus (MRSA) isolated from intensive care unit (ICU). Methods MRSA strains were isolated from patients, medical staff and environment of hospital ICUs. Disk diffusion (K-B method) was used for drug resistance testing; Staphylococcal cassette chromosome mec (SCCmec) and Staphylococcal protein A (spa) typing methods were used for genotyping and identifying the homology. Results There were 78 strains of Staphylococcus aureus isolated including 62 isolates of MRSA, which were mainly from the burn ICU (22, 35.48%). Among 62 MRSA strains, 50 were hospital acquired strains, in which 43 isolates were of SCCmec Ⅲ, 4 of SCCmec Ⅰ and 3 of SCCmec Ⅱ. Twelve isolates could not be typed. Twenty-eight out of 37 hospital acquired isolates were typed by spa typing as SCCmec Ⅲ-t030, which belonged to the same clone. Conclusion MRSA in ICU is multi-drug resistant and SCCmec Ⅲ-t030 is the most prevalent genotype, which indicates that clinical MRSA strains and environmental MRSA strains may be homologous.

Expression, purification and application of staphylococcal protein A fused to SUMO / 中华微生物学和免疫学杂志

Objective To clone the full length staphylococcal protein A(SPA) gene from Staphylococcus aureus (ATCC6538), and subsequently study the gene structure and antibody binding ability.Methods The full length and the functional region of the SPA gene were cloned into pHisSUMO vector respectively, and expressed in E. coli. The full length and the functional fragment of the SPA protein were detected for antibody binding ability and stability. The functional fragment of the SPA protein fused with SUMO was coupled to the CNBr-activated agarose for antibody purification from rabbit serum. Results A variant of the full length SPA gene was cloned, which has been submitted to GenBank (the accession number is EU695225). Two fusion proteins had the same antibody binding ability as the untagged SPA protein. However, the formers was more stable than the latter at the tested conditions. SUMO-SPA conjugated-agarose kept high efficiency for antibody binding. Conclusion To our knowledge, the full length SPA gene of S.aureus(ATCC6538) is a novel variant. The SUMO tag can improve the stability of the functional region of the SPA protein without damaging the antibody binding ability. This fusion protein has been used for antibody purification successfully.

Study and application of immunogold filtration assay for detecting serum antibodies against helicobacter pylori / 中国实用内科杂志

Objectives To establish a simple,quick,accurate serodiagnostic assay for detecting serum antibodies against Helicobacter pylori(H.pylori).Methods We established quick immunocolloidal gold filtration assay(DIGFA) by combining colloidal gold labelling technique with filtration method.145 sera from cases with H.pylori infection were detected by DIGFA,and the results were compared with that of ELISA and pathological examinations.In addition,blocking test and human IgG protein detection were conducted.The sensitivity,specificity of DIGFA were analysed.Besides,DIGFA was applied to investigate the H.pylori infection situation among persons of different ages.Results The coincidence rate reached 95 1% compared with the ELISA,and it was 88 8% compared with pathological the examination.The detectable minimum amount of IgG was 10?g/mL.The antibodies detected by DIGFA proved to be specific antibodies by blocking test.The human H.pylori infection rate among 216 persons reached 42 5% by DIGFA.Conclusions DIGFA can be applied effectively in clinical diagnosis and epidemiological survey.

The Clinical Significance of Anti-LSP Antibodies in the Sera of Patients with Viral Hepatitis: An Evaluation with SPA-RIA & ELISA / 第三军医大学学报

Antibodies against LSP in the sera of 168 patients with various types of viral hepatitis were determined with SPA-RIA. The sera of another group of 178 patients (109 of them were from the first group) with viral hepatitis were studied with ELISA for the same antibodies, which were further divided into three categories, that is, IgG, IgM and IgA classes. The results of 109 patients examined with both of the two methods, indicated that anti-LSP antibodies measured by SPA-RIA might mainly represent anti-LSP IgG class. It was found that circu-lating anti-LSP antibodies could easily be detected in most patients with either acute or chronic hepatitis. After analyzing the-results, the authors suggest that the humoral immune response against LSP might not be the sole initiating factor in the pathogenesis of viral hepatitis, they are more likely the result of the antigen variation of the injured liver cells.