Development of a liquid chromatography-tandem mass spectrometry method to determine colistin, bacitracin and virginiamycin M1 at cross-contamination levels in animal feed.

Cross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6-107.8% and precision ranged 12.6-21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on "real" samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin.

A portable fluorescent microsphere-based lateral flow immunosensor for the simultaneous detection of colistin and bacitracin in milk.

The polypeptide antibiotics colistin (COL) and bacitracin (Baci) are extensively used as veterinary drugs and feedstock additives in the livestock industry, which inevitably causes residues in animal-origin food, which can accelerate human tolerance to antibiotics. In this study, a portable lateral flow immunoassay (LFIA) for the simultaneous determination of COL and Baci residues in milk was developed. The replacement of gold nanoparticles used in the traditional LFIA with fluorescent microspheres (FMs) to label monoclonal antibodies (mAbs) allowed qualitative and quantitative analyses within a few minutes. Based on the principle of competitive binding to FM-labelled mAbs between analytes in samples and fixed antigens on the membrane, the assay provided qualitative cut-off values of 100 and 50 ng mL-1 for Baci and COL in milk samples. Furthermore, a strip reader-based semi-quantitative detection system could detect lower limits of 7.85 and 1.89 ng mL-1 for Baci and COL, respectively. In conclusion, the proposed multiplex LFIA immunosensor provides an auxiliary analytical tool for the rapid and simultaneous screening of COL and Baci in large cohorts of samples.

UVC- and UVC/H2O2-Driven nonribosomal peptide antibiotics degradation: application to zinc bacitracin as a complex emerging contaminant.

Zinc bacitracin (Zn-Bc) belongs to the group of nonribosomal peptide antibiotics (NRPA), comprising a mixture of non-biodegradable congeners characterized by complex structures containing cyclic, polycyclic, and branched chains. However, reports on the use of AOPs for the degradation of NRPA are non-existent. In this context, the present work investigated the photodegradation of Zn-Bc in aqueous solution by direct photolysis and the UVC/H2O2 process. The effects of the specific UVC photon emission rate and initial H2O2 concentration were studied following a Doehlert-design response surface approach. The results showed that all congeners photolyzed at the highest UVC doses in the absence of hydrogen peroxide, with a calculated quantum yield of 0.0141 mol Zn-Bc mol photons-1. However, no TOC removal was observed after 120 minutes of irradiation, suggesting the disruption of the peptide bonds in the antibiotic molecules without significant changes in the amino acid residues. The addition of H2O2 substantially accelerated Zn-Bc photodegradation, resulting in a remarkable removal of up to 71% of TOC. Most importantly, the antimicrobial activity against Staphylococcus aureus could be completely removed by both treatments. These findings point out that the UVC/H2O2 process can be straightly engineered for the treatment of metalloantibiotics-containing wastewater in pharmaceutical facilities.

Colloidal gold-based immunochromatographic strip assay for the rapid detection of bacitracin zinc in milk.

In this study, a murine monoclonal antibody (mAb) of 6D2-G10 against bacitracin zinc (BAC) was produced and applied to an immunochromatographic strip (ICS) for the initial detection of BAC in milk. The ICS with a cut-off value of 25 ng/mL could be perceived by the naked eye within 10 min. With the assist of the strip reader, the limit of detection (LOD) was measured as 0.82 ng/mL, the half-maximal inhibitory concentration (IC50) was recorded as 3.16 ng/mL, and the linear detection range was from 0.97 to 10.30 ng/mL. The recoveries ranged from 87.7% to 96.0% with the highest coefficient of variation (CV) of 9.1% in the intra-assay and from 84.3% to 90.2% with the highest CV of 10.7% in the inter-assay. In short, the established ICS provided a serviceable analytical tool for qualitatively and quantitatively monitoring BAC in milk.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

Simultaneous detection of bacitracin and polymyxin B in livestock products using liquid chromatography with tandem mass spectrometry.

