Development and verification of kinetic chromogenic quantitative detection method for endotoxin content in intermediate of component pertussis antigen / 中国生物制品学杂志

@#Objective To develop a kinetic chromogenic quantitative method for the determination of endotoxin content in intermediate of pertussis antigen,and to verify the method so as to better control the quality of diphtheria,tetanus,and pertussis vaccine(DTP vaccine).Methods A kinetic chromogenic assay[Limulus Amebocyte Lysate(LAL)]was developed to detect the endotoxin content in the intermediate products of pertussis antigens after detoxification,and verified for the linearity,specificity,accuracy,reproducibility and intermediate precision. The quantitative detection results of kinetic chromogenic assay were compared with those of gel method.Results The absolute value of the linear correlation coefficient（|r|）of the kinetic chromogenic assay was more than 0. 99;under the maximum effective multiple dilution,the interference test recovery of the intermediate was within 50% — 200%,and pertussis toxin(PT)diluted to 10,100 and 1 000 times,filamentous hemagglutinin(FHA)diluted to 3 000,5 000 and 10 000 times,and pertussis adhesin(PRN)diluted to 50,75 and 100 times had no interference effect on the experiment after detoxification;the accuracy verification recovery rates of PT,FHA and PRN were 125%,110% and 99% respectively;and the CVs of reproducibility verification were 7. 21%,8. 31% and 5. 84%,and the CVs of intermediate precision verification were 6. 04%,16. 29% and 12. 23%,respectively.The bacterial endotoxin content of the three batches of pertussis antigen intermediates detected by kinetic chromogenic assay was consistent with that verified by gel method,both of which were less than the limit of bacterial endotoxin in the intermediates of pertussis antigen after detoxification.Conclusion The developed kinetic chromogenic assay has good linearity,specificity,accuracy and precision with accurate detection results,which can be used to detect the endotoxin content in intermediate products of component pertussis antigen after detoxification.

Current status and development trend of coagulation factor Ⅷ activity / 中华检验医学杂志

The detection of coagulation factor Ⅷ activity plays an important role in the diagnosis, typing, efficacy monitoring and detection of inhibitor titer in hemophilia A, acquired hemophilia A and von Willebrand disease. However, due to the diversity of detecting systems, the difference of reagent composition, the existence of interfering substances and other influence factors, the detection of coagulation factor Ⅷ activity in the laboratories in China still needs to be improved.

Study on quantitative detection of bacterial endotoxin in recombinant novel coronavirus vaccine (CHO cell) by micro-dynamic chromogenic / 中国药理学通报

Aim To explore the feasibility of the micro- dynamic chromogenic method for quantitative detection of bacterial endotoxin in recombinant novel coronavirus vaccine ( CHO cell).Methods The micro-dynamic color method of Limulus reagent was used to establish a bacterial endotoxin standard curve.The dilution factor was determined through interference pre -experiment, the recoverv rate of the endotoxin added to the test so- J lution was determined, and the interference test to complete the quantitative detection test of the bacterial endotoxin content in the test product was performed, and the results were compared with those of the gel-clot method.Results Hie linear range of the concentration of the standard curve was 0.02 to 2.0 EU • mL 1 , and the regression equation of the standard curve was lgT =-0.302 7 lgC +2.858 7( r = 0.998 9).When recombinant novel coronavirus vaccine ( CHO cell) was cliluted 40 times or below, the micro -dynamic chromogenic reagent did not interfere with the bacterial endotoxin agglutination reaction, and the recovery rate was 50% to 200%.The test results were consistent with the gel- clot method.Conclusions The micro-dynamic chromogenic method can be used for the quantitative detection of bacterial endotoxins in recombinant novel coronavirus vaccine ( CHO cell) with accurate results, high sensitivity, and process monitoring.

Study on equivalence between micro kinetic chromogenic assay and kinetic chromogenic assay / 中国药理学通报

Aim To evaluate the equivalence between micro kinetic chromogenic assay anrl kinetic chromogenic assay in order to provide data support for the use of alternative methods.Methods Detection conditions; micro kinetic chromogenic assay and kinetic chromogenic assay limulus reagent were used, sample amount of each well and limulus reagent was 25 jxL ( kinetic chromogenic assay was 100 jxL) , detection wavelength was 405 nm, ONSET OD value was 0.03, and half- well elisa plate was used for detection ( kinetic chromogenic assay was ordinary ELISA plate).The equivalence of the two methods was evaluated by various statistical methods, such as equivalence test, in collaboration with four laboratories in China.Results The results of one-way an OVA, paired T test and equivalence test were consistent, indicating that there were some differences between the existing kinetic chromogenic assay of different manufacturers, while there was no significant difference between the trace or conventional amount of reagent used by each manufacturer.Conclusions Micro kinetic chromogenic assay is e- quivalent to existing reagents in terms of accuracy and recoverv.J.

