Single Cell Traction Force Measured by Foldable Microplates / 医用生物力学

Objective To fabricate a foldable microplate for single cell culture and establish finite element model of the folding microplate, so as to calculate traction force of single cells during contraction in three-dimensional (3D) state.Methods The folding angle of the microplate casued by cell traction force was calculated. Then the relation between bending moment and folding angle as well as the relation between traction force and bending moment were derived by using finite element simulation, so as to realize the characterization of traction force for singel cell in 3D state.Results The folding angles of the microplate with HSF and MC3T3-E1 cells in 3D state were 73°-173° and 49°-138°, respectively. The single cell traction forces of HSF and MC3T3-E1 cells were 55-210 nN and 52-161 nN, respectively.Conclusions The proposed method for measuring traction force of single cells in 3D state by fabricating the foldable microplate for single cell culture will provide some references for further development of calculating traction forces in 3D cell adhesion, spreading and migration.

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes / Formação de biofilme por Prototheca zopfii isolados de mastite bovina clínica e subclínica em distintas condições de crescimento sob diferentes corantes

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

RESUMO: Prototheca spp. tem sido relatado como um patógeno ambiental causador de mastite bovina em vários países. A formação de biofilme é um fator associado a diferentes graus de virulência desenvolvidos por muitos microrganismos, incluindo Prototheca zopfii. O presente estudo teve como objetivo comparar duas condições de crescimento e dois corantes para determinar a combinação mais adequada para avaliar qualitativa e quantitativamente a produção de biofilme por P. zopfii. A formação de biofilme foi avaliada em microplacas de poliestireno sob condições estáticas e dinâmicas de crescimento e coloração com cristal violeta ou azul de algodão. Todos os isolados de P. zopfii de vacas com mastite foram caracterizados como produtores de biofilme, independentemente das condições de crescimento e coloração. O corante azul de algodão demonstrou ser o método mais adequado para classificar a intensidade de produção de biofilme de P. zopfii.

Headless double-threaded compression screws plus microplate for capitellar fracture of Ring type Ⅱ / 中华创伤骨科杂志

Objective To investigate the clinical effects of headless double-threaded compression screws plus microplate for treatment of capitellar fracture of Ring type Ⅱ.Methods In the period from March 2015 to February 2018,12 capitellar fractures of Ring type Ⅱ were treated at Department of Upper limb Orthopedics,Zhengzhou Orthopaedics Hospital.There were 8 men and 4 women,aged from 25 to 57 years (average,35.4 years).There were 5 left and 7 right fractures.The fractures were exposed and reduced through the posterolateral elbow approach in 6 patients and through the lateral elbow approach in the other 6 ones.Fixation of all the fractures was conducted with headless double-threaded compression screws plus microplate.At the final follow-up,the elbow function was evaluated by the Mayo elbow performance score and the severity of traumatic arthritis by the Broberg & Morrey imaging criteria.Results All the patients were followed up for 9 to 36 months (average,23 months).The fracture healing time ranged from 9 to 12 weeks (average,10.6weeks).The final follow-ups revealed fine stability of the affected elbow joint in all the patients,and no loss of reduction,loosening or breakage of internal fixators or ischemic necrosis in any case.At the final follow-ups,the elbow inflection angles ranged from 90° to 130° (average,114.3°),the elbow extension angles from 0° to 20°(average,12.4°),the forearm pronation angles from 50° to 85° (average,70.5°) and the forearm supination angles from 45° to 80° (average,64.3°).The Mayo elbow performance scores at the final follow-up averaged 93.2points (from 85 to 100 points),yielding 8 excellent and 4 good cases.According to the Broberg & Morrey imaging criteria,10 patients showed no regressive change and 2 patients change by one grade.Conclusions A proper surgical approach should be chosen according to the preoperative imaging findings.The fracture of articular surface can be firmly fixated by head-less double-threaded compression screws and the integrity of the distal lateral column of the humerus can be restored with a microplate to achieve a strong biomechanical fixation.Fine clinical outcomes can be obtained by early functional exercise.

Effectiveness analysis of distal radius microplate locking plate for treatment of displaced fracture of medial clavicle / 中国修复重建外科杂志

Objective: To investigate and evaluate the effectiveness of the distal radius microplate locking plate for the treatment of displaced fracture of medial clavicle. Methods: Between January 2013 and June 2017, 18 cases of obvious displaced fracture of medial clavicle were treated with distal radius microlocking plate. There were 10 males and 8 females, with an average age of 51.4 years (range, 18-88 years). Causes of injury included traffic accident injury in 15 cases, heavy object injury in 3 cases; all of them were closed injury. According to Edinburgh classification, 15 cases were ⅠB1 type and 3 cases were ⅠB2 type. Fracture displacement was 12-21 mm (mean, 16.3 mm). The time from injury to operation was 3-7 days (mean, 4.3 days). After operation, the clinical healing and complications of fracture were observed, and shoulder function was evaluated according to Rockwood's scoring criteria. Results: No incisional infection, neurovascular injury, or other early complications occurred. All 18 patients were followed up 8-15 months (mean, 12 months). All fractures reached clinical osseous union, and the healing time was 8-24 weeks (mean, 16.6 weeks). Postoperative plate loosening occurred in 1 case, which was removed surgically, while other patients did not suffer from complications such as bone nonunion, displacement, internal fixator loosening, and loss of reduction. At last follow-up, according to Rockwood's scoring criteria, the results were excellent in 12 cases and good in 6 cases. Conclusion: The distal radius microplate locking plate is effective for the treatment of displaced medial clavicle fracture, which has few complications, and is feasible for early functional exercise, and is helpful for the recovery of shoulder joint function.

Effect of different types of microplate and loading volumes on the detection results of multi-function microplate reader / 中国职业医学

OBJECTIVE: To evaluate the effect of different types of microplate and loading volumes on the detection results of multi-function microplate reader, and to optimize the analysis method. METHODS: A multi-function microplate reader was used to perform spectrum scanning on each of 5 detection holes of common and ultraviolet(UV) microplates, and the applicable detection wavelength range was those with light transmittance greater than 80.00%. The optical density measurement was carried out on each 12 detection holes of common and UV microplates at different wavelengths, then the matching of the detection holes was compared. Potassium permanganate was quantitatively analyzed by common microplate and UV microplate, while acetone was analyzed by UV microplate, and then detection limit, lower limit of quantitation(LLQ), accuracy and precision at different loading volumes and concentrations were calculated and compared. RESULTS: The shortest applicable analyzing wavelengths for common and UV microplates were(362±2) and(230±3) nm respectively, while the longest applicable analyzing wavelengths were both 1 000 nm. The light transmittance of UV microplate was higher than that of common microplate when the analyzing wavelengths were lower than 400 nm(P<0.01). The deviation and range of light transmittance of detection holes analyzed by UV microplates were smaller than that of common microplates when the analyzing wavelengths were 350-1 000 nm(P<0.05). The detection limit and LLQ of potassium permanganate by multi-function microplate reader was not associated with the types of microplate. The adding standard recoveries of potassium permanganate by UV microplate was higher than that by common microplate(P<0.05). The adding standard recoveries of potassium permanganate by loading volumes of 200 and 250 μL was lower than that by loading volumes of 150 μL(P<0.01), while adding standard recovery of acetone by loading volumes of 200 μL was lower than that by loading volumes of 150 μL(P<0.05). CONCLUSION: When using a multi-function microplate reader to detect chemicals, it is recommended to use UV microplate with wavelengths at the range of 230-1 000 nm, and loading volumes of 200-250 μL.

Microplate silica allochroic gel assay for detecting rifampicin resistance of Mycobacterium tuberculosis / 重庆医学

Objective To discuss the clinical application value of microplate allochroic silica gel assay for rapidly detecting rifampicin(RIF)-resistance of Mycobacterium tuberculosis(MTB).Methods Fifty MTB clinical isolates preserved in the tuberculosis(TB) laboratory of Nantong Municipal Sixth People's Hospital were detected RIF-resistance by using the microplate allochroic silica gel assay and compared with the Bactec MGIT960 method.Then,the RIF susceptibility test in 40 clinical sputum smear-positive specimens were simultaneously detected by using the microplate allochroic silica gel assay and Bactec MGIT960.Finally the sensitivity,specificity and accuracy of the test results were compared.Results The optimal inoculation volume of MTB was 10-3 mg/mL,the optimal detection time was 7-10 d and the judging critical value of the RIF minimum inhibitory concentration(MIC) Was 1.00 μg/mL by microplate allochroic silica gel assay.With the Bactec MGIT960 test as the gold standard,the sensitivity,specificity and accuracies of microplate allochroic silica gel assay for RIF-resistance susceptibility test of smear-positive specimens were 94.12％,100％ and 97.37％ respectively.Conclusion Microplate Allochroic silica gel assay can be used for directly detecting the MTB sensitivity to RIF of in sputum specimens.

Variety methods study on hemolytic effect of colored injection doxorubicin hydrochloride in vitro / 药物评价研究

Objective The visual inspection method were not appropriate to perform a hemolysis evaluation for colored injection like doxorubicin hydrochloride,this article adopted three methods to evaluate the hemolysis test of doxorubicin hydrochloride in vitro and provide reference for clinical drug safety.Methods Using rabbit erythrocytes as experimental object,the durg concentration 4.0 and 2.0 mg/mL was chosen which range of clinical concentration and preclinical safety evaluation concentration,to evaluate the hemolysis test of doxorubicin hydrochloride injection with blood analyzer test,direct colorimetric assay,and indirect colorimetric assay.Results The evaluation results of three different methods were very consistent.The tube's hemolysis rate of 4.0 mg/mL dose was far greater than 5％,which means serious hemolysis;Only 0.1 mL tube of 2.0 mg/mL dose (according to the drug concentration equal to 0.5 mL tube of 0.4 mg/mL drug concentration) without hemolysis occurring,the other tubes' hemolysis rates were far greater than 5％,which means serious hemolysis.Conclusion The hemolysis phenomenon may occur when 2.0 mg/mL dose of doxorubicin hydrochloride solution for iv injection is used in clinic and dilution (final concentration not more than 0.4 mg/mL) is recommended.

Investigation of irregular antibodies in blood samples from donors in Shanghai area / 中国输血杂志

Objective With the purpose of further improving blood safety and providing the reference basis for a strategy suitable for large-scale routine screening of irregular antibodies in blood service,the irregular antibodies in blood samples from donors in Shanghai area were investigated.Method Irregular blood antibodies were screened in 824 072 blood donors through combined method of saline medium and microplate papain.Result 1 246 samples of blood donors were detected with irregular antibodies among the total 824 072 samples which indicate the positive rate of 1.51‰.Conclusion Screening of irregular antibodies in blood samples from donors can effectively reduce or avoid hemolytic transfusion reactions.The combined method of saline medium and microplate papain is an effective method for large-scale routine screening of irregular antibodies in blood service.

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

Screening antimycobacterial activity of Baccharis dracunculifolia, Centella asiatica, Lantana camara and Pterodon emarginatus / Atividade anti-micobacteriana de Baccharis dracunculifolia, Centella asiatica, Lantana camara e Pterodon emarginatus

ABSTRACT The permanent investigation of new antimycobacterial drugs is necessary for the eradication programs of tuberculosis and other mycobacterium-related diseases. The aim of the present study is to search for new sources of antimycobacterial drugs using plant materials. In this study, 11 plant materials (extracts, essential oils and some fractions) obtained from 4 species of medicinal plants traditionally used as general therapeutics for different illnesses and specifically as treatment of tuberculosis, were evaluated using the microplate resazurin assay against 2 species of the Mycobacterium tuberculosis Complex and 3 nontuberculous mycobacteria. The results showed the hexane extract and the essential oil from fruits of Pterodonemarginatus (Vogel) as potential sources of antimycobacterial drugs against 4 species of tested mycobacteria. The hexane fraction of methanol extract from leaves of Centella asiatica also presented significant mycobacterial growth inhibition, but against M. chelonae only. In conclusion, it was possible to contribute to the antimycobacterial investigations by presenting three new samples of plants with significant antimicrobial activity against four Mycobacteriumspp and suggest future studies about the antimycobacterial properties of fruits from P. emarginatus.

RESUMO A investigação permanente de novas drogas antimicobacterianas é necessária no programa de erradicação da tuberculose e de outras doenças relacionadas com micobactérias. O objetivo deste estudo foi buscar novas fontes de drogas antimicobacterianas usando material vegetal. Neste estudo, 11 materiais de base vegetal (extratos, óleos essenciais e algumas frações) foram avaliados contra 5 espécies de micobactérias. Estes materiais foram obtidos a partir de 4 espécies de plantas medicinais tradicionalmente utilizadas como terapêutica geral para diferentes doenças e, especificamente, no tratamento de tuberculose (Baccharis dracunculifolia, Centella asiatica, Lantana camara, Pterodon emarginatus). Os ensaios foram realizados em microplacas com resazurina contra duas espécies do Complexo Mycobacteriumtuberculosis e 3 espécies de micobactérias não tuberculosas. Os resultados mostraram o extrato hexânico e o óleo essencial de frutos de P.emarginatus como potenciais fontes para drogas antimicobacterianas contra quatro espécies de micobactérias testadas. A fração hexânica do extrato metanólico das folhas de C. asiatica também apresentou significativa inibição do crescimento de micobactérias apenas contra M.chelonae. Em conclusão, foi possível contribuir para as investigações de antimicobacterianos por apresentar três novas amostras de plantas com atividade antimicrobiana significativa contra quatro Mycobacterium spp e sugerir a realização de estudos futuros sobre as propriedades antimicobacterianas de frutos de P. emarginatus.

Assessment of the Activity of Selected Indian Medicinal Plants against Mycobacterium tuberculosis: A Preliminary Screening Using the Microplate Alamar Blue Assay.

Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against sensitive and multidrug resistant (MDR) strains. Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants, which have been reported in traditional literature for various uses including respiratory ailments. Place and Duration of Study: Mumbai, India; May 2009 – December 2011. Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts (prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees. (leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC. (tuber) were tested at 1, 10 and 100 μg/ml using the Microplate Alamar Blue Assay. The active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549) using the neutral red assay and a phytochemical analysis was made using High Performance Thin Layer Chromatography (HPTLC). Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 μg/ml. The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%. A combination of acetone and ethanol extracts at 50 μg/ml each was effective against all isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and piperine were not cytotoxic to the A549 cell line. Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract may have active components in addition to piperine. It is possible that the class and expression of efflux pumps in H37Rv is different from that in the clinical isolates, and hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical isolates in addition to reference strains. The observation of the increased efficacy of the combination of acetone and ethanol extracts is interesting.

In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China

Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biofilms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.

Acetylcholinesterase inhibition by somes promising Brazilian medicinal plants / Plantas medicinais brasileiras promissoras para inibição da acetilcolinesterase

A microplate assay and a thin-layer chromatography (TLC) "in situ" assay based on the Ellman assay was used to screen for acetylcholinesterase inhibitors from ethyl acetate and methanol extracts of Brazilian medicinal plants of families that, according to the literature, have traditional uses that might be connected with acetylcholinesterase inhibition. Eighteen species belonging to Convolvulaceae, Crassulaceae, Euphorbiaceae, Leguminosae, Malvaceae, Moraceae, Nyctaginaceae and Rutaceae families were tested. The most active plants were Ipomoea asarifolia (IC50 = 0.12 mg/mL), Jatropha curcas (IC50 = 0.25 mg/mL), Jatropha gossypiifolia (IC50 = 0.05 mg/mL), Kalanchoe brasiliensis (IC50 = 0.16 mg/mL) and Senna alata (IC50 = 0.08 mg/mL). The most promising extracts were the Jatropha gossypiifolia and Senna alata species assuming there were compounds with a similar activity to galanthamine, which should contain about 1 percent of an active compound, or if present at lower levels even more active compounds than galanthamine (IC50 = 0.37 x 10-3 mg/mL) should be present.

Os ensaios de microplaca e cromatografia em camada delgada com base no ensaio de Ellman foram usados para triagem de inibidores da acetilcolinesterase dos extratos acetato de etila e metanol de plantas medicinais brasileiras de famílias que, segundo a literatura, tem usos tradicionais que podem estar relacionadas com a inibição da acetilcolinesterase, enzima associada ao mal de Alzheimer. Dezoito plantas das famílias: Convolvulaceae, Crassulaceae, Euphorbiaceae, Leguminosae, Malvaceae, Moraceae, Nyctaginaceae e Rutaceae foram testadas. As espécies mais ativas foram Ipomoea asarifolia (CI₅₀ = 0,12 mg/mL), Jatropha curcas (CI₅₀ = 0,25 mg/mL), Jatropha gossypiifolia (CI₅₀ = 0,05 mg/mL), Kalanchoe brasiliensis (CI₅₀ = 0,16 mg/mL) e Senna alata (CI₅₀ = 0,08 mg/mL). Os extratos mais promissores foram os das espécies Jatropha gossypiifolia e Senna alata, assumindo a presença de compostos com atividade semelhante à galantamina que deve conter cerca de 1 por cento de um composto ativo, ou se presentes em menores níveis ainda mais compostos ativos que a galantamina (CI₅₀ = 0,37 x10^–3 mg/mL) devem estar presentes.

A Case of Paragonimus westermani Infection Diagnosed by Serological Testing / General Medicine

<b>BACKGROUND</b>: We describe a 40-year-old Thai woman living in Japan who was transferred to Juntendo University Hospital after lung cancer was suspected. Chest X-ray showed a nodular lesion and pleural effusion in the left lung. Laboratory data showed eosinophilia. She denied having consumed raw or undercooked food at the initial interview. Microplate enzyme-linked immunosorbent assay (ELISA) for Paragonimus westermani specific immunoglobulin (Ig) G antibody was positive at a high titer, confirming the diagnosis of P. westermani infection. She was successfully treated with oral praziquantel. All primary practitioners should be aware that paragonimiasis is an important pulmonary disease that can cause nodular lesions on chest X-ray.

Modelo experimental de reconstrução em falha mandibular em coelhos / Experimental model of reconstruction in mandibular defect in rabbit

Este trabalho tem como objetivo apresentar um novo modelo experimental de reconstrução em falha mandibular em coelhos. Foram utilizados 14 animais, nos quais, em uma primeira etapa, extraiu-se cirurgicamente o dente incisivo inferior esquerdo permanente. Após um período de 50 dias para o preenchimento ósseo do alvéolo dentário, foi realizada a cirurgia. Inicialmente, coletou-se 10x5x5mm de enxerto ósseo autógeno da crista ilíaca e, em seguida, foi procedida uma falha mandibular parcial de mesmo tamanho, que foi preenchida com o enxerto e fixada com microplaca de titânio. Os coelhos foram submetidos a avaliações clínicas e radiográficas, sendo sete submetidos a eutanásia aos 15 dias e os demais aos 30 dias para análise macro e microscópica. A ingestão de água e ração não foi comprometida, e os animais não apresentaram algia, dificuldade de mastigação e de apreensão. Somente um animal apresentou rejeição a um parafuso, sem ocorrer o deslocamento da placa nem do enxerto. Esse método mostrou-se eficaz como modelo experimental de reconstrução em falha mandibular de coelhos, demonstrando a evolução cicatricial óssea do enxerto por meio dos exames radiográficos, macroscópicos e microscópicos aos 15 e 30 dias.

This paper aims to present a new experimental model of mandibular defect reconstruction in rabbits. It was used 14 animals, in which, in a first stage the left permanent inferior incisive tooth was extracted surgically. After a 50 days period for the bone to fulfill the dentary alveolus, surgery was performed. At first, a 10x5x5mm autograft was removed form the iliac crest, following a partial mandibular failure in the bone with the same auto graft size which was filled with the graft and fixed using titanium microplate. The rabbits were submitted to clinical and radiographic evaluation and 7 of them were euthanasiated at 15 days and the other 7 at 30 days to macro and microscopic analysis. The water and commercial food ingestion was not compromised, and the animals did not showed any chewing or apprehension difficulties, neither pain. Only one animal presented rejection to one screw, without occurring micro plate nor auto graft dislocation. This method showed to be efficient for an experimental model of reconstruction in mandibular defects of rabbits, demonstrating a healing evolution of the graft through radiographic, macroscopic and microscopic exams in 15th and 30th days.

Application of microplate almar blue assay for rapid detection of drug resistance of M. tuberculosis strains

Background: Recently, microplate almar blue assay has been used commonly in detecting gen related to tuberculosis drug resistance, which provides results after 5-7 days with lower cost compared to traditional methods. Objective: To evaluate the application of microplate almar blue assay for rapid detection of drug resistance of M. tuberculosis strains. Subject and Method: A microplate-based assay which uses Alamar blue reagent - an oxidation reduction dye (MABA), was used for the determination of the anti-tuberculosis drug (isoniazid-INH, rifampicine-RIF, streptomycine-STR and ethabuton=EMB) resistance of 96 M. tuberculosis strains isolated from Vietnamese patients in comparison to those obtained by conversional method. Result: MABA showed to have high sensitivity and specificity in testing the sensitivity to individual anti-tuberculosis drugs (from 82.4% for STR to 93.3% for - INH and from 82.5% for EMB to 98.4% for STR; respectively), as well as for the multi-drug resistant M.tuberculosis (86.4% of sensitivity), highly correlated with the result determined by proportion method. Conclusion: MABA reveals the advantage in shortening test time, in simple performance and lower cost compared with the conversional culture based methods.

Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis / Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis

La tuberculosis multidrogorresistente (MDR), originada por aislamientos de cepas simultáneamente resistentes a isoniacida y rifampicina, es reconocida en la actualidad como un problema sanitario mundial. Nuevas técnicas que permitan detectar en forma temprana cepas MDR son necesarias para evitar su dispersión en la comunidad. En este trabajo hemos evaluado el empleo de un micrométodo colorimétrico para determinar las concentraciones inhibitorias mínimas (CIM) de las drogas de primera línea frente a dichas cepas, usando el indicador: bromuro de 3-(4,5 dimetiltiazol-2-yl)-2,5 difeniltetrazolio (M-MTT). Junto a la cepa de referencia H37Rv se procesaron 603 aislamientos clínicos, 507 provenientes de casos pulmonares (84,1%) y 96 de extrapulmonares (15,9%). Como estándar de referencia se utilizó el método de proporciones en medio de Löwenstein-Jensen (MP), con isoniacida (INH), 0,20 µg/ml; estreptomicina (SM), 4,00 µg/ml; etambutol (EMB), 2,00 µg/ml y rifampicina (RMP), 40,00 µg/ml. Los intervalos de concentraciones de las drogas empleadas en el M-MTT fueron: INH, 1,00-0,03 µg/ml; SM, 8,00-0,25 µg/ml; EMB, 32,00-1,00 µg/ml y RMP: 2,00-0,06 µg/ml. El resultado de la CIM por el M-MTT fue obtenido en un tiempo promedio de 8 días (rango total: 7 a 12 días). Los puntos de corte para cada una de las drogas, calculados mediante el análisis de la curva ROC, fueron: INH, 0,25 µg/ml; RMP, 0,50 µg/ml; SM, 4,00 µg/ml y EMB, 4,00 µg/ml. Los valores de sensibilidad y especificidad fueron 100% en el caso de RMP; y 97,8% y 99,5%, respectivamente, para INH. El análisis estadístico de los resultados permitió concluir que el M-MTT es un método seguro para la rápida detección de MDR. Por su sencillez y bajo costo, podría ser aplicado en los países en vías de desarrollo.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 µg/ml; streptomycin (SM), 4.00 µg/ml; ethambutol (EMB), 2.00 µg/ml and rifampin (RMP), 40.00 µg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 µg/ml; SM, 8.00-0.25 µg/ml; EMB, 32.00-1.00 µg/ml and RMP, 2.00-0.06 µg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 µg/ml, RMP, 0.50µg/ml, SM, 4.00 µg/ml and EMB, 4.00 µg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Evaluation of HBs Ag, HCV and HIV Ag-Ab Assays using Bio-Rad Elite Microplate Analyzer / 대한진단검사의학회지

BACKGROUND: In this study, we evaluated the performance of Elite microplate analyzer (Bio-Rad Laboratories, France) and the related assays (ULTRA line) for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). METHODS: Seroconversion panels, HBsAg positive/HBsAg negative (n=99/n=1,422), anti-HCV positive/negative (n=97/n=1,670), and anti-HIV positive/negative (n=112/n=1,704) samples were used to evaluate the performance of Monolisa HBsAg ULTRA, Monolisa HCV Ag-Ab ULTRA, and Genscreen ULTRA HIV Ag-Ab, respectively. The agreement of Elite microplate analyzer with CODA analyzer (Bio-Rad Laboratories, CA, USA) was also evaluated. RESULTS: The detection limit of Monolisa HBsAg ULTRA was 0.034 IU/mL. For Monolisa HBsAg ULTRA, Monolisa HCV Ag-Ab ULTRA, and Genscreen ULTRA HIV Ag-Ab, diagnostic sensitivities were all 100%, diagnostic specificities were 100%, 99.8% and 99.9%, and total CVs (coefficients of variation) were 13.8-17.5%, 3.4-5.2%, and 7.5-9.5%, respectively. The agreement of Elite microplate analyzer with CODA analyzer was 99.5%. CONCLUSIONS: The performance of Elite microplate analyzer and the related assays on analytical sensitivity, precision, early detection, diagnostic sensitivity and specificity was all adequate for a mass screening. However, further large multi-center studies should be performed to validate our results.

Surgical Results of Zygomatico-Orbital Complex Fractures according to the 3-dimensional Classification

PURPOSE: To classify zygomatico-orbital fractures based on Zingg's classification and to evaluate the results of treatment. METHODS: Thirty patients who were diagnosed with zygomatico-orbital fracture between 2000 and 2004 were classified based on Zingg's classification, were treated and followed up for more than 3 months. Of the 30 patients, 28 received surgical treatment, including Gillies reduction or open reduction and rigid fixation with a microplate. RESULTS: The mean follow-up period was 6 months. Most of the patients were satisified with the results of surgery. There were no secondary operations due to facial asymmetry, secondary wound infection, microplate exposure or paresthesia. However, there were 3 re-operations for diplopia, enophthalmos, and lower eyelid retraction, respectively; the conditions had persisted until postoperative 2 weeks. CONCLUSIONS: The use of 3-dimensional Zingg's classification for zygomatico-orbital fractures appeared to be beneficial in making more accurate diagnoses and in determining surgical treatment. Oculoplastic approaches, including subciliary or transconjunctival incision, Gillies reduction and rigid fixation with a microplate, are considered to be good methods for the treatment of zygomatico-orbital fractures, as they are associated with exact anatomical and functional restoration and few complications.