With the overarching aim to develop a simple and reliable method for the quantitative analysis of polypeptide antibiotics in various livestock products, the content of bacitracin, and polymyxin B in pork, beef, chicken, milk, and eggs was analyzed using colistin sulfate as an internal standard. The extracted samples were eluted via solid-phase extraction using 2% formic acid in acetonitrile/methanol (1:1, v/v). The two polypeptides were identified and quantified based on the intensities of mass fragments from the respective triply charged precursor ions (bacitracin: 474.97 amu and polymyxin B: 402 amu) at the defined retention time windows using liquid chromatography with electrospray ionization tandem mass spectrometry in time-scheduled multiple reaction monitoring mode. The calibration curves showed good linearity over the concentration range 50-2500 ng/mL with determination coefficients ≥ 0.991. The mean recoveries were in the range 80.3-88.8% with relative standard deviations <13% for all samples. The limits of quantitation ranged from 30-250 ng/g. The developed method was applied to market samples, but the target analytes were not detected in any of the samples. The developed method is reliable for the simultaneous detection of bacitracin and polymyxin B in pork, beef, chicken, milk, and eggs.

Uso de óleo essencial de orégano, salinomicina e bacitracina de zinco na dieta de frangos de corte / Use of oregano essential oil, salinomycin and bacitracin zinc in diets of broiler chicken

O presente trabalho teve como objetivo avaliar dietas com óleo essencial de orégano, associado ou não com salinomicina, como alternativa à bacitracina de zinco sobre o desempenho zootécnico de frangos de corte. Foram utilizados 600 pintos de um dia de idade, machos, da linhagem Cobb® 500, criados até 42 dias de idade em boxes com cama de casca de arroz providos de comedouros tubulares e bebedouros nipple. O delineamento foi inteiramente casualizado, com seis dietas e 10 repetições de 10 aves cada. As dietas experimentais à base de milho e farelo de soja foram: controle positivo – antibiótico (bacitracina de zinco) + 0,05% de anticoccidiano (salinomicina), controle negativo – dieta basal (DB) sem aditivos, DB + 0,05% de salinomicina e 0,03% de óleo essencial de orégano (Orego-Stim®), DB + 0,03% de óleo essencial de orégano, DB + 0,05% de salinomicina e 0,05% de óleo essencial de orégano, DB + 0,05% de óleo essencial de orégano. Não foi encontrado efeito da utilização do óleo de orégano até 21 dias no desempenho das aves. Nos demais períodos, aos 35 e 42 dias, o desempenho das aves tratadas com 0,03% de óleo essencial de orégano + salinomicina apresentou resultados semelhantes ao controle positivo, levando à conclusão de que a dose de 0,03% de óleo essencial de orégano + salinomicina pode substituir a bacitracina de zinco + salinomicina em dietas para frangos de corte.

The inclusion of oregano essential oil, alone or associated with salinomycin, was evaluated as an alternative to zinc bacitracin on the performance of broiler chickens. This study used 600 male Cobb 500® day-old chicks, raised 42 days in boxes with rice hulls, provided with tubular feeders and nipple drinkers. The experimental design was completely randomized with six diets and 10 replications with 10 birds per experimental unit. The diets were based on corn and soybean meal: positive control - antibiotic (zinc bacitracin) + 0.05% anticoccidial (salinomycin), negative control - basal diet (BD) without additives, DB + 0.05% of salinomycin and 0.03% of oregano essential oil (Orego-Stim®), DB + 0.03% of oregano essential oil, DB + 0.05% of salinomycin and 0.05% of oregano essential oil, DB + 0.05% of oregano essential oil. There were no treatment effects on broiler performance until 21 days of age. In the other periods, at 35 and 42 days, the oregano essential oil at 0.03% combined with salinomycin presented similar effects as the positive control, leading to the conclusion that 0.03% of oregano essential oil associated with the salinomycin can replace zinc bacitracin + salinomycin in broiler chicken diets.

Role of three-body recombination for charge reduction in MALDI process.

Ions in Matrix-Assisted Laser Desorption/Ionization (MALDI) are predominantly singly charged for small analyte molecules. With the estimated high number density and low temperature of electrons, the three-body recombination mechanism is attractive and should be considered as an important cause for the charge reduction in the plume. Theoretical calculations indicate that the rate coefficient of the three-body recombination is about 50 times higher than that of the two-body recombination if the analyte molecule has insufficient degrees of freedom. Experimental results show that, for small analyte molecules, the ratio of AH2(2+)/AH(+) is close to the theoretical 5% value from the three-body recombination modeling and this ratio declines with the increasing electron and matrix molecule number density caused by greater laser irradiance. The ratio of [A + 2](+)/[A + 1](+) is higher than the theoretical isotopic value, and the excess [A + 2](+) could exclusively result from the three-body recombination collisions. Further evidence demonstrates that [A + 2](+)/[A + 1](+) increases with electron number density, which is in correspondence with the model. All of these theoretical and experimental results indicate that three-body recombination is an essential charge reduction mechanism for small molecules in the MALDI plume.