Proficiency testing for determination of photometric method of bacterial endotoxin / 中国药理学通报

Aim To design and implement the bacteri¬al endotoxin test proficiency testing plan to evaluate the laboratory's ability and level to determine bacterial en¬dotoxin. Methods According to the Chinese Pharma-copoeia (2015 edition, Vol IV) -1143 Bacterial Endo¬toxin Test-Photometric Method ( Method 2 ) , each la¬boratory used any of these methods to determine the endotoxin content of the sample to be tested. The stati- stical software JMP13 was used for statistical analysis of the feedback results of the participating laboratories. The consensus value of the participants, namely the ro¬bust average value of the effective test results of all the participating laboratories, was used as the assigned value X of the endotoxin content of the samples to be tested in the proficiency testing of the round. The re¬sults of participating laboratories were evaluated ac¬cording to the following criteria: (1) the laboratory test results were within 50% to 200% of the assigned value of the sample, which was evaluated satisfactory; ( 2 ) the laboratory test results were not within the 50 -200% range of the assigned value of the sample, which was evaluated dissatisfactory. Results A total of laboratories participated in this capacity verification plan, with 45 in the laboratory with satisfactory re¬sults, with a satisfaction of 91. 8% ; There were 4 la¬boratories that received "dissatisfaction" results, all of which were not within the range of 50% -200% of the assigned value of samples, and the dissatisfaction of 8. 2% . Conclusions Most of the participating labora¬tories can accurately detect the bacterial endotoxin con¬tent in the sample to be tested, indicating that the level of bacterial endotoxin detection in our country is gener¬ally good at present.

Bacteriological Profile of Wound Swab and Pus Samples Using Conventional Media and Chromogenic Medium.

Background: Wound infections continue to be a causeof concern as they can delay healing and cause woundbreakdown. Their effective treatment demands quickisolation and identification of causative organisms withappropriate antibiotic sensitivity pattern. Material andMethods: Wound swab and pus samples received frominpatient as well as outpatient department of all agegroups and both genders were processed usingconventional media as well as chromogenic medium(HiCrome UTI) and results of both were compared.Antibiotic sensitivity testing was done on Vitek 2Compact automated system. Results: Among 342samples, 77% showed growth. Fifty eight percentagewere Gram negative and 42% were Gram positiveorganisms. Polymicrobial growth was seen in 11% ofsamples. HiCrome UTI isolated all organisms inculture. Colony characteristics and colour of all isolateson HiCrome UTI were comparable to theiridentification on Vitek 2 Compact. Among the Grampositive organisms, commonest was MethicillinSensitive Staphylococcus aureus (MSSA 42%)followed by Methicillin Resistant Staphylococcusaureus (MRSA 33%), Enterococcus faecalis (10%),Staphylococcus epidermidis (8%), Staphylococcushaemolyticus (3%), Streptococcus pyogenes (2%) andStreptococcus agalactiae (2%). Most of the Grampositive organisms were sensitive to vancomycin,teicoplanin, linezolid and clindamycin The commonGram negative organisms were E. coli (36%),Klebsiella pneumoniae (20%), Pseudomonasaeruginosa (18%), Proteus mirabilis (7%),Enterobacter cloacae (6%) and Acinetobacterbaumannii (4%). Most of the Gram negative organismswere sensitive to cefepime, beta lactams-betalactamase inhibitors, aminoglycosides and fluoroquinolones. Conclusion: Gram-negative organismspredominated in our study. HiCrome UTI agar can beused as a cost-effective approach for rapid isolation ofall organisms. It gives definite identification ofcommon organisms and thus reduces turn-around-timefor the same. It provides presumptive identification ofinfrequent organisms which can be further confirmedby simple biochemical tests. Hence these properties ofHiCrome UTI agar help serve the purpose especiallyfrom mixed cultures and in resource constraint settings

Validación del ensayo FVIII: C cromogénico en una plataforma automatizada

El objetivo de este trabajo fue realizar la validación analítica del método cromogénico (FVIII:Ccro) en la plataforma ACL TOP y correlacionarlo con el método coagulable en una etapa (FVIII:Ccoag). El estudio de validación (EP5-A2, EP6-A2 y comparación de métodos por EP-9) se realizó para la curva de rango normal-bajo (CRNB): aproximadamente entre 10-150 UI/dL de FVIII y de rango muy bajo (CRMB): aproximadamente entre 0-10 UI/dL. Los resultados de repetitividad (CVr) y precisión intermedia (CVi) fueron menores del 6% y comparables a los informados por el fabricante para otras plataformas. El rango de medición analítica fue de 11-129 UI/dL con CRNB, y se extrapoló a 0,3 UI/dL al utilizar la CRMB. Para la CRNB FVIII:Ccro mostró buena correlación con FVIII:Ccoag: r: 0,98, pendiente: 0,982 (0,961-1,003), ordenada al origen: -0,3 (-1,1-0,5), sesgo: -2,0%. Para CRMB se obtuvo un r de 0,96, pendiente: 0,921 (0,855-0,988), ordenada al origen: -0,07 (-0,35-0,20), sesgo: -10,2%. Sólo 4 pacientes presentaron niveles discrepantes entre ambos métodos. La determinación de FVIII:C por el método cromogénico automatizado en la familia ACL TOP fue comparable con FVIII coagulable en una etapa en el rango analítico evaluado. El FVIII:Ccro automatizado puede utilizarse para el diagnóstico y seguimiento del tratamiento de los pacientes hemofílicos.

The objective of this work was to perform the analytical validation of the chromogenic method (FVIII:Ccro) on the ACL TOP platform correlating with one stage assay (FVIII:Ccoag). The validation study (EP5-A2, EP6-A2 and comparison of methods by EP-9) was performed for the low-normal range curve (CRNB): approximately between 10-150 IU/dL of FVIII and very low range (CRMB): approximately between 0-10 IU/dL. The results of CVr (repeatability) and CVi (intermediate precision) were lower than 6% and comparable to those reported by the manufacturer for other platforms. The analytical measurement range was 11-129 IU/dL, extrapolated to 0.3 IU/dL using the CRMB. For CRNB FVIII:Ccro showed good correlation with FVIII:Ccoag: r: 0.98, slope: 0.982 (0.961-1.003), intercept: -0.3 (-1.1-0.5), bias: -2.0%. For CRMB: r: 0.96 was obtained, pending: 0.921 (0.855-0.988), intercept: -0.07 (-0.35-0.20), bias: -10.2%. Only 4 patients presented discrepant levels between both methods. The automated chromogenic FVIII assay in the ACL TOP family is comparable with one stage coagulable FVIII in the analytical range studied. The FVIII:Ccro automated can be used for the diagnosis and monitoring of the treatment of hemophilic patients.

O objetivo deste trabalho foi a validação analítica do método cromogênico (FVIII:Ccro) na plataforma ACL TOP correlacionando-se com o método coagulável numa etapa (FVIII:Ccoag). O estudo de validação (EP5-A2, EP6-A2 e comparação de métodos por EP-9) foi realizado para a curva de faixa normal-baixa (CRNB) aproximadamente entre 10 e 150 Ul/dL de FVIII e de faixa muito baixa (CRMB): aproximadamente entre 0 e 10 UI/dL. Os resultados de Repetitividade (CVr) e precisão intermediária (CVi) foram inferiores a 6% e comparáveis aos descritos pelo fabricante para outras plataformas. A faixa de medição analítica foi de 11-129 UI/dL com CRNB extrapolando-se para 0,3 UI/dL utilizando a CRMB. Para a CRNB FVIII:Ccro houve boa correlação com o FVIII: Ccoag: r: 0,98, inclinação: 0,982 (0,961-1,003), ordenada na origem: -0,3 (-1,1-0,5), Viés: -2,0%. Para CRMB: foi obtido um r: 0,96, pendente: 0,921 (0,855-0,988), ordenado na origem: -0,07 (-0,35-0,20), Viés: -10,2%. Apenas quatro pacientes apresentaram níveis discrepantes entre os dois métodos. A determinação de FVIII:C pelo método cromogênico automatizado na família ACL TOP foi comparável ao FVIII coagulável em um estágio na faixa analítica avaliada. FVIII: O FVIII:Ccro automatizado pode ser utilizado para o diagnóstico e seguimento do tratamento dos pacientes hemofílicos.

Incidencia de Escherichia coli O157: H7 en heces de rumiantes lactantes con síndrome diarreico / Incidence of Escherichia coli 0157: H7 in feces of lactating ruminants with diarrheal syndrome

RESUMEN Objetivo. Identificar Escherichia coli O157:H7 presente en heces diarreicas de rumiantes lactantes con síndrome diarreico y seguridad de ingesta de calostro. Materiales y métodos. Se realizó un muestreo de 316 rumiantes durante el período de agosto 2015 a marzo 2016 en los municipios de Río Grande, General Enrique Estrada, Morelos y Calera de Víctor Rosales del estado de Zacatecas, 67 de bovinos, 183 de ovinos y 66 de caprinos. Resultados. Se identificaron en medio cromogénico CHROMagarTM: 260 coliformes, 78 Escherichia coli O157:H7, 16 Proteus spp., y 25 colonias de bacterias sin identificar con este medio, encontrándose una incidencia de Escherichia coli O157:H7 de 22.03% en los cuatro municipios. Conclusiones. Escherichia coli O157:H7 es la segunda bacteria encontrada en heces de rumiantes con un 22% de incidencia, la cual es un factor de riesgo de muerte en rumiantes lactantes (menos de 21 días de nacidos) causando pérdidas económicas y riesgo para la salud de la población del estado de Zacatecas.

ABSTRACT Objective. To identify Escherichia coli 0157:H7 present in diarrheal feces of lactating ruminants with diarrheal syndrome and safety of colostrum intake. Materials and methods. A feces sampling of 316 ruminants was carried out during the period of August 2015 to March 2016 in the municipalities of Río Grande, General Enrique Estrada, Morelos and Calera de Victor Rosales of the state of Zacatecas, obtained from 67 cattle, 183 sheep and 66 goats. Results. The following were identified in CHROMagarTM chromogenic medium: 260 coliforms, 78 Escherichia coli 0157:H7, 16 Proteus spp. and 25 colonies of unidentified bacteria, finding an incidence of Escherichia coli 0157:H7 of 22.03% in the four municipalities. Conclusions. Escherichia coli 0157:H7 is the second bacteria found in ruminant feces with an incidence of 22%, which is a mortality risk factor in lactating ruminants (less than 21 days old), causing economic loss and health risk for the population of the state of Zacatecas.

Recurrent black stains and periodontal disease

Objectives: The objective of this study was to identify the anaerobic pigment-forming bacteria present in black stain and correlate its occurrence with dental caries incidence and periodontal destruction. Materials and Methods: A total of 50 healthy subjects with the chief complaint of recurrent black stains were selected based on the inclusion and exclusion criteria. decayed/missing/filled surfaces score, community periodontal index, Gingival Crevicular Fluid (GCF), black stain score, and microbial analysis were done. Results: The data presented indicate that black stain has a constant microflora, dominated by various gram-negative rods, gram-positive cocci and rods (P ≤ 0.1). However, the incidence of gram-positive rods decreased with the increase in plaque score and probing depths and decrease in black stain score. Conclusions: Presence of black stains is predominated by various gram-positive and negative rods, and gram-positive cocci. Increase in the plaque score decreases the severity of black stains, thereby increasing the probability of periodontal destruction and dental caries incidence in adult subjects. Further studies are required to corroborate the results.

Shorter Incubation Times for Detecting Multi-drug Resistant Bacteria in Patient Samples: Defining Early Imaging Time Points Using Growth Kinetics and Total Laboratory Automation

BACKGROUND: The transition from manual processing of patient samples to automated workflows in medical microbiology is challenging. Although automation enables microbiologists to evaluate all samples following the same incubation period, the essential incubation times have yet to be determined. We defined essential incubation times for detecting methicillin-resistant Staphylococcus aureus (MRSA), multi-drug resistant gram-negative bacteria (MDRGN), and vancomycin-resistant enterococci (VRE). METHODS: We monitored the growth kinetics of MRSA, MDRGN, and VRE between two and 48 hours on chromogenic media to establish the time points of first growth, single colony appearance, and typical morphology for 102, 104, 106, and 108 colony forming units/mL. Subsequently, we imaged plates inoculated with 778 patient samples after 20, 24, and 36 hours. RESULTS: The first growth, single colony appearance, and typical morphology time points were inoculum-dependent. First growth appeared after 6–18 hours, 4–18 hours, and 8–48 hours for MRSA, MDRGN, and VRE, respectively, and single colonies appeared at 12–18 hours, 6–20 hours, and 12–48 hours, respectively. Typical morphology was visible at 14–22 hours and 12–48 hours for MRSA and VRE, but was not determined for MDRGN. By examining patient samples, ≥98% of MRSA and MDRGN were visible 20 hours after the start of incubation. Following 24 hours of incubation, only 79.5% of VRE were clearly visible on the respective plates. CONCLUSIONS: An incubation time of 20 hours is sufficient for detecting MRSA and MDRGN. VRE growth is much slower and requires additional imaging after 36 hours.

Evaluation of a new chromogenic media for the isolation and culture of Clostridium difficile / 临床检验杂志

Objective@#To evaluate the performance of a chromogenic agar developed by our laboratory for the isolation and culture of Clostridium difficile (CDCA). @*Methods@#The chromogenic specificity of CDCA was evaluated by inoculation of C. difficile and other standard strains, and the sensitivities of CDSA (BD), CDIF (BioMérieux) and CDCA were determined by the C. difficile standard strains respectively. A total of 120 clinical stool specimens were cultured for C. difficile by three chromogenic media respectively. The colonies were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tpi gene was also detected. The sample which could be identified as C. difficile in any of the three chromogenic medium was defined as true positive. @*Results@#Most of standard strains were inhibited by CDCA, however some Clostridium species including C. clostridiiforme, C. bifermentans, C. tertium and Bacteroides fragilis grew lightly with chromogenic reaction. The sensitivities of CDSA, CDIF and CDCA were 2.0×105 CFU/mL, 8.0×101 CFU/mL and 4.0×10 2 CFU/mL, respectively. Among the 120 samples, 31 (25.8%) were defined as true C. difficile positive samples, while the positive rate of CDSA, CDIF and CDCA were 25 (20.8%), 28 (23.3%) and 26 (21.7%), respectively. There was no significant difference for clinical diarrhea specimens among the three chromogenic media (χ 2 =0.418, P=0.811). In comparison to the standard, the sensitivity, specificity, positive predictive value and negative predictive value were 83.8%, 100%, 100% and 94.7% for CDCA; 90.3%, 98.9%, 96.6% and 96.7% for CDIF; and 80.6%, 100%, 100% and 93.7% for CDSA. @*Conclusion@#The CDCA developed by our laboratory could be used to preliminarily isolate C. difficile with good specificity and sensitivity.

Efecto de una combinación de nutrientes e inhibidores del crecimiento bacteriano sobre el desarrollo de Salmonella / Effect of a combination of nutrients and inhibitors of bacterial growth on the development of Salmonella

La selección de bases nutritivas para los medios de cultivo está relacionada con el microorganismo objeto de estudio y el propósito del medio. Los inhibidores del crecimiento bacteriano en los medios selectivos y diferenciales pueden interferir en el desarrollo del microorganismo de interés. Por ello se requiere un balance entre sustancias promotoras e inhibidoras del crecimiento bacteriano, sobre todo en bacterias que pueden encontrarse a muy bajas concentraciones o sometidas a diferentes condiciones de estrés durante el almacenamiento de las muestras que las contiene, como Salmonella. El objetivo consistió en evaluar el efecto de una combinación de nutrientes de diferentes orígenes y de inhibidores del crecimiento de bacterias grampositivas sobre el desarrollo de Salmonella. Se seleccionaron 52 cepas: incluyendo Salmonella, otras bacterias y levaduras. Se determinó la capacidad nutricional de mezclas de bases nutritivas registrando el incremento de la biomasa mediante técnica espectrofotométrica. Se comprobó la capacidad de recuperación e inhibición de dos variantes experimentales con diferentes inhibidores mediante la determinación de parámetros cuantitativos, y se comparó la productividad de la formulación final con un medio cromogénico. Salmonella mostró un crecimiento abundante en las variantes con diferentes combinaciones nutricionales, se logró la inhibición de un grupo de microorganismos y la productividad de la composición final fue superior a 0.80. La mezcla de bases nutritivas de diferentes orígenes y las sales biliares como inhibidor de crecimiento de bacterias grampositivas resulta una combinación eficaz para promover el crecimiento de Salmonella cuando estas bacterias se encuentran en baja concentración.

The selection of nutrient bases depends on the microorganism and the purpose of the medium. Inhibitors of bacterial growth are of great importance in selective and differential media and may interfere in the growth of the microorganism of interest. An adequate balance between promoter substances and inhibitors of bacterial growth is thus required, especially for bacteria that could be found either at very low concentrations or those subject to different stress conditions during storage of the samples containing them, such as Salmonella. The aim was to evaluate the effect of a combination of nutrients of different origins and inhibitors of grampositive bacteria on the development of Salmonella serotypes. 52 Salmonella strains and a representation of other bacteria and yeasts were selected. The nutritional capacity of the composition was determined by spectrophotometric technique formulating variants with mixtures of nutritive bases, and recording the increase in biomass. Recovery capacity and inhibition of two experimental variants with different inhibitors were quantitatively tested. The productivity of the final formulation was compared with a chromogenic medium (Oxoid, England). Salmonella showed abundant growth in the variants made with different nutrient combinations. Both experimental formulations showed their ability to recover microorganisms of interest. The final composition showed productivity values higher than 0.80 in both variants. The mixture of nutrient bases and bile salts as an inhibitor of growth of grampositive bacteria was an effective combination, capable of stimulating the growth of Salmonella genus when these bacteria are found at low concentration.

Development of Semi-quantitative Solid Sampling Mercury Analyzer for Cosmetics / 分析化学

On the basis of the chromogenic reaction between Hg and CuI, a semi-quantitative solid sampling Hg analyzer comprising the catalytic furance, Hg testing tube, air pump and smart cellphone was developed. White carrier 101 was chosen as the adsorbent for CuI to react with Hg from the catalytic furnace. The established Hg analyzer can not only visually recognize the coloration when Hg exceeding the limit standard, but also semi-quantitatively detect the Hg content in cosmetics fast using a smart cellphone and RGB analysis software, after direct solid sampling introduction of cosmetics sample. The instrumental detection limit ( LOD) of mercury was 50 ng, the linearity ranged from 50 ng to 2500 ng, the linear regression coefficient ( R2) was higher than 0. 97, and the RSD of the corresponding RGB values was 6% ( n=11 ) . Nine real cosmetics samples were measured by the established method, whose relative differences of Hg contents with that by the standard method (Safety Technical Specification for Cosmetics, 2015 edition) were less than 10% . The whole analytical time can be controlled within 5 min. The established instrumental method is simple, fast, accurate and visual, and extremely suitable to fast and on-site monitoring of Hg in cosmetics samples.

Determinación de hierro sérico: Evolución del desempeño de los laboratorios participantes del Sub-Programa PEEC-Hematología / Iron serum determination: evolution of performance of participating laboratories of the PEEC-Hematology Sub-Program / Determinação de ferro sérico: Evolução do desempenho dos laboratórios participantes do Sub-Programa PEEC-hematologia

El objetivo de este trabajo fue determinar la evolución del desempeño analítico en la determinación de hierro sérico, de los laboratorios participantes del Sub- Programa PEEC-Hematología (PEEC-H) del Programa de Evaluación Externa de Calidad Prof. Dr. Daniel Mazziotta de la Fundación Bioquímica Argentina, mediante el análisis de los resultados de ferremia en 6 encuestas (E) realizadas en los meses de julio entre los años 2010 y 2015 (E 77, 81, 85, 89, 93 y 97). Hasta el 2011 se utilizaban métodos con y sin desproteinización, siendo estos últimos los más utilizados (94%). En 2015 en la red de laboratorios se emplearon solamente métodos directos sin desproteinización, siendo los colorimétricos los más utilizados (aproximadamente 95%). El Desvío Relativo Porcentual aceptable (DRPa) fue de ±10% en todas las encuestas analizadas. El 56% de los laboratorios tuvieron un desempeño promedio aceptable en las E 77, 81 y 85, evolucionando 3 años después, a 70% en las E 89, 93 y 97. Según estas consideraciones, al presente no es necesario ajustar el DRPa para el analito hierro, ya que con este valor los laboratorios aún deben trabajar para lograr una mejoría en su desempeño.

The aim of this work was to evaluate the evolution of the analytical performance of serum iron determination by the laboratories participating in the Sub- Program PEEC-Hematology (PEEC-H) EQAS Program Prof. Dr. Daniel Mazziotta of the Argentine Biochemical Foundation. To this end, results of serum iron determinations from July 2010 to July 2015 (surveys #77, 81, 85, 89, 93 and 97) were used. Up to 2011, there were methods both with and without deproteinization, the latter being the most used (94%). In 2015, only one commercial method without deproteinization was used, with colorimetric methods employed in 95% of the cases. In all the surveys analyzed, the acceptable DRP was ±10%. In surveys 77, 81 and 85, 56% of the laboratories had an acceptable performance percentage, and it evolved to a 70% in the surveys 89, 93 and 97, three years later. According to these considerations, there is no need to adjust the acceptable DRP for the iron analyte. In this way, laboratories will continue to work in order to improve their performance.

O objetivo deste estudo foi determinar a evolução do desempenho analítico na determinação de ferro sérico, dos laboratórios participantes no Sub-Programa PEEC-Hematologia (PEEC-H) do Programa de Avaliação Externa de Qualidade Prof. Dr. Daniel Mazziotta da Fundación Bioquímica Argentina, através da análise dos resultados de ferremia em 6 pesquisas de opinião (E) realizadas nos meses de julho entre os anos 2010 a 2015 (Pesquisa No. 77, 81, 85, 89, 93 e 97). Até 2011 eram empregados métodos com e sem desproteinização, sendo os colorimétricos os mais utilizados (aproximadamente 95%). O Desvio Relativo Percentual aceitável (DRPa) foi de ±10% em todas as pesquisas analisadas. 56% dos laboratórios tiveram desempenho médio aceitável nas pesquisas 77, 81 e 85, progredindo para 70% nas pesquisas de 89,93 e 97, 3 anos mais tarde. De acordo com estas considerações, hoje não é necessário ajustar o DRPa para o analito ferro, visto que com esse valor os laboratórios ainda devem trabalhar para alcançar uma melhoria no seu desempenho.

Application of multiplex PCR combined with invasive reaction and chromogenic reaction catalyzed by gold nanoparticles in detection of encephalitis and meningitis virus / 中国人兽共患病学报

We developed a method for detecting encephalitis and meningitis virus by using multiplex PCR combined with invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles.Primers were designed based on the conservative regions of encephalitis and meningitis virus (Eastern equine encephalitis virus,EEEV;Western equine encephalomyelitis virus,WEEV;West Nile virus,WNV;Nipah virus,NiPA;Japanese encephalitis virus,JEV).Multiplex PCR system,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were established to detect different encephalitis and meningitis virus in one reaction.Tick-borne encephalitis virus (TBEV),St Louis encephalitis virus (StLEV),Chikungunya virus (CHIKV) and Dengue virus(DV) were used to test its specificity.Quantitative RNA transcribed in vitro and PCR fragments were used to assess its sensitivity.Clinical specimens collected from JEV patients were detected by this method.A method for detecting encephalitis and meningitis virus by using multiplex PCR,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were successfully established.This method can detect targeted pathogens specifically,and it has no cross reaction with TBEV,StLEV,CHIKV and DV.The detecting limitation for different targets was 103 copies/μL.Clinical samples were positive for JEV nucleic acids for above assay.The method presented here has characteristic of high specificity,sensitivity and throughput.The results can be observed by visual inspection.This method has broad application prospects in pathogen detection.

Determination of Bacterial Endotoxin in Fat Emulsion(10%)/Amino Acid (15)/Glucose (20%) Injection by Gel Method / 中国药房

OBJECTIVE:To adopt gel method for the determination of bacterial endotoxin in Fat emulsion(10%)/amino acid (15)/glucose (20%) injection. METHODS:According to the gel method in term ofbacterial endotoxin test methodin Chinese Pharmacopeia(2015 edition),the maximal valid dilution(MVD)of samples were determined through interference test and the vali-dated. The results were compared with chromogenic method. RESULTS:In gel method,the interference to agglutination reaction of TAL and bacterial endotoxin can be excluded when samples were diluted 24 times or less. In chromogenic method,the samples should be diluted 76 times or less. CONCLUSIONS:Gel method can be used for bacterial endotoxin test of Fat emulsion(10%)/amino acid(15)/glucose(20%)injection.

Recuperación y diferenciación de aeromonas con CromoCen AE y CromoCen AGN / Recovery and differentiation of Aeromonas with CromoCen AE and CromoCen AGN

INTRODUCCIÓN: en la actualidad las especies del género Aeromonas han emergido como un problema de salud pública, son ellas los agentes etiológicos de las enfermedades diarreicas con el aumento de la atención médica por años. Los procedimientos convencionales para su diagnóstico son muy engorrosos, laboriosos y duraderos. Una nueva metodología que emplea medios de cultivo cromogénicos ha permitido la simplificación y aceleración de su diagnóstico, que ofrece resultados altamente específicos. OBJETIVO: estudiar el efecto de la combinación de diferentes agentes selectivos de los microorganismos grampositivos sobre el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. MÉTODOS: se estudió el efecto inhibidor de la combinación de agentes selectivos (desoxicolato de sodio (0,05-0,2 g·L-1), sales biliares (0,65 g·L-1), verde brillante (0,025-0,03 g·L-1), cristal violeta (0,001-0,01 g·L-1) y sulfito de sodio (0,8 g·L-1) sobre los microorganismos grampositivos, así como la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. Como base se utilizó la formulación de CromoCen AGN, sin el desoxicolato de sodio. RESULTADOS: los valores de las productividades de los medios CromoCen AE y CromoCen AGN a partir del inóculo 1,5 × 102 UFC·mL-1 resultaron, para: A. hydrophila 116,8 % y 23,9 %, A. caviae 100,8 % y 3,95 %, A. bestiarium 93,6 % y 28,8 %, A. culicicola 85,1 % y 66,12 %, A. veronii 116,7 % y 59,2 %, A. popoffi 86,56 % y 13,2 %, A. trota 94,8 % y 11,25 % y para A. eucrinophila 103,9 % y 2,80 %. La nueva composición cromogénica logró la diferenciación de los microorganismos por sus características culturales: color, forma, superficie, bordes en las colonias y proteólisis del medio circundante. CONCLUSIONES: la combinación de diferentes agentes selectivos para la inhibición de los microorganismos grampositivos coadyuvo el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas.

INTRODUCTION: Species of the genus Aeromonas are a current public health problem, for they are the etiological agents responsible for the growing incidence of diarrheal diseases requiring medical care. Conventional procedures for their diagnosis are very complicated, laborious and time-consuming. A new methodology based on the use of chromogenic culture media allows diagnostic simplification and acceleration, yielding highly specific results. OBJECTIVE: Study the effect of combining several selective agents for gram-positive microorganisms upon an increased capacity for recovery, quantification and differentiation of Aeromonas species. METHODS: Assessment was conducted of the inhibiting effect of combined selective agents (sodium deoxycholate (0.05-0.2 g·L-1), bile salts (0.65 g·L-1), brilliant green (0.025-0.03 g·L-1), crystal violet (0.001-0.01 g·L-1) and sodium sulfite (0.8 g·L-1)) on gram-positive microorganisms, as well as their capacity for recovery, quantification and differentiation of Aeromonas species. The base used was the CromoGen AGN formulation without sodium deoxycholate. RESULTS: Productivity values for the media CromoCen AE and CromoCen AGN based on inoculation of 1.5 × 102 CFU·mL-1 were 116.8 % and 23.9 % for A. hydrophila, 100.8 % and 3.95 % for A. caviae, 93.6 % and 28.8 % for A. bestiarium, 85.1 % and 66.12 % for A. culicicola, 116.7 % and 59.2 % for A. veronii, 86.56 % and 13.2 % for A. popoffi, 94.8 % and 11.25 % for A. trota, and 103.9 % and 2.8 0% for A. eucrinophila. The new chromogenic composition enabled differentiation of microorganisms based on their cultural characteristics: color, shape, surface, colony borders and environmental proteolysis. CONCLUSIONS: Combination of various selective agents for the inhibition of grampositive microorganisms led to an increased capacity for recovery, quantification and differentiation of Aeromonas species.

Establishing the heparin therapeutic range using aPTT and anti-Xa measurements for monitoring unfractionated heparin therapy

BACKGROUND: Unfractionated heparin (UFH) has unstable pharmacokinetics and requires close monitoring. The activated partial thromboplastin time (aPTT) test has been used to monitor UFH therapy for decades in Korea, but its results can be affected by numerous variables. We established an aPTT heparin therapeutic range (HTR) corresponding to therapeutic anti-Xa levels for continuous intravenous UFH administration, and used appropriate monitoring to determine if an adequate dose of UFH was applied. METHODS: A total of 134 ex vivo samples were obtained from 71 patients with a variety of thromboembolisms. All patients received intravenous UFH therapy and were enrolled from June to September 2015 at Gyeongsang National University Hospital. All laboratory protocols were in accordance with the Clinical and Laboratory Standards Institute guidelines and the College of American Pathologist requirements for aPTT HTR. RESULTS: An aPTT range of 87.1 sec to 128.7 sec corresponded to anti-Xa levels of 0.3 IU/mL to 0.7 IU/mL for HTR under our laboratory conditions. Based on their anti-Xa levels, blood specimen distribution were as follows: less than 0.3 IU/mL, 65.7%; 0.3–0.7 IU/mL (therapeutic range), 33.6%; and more than 0.7 IU/mL, 0.7%. No evidence of recurring thromboembolism was observed. CONCLUSION: Using the conventional aPTT target range may lead to inappropriate dosing of UFH. Transitioning from the aPTT test to the anti-Xa assay is required to avoid the laborious validation of the aPTT HTR test, even though the anti-Xa assay is more expensive.

Determination of Anti-FIIαActivity in Compound Heparin Sodium Cream by a Trace Chromogenic Sub-strate Method / 中国药师

Objective:To establish a trace chromogenic substrate method for the determination of anti-FIIαactivity in compound heparin sodium cream .Methods: The anti-FIIαactivity in compound heparin sodium cream was determined by a trace chromogenic substrate method according to the completely random design of experiment based on the amount reaction principle of 4*4 parallel lines in the biological test statistics method .Results:The calibration curve was linear within the range of 0.005 04 IU· ml-1-0.021 IU· ml-1(r=0.992).The average recovery was 101.6% with RSD of 2.76% (n=9).Conclusion: The method is accurate, reliable and reproducible , and can be used for evaluating the quality of compound heparin sodium cream .

Characterization of Bacteria Community Isolated from Wood Decay.

In order to discover enzymes having potential for wood fibre modification, bacteria (fourteen strains designated MMB1 to MMB14) were isolated from a decomposing stump from a resinous tree. Phylogenetic analysis and biochemical characterization indicated that these isolates were related to Microbacterium, Chryseobacterium, Lysinibacillus, and Bacillus gene; although most demonstrated phenotypic differences compared to previously characterized relatives. Only the Bacillus strains showed cellulolytic activity (as CMCase detected with Congo red) and only Bacillus subtilis strains (MMB10 to MMB14) displayed cellulolytic and secreted xylanase activity. Phenotypic characterization of two strains (MMB8 and MMB9) related to a previously characterized isolate (Bacillus sp. JU2), supported their reassignment to the genus Lysinibacillus. The Microbacterium strain MMB1 produced a green pigment when grown in the presence of light. Some microbes from the consortium were devoid of wood polymer modifying enzymes, and may be dependent on other organisms for their survival in this biotope.