Production of peptide antibiotics by Bacillus sp: GU 057 indigenously isolated from saline soil

A total of 112 soil samples were taken from differents areas of district D.I.Khan and Kohat (KPK) Pakistan and screened for production of antibiotics against the Micrococcus luteus and Staphylococcus aureus. Widest zone of inhibition (18mm) was produced by microorganism isolated from saline soil. The strain was later identified as Bacillus GU057 by standard biochemical assays. Maximum activity (18mm inhibition zone) was observed against Staphylococcus aureus after 48 hours of incubation at pH 8 and 4% concentration of glucose. The antibiotic was identified by autobiography as bacitracin. The Bacillus strain GU057 was confirmed as good peptide antibiotic producer and can effectively be indulged as biocontrol agent.

Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifier.

Any product marketed in the United States and labeled bacitracin must comply with the tests, procedures, and acceptance criteria in the relevant monograph published in the US Pharmacopeia (USP). The test procedure relies on accurate recovery of Bacitracin A and many other bacitracin components. The authors determined that the current USP procedure does not recover Bacitracin A at low concentration levels. They postulate that this low recovery is the result of bacitracin's known ability to chelate metal ions, e.g. in bacitracin-zinc complexes. Thus the ubiquitous metal ions in HPLC systems may be responsible for sequestering bacitracin and giving low recoveries. Addition of edetate disodium (EDTA) to the mobile phase improved the recovery. The method validation results include precision, accuracy, linearity, specificity, and robustness.

Spectrophotometric determination of bacitracin in bulk drug as dabsyl derivative in a range of visible light.

A fast spectrophotometric method has been developed for bacitracin identification and determination after condensation reaction with dabsyl chloride. In addition, determination of dye stability of sulfonamide derivative and identification of the molar ratio of reagents was done at various time-points. The developed method has a good linearity with very broad spectrum, correlation coefficient of r = 0.9972, good precision (RSD = 1.54 +/- 0.11%), and recovery at three different levels of concentration was found between 98.33% and 103.47%. Usefulness of the method was demonstrated by positive results obtained during determination of bacitracin concentration in bulk drug.

A novel cell-associated protection assay demonstrates the ability of certain antibiotics to protect ocular surface cell lines from subsequent clinical Staphylococcus aureus challenge.

In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 µg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 µg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 µg/ml, whereas AZM displayed toxicity to one cell line at 512 µg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Mobile phase selection for the combined use of liquid chromatography-inductively coupled plasma mass spectrometry and electrospray ionisation mass spectrometry.

Four different organic solvents: dimethylformamide, 1,4-dioxane, n-propanol and ethanol were evaluated as alternative organic modifiers to acetonitrile for liquid chromatography (LC) separations. The aim was to establish common sets of chromatographic conditions that could be applied for LC hyphenation to inductively coupled plasma mass spectrometry (ICPMS) as well as to electrospray ionization MS (ESIMS). The approach was to evaluate candidate solvents that, compared to acetonitrile, potentially could give improved analytical performance (low solvent vapor loading, maximized analyte sensitivity and minimized carbon depositions on instrumental parts) in ICPMS analysis while retaining chromatographic and ESIMS performances. The study showed that dimethylformamide, 1,4-dioxane, n-propanol and ethanol all can be advantageous chromatographic modifiers for LC-ICPMS analysis, giving superior performance compared to acetonitrile. For the combined use of LC-ICPMS and LC-ESIMS with a common set of chromatographic conditions, n-propanol gave the best overall performance. The 195Pt+ signal in ICPMS was continuously monitored during a 0-60% organic solvent gradient and at 25% of organic modifier, 100% of the signal obtained at the gradient start was preserved for n-propanol compared to only 35% of the signal when using acetonitrile. Platinum detection limits were 5-8 times lower using n-propanol compared with acetonitrile. Signal-to-noise ratio in continuous ESIMS signal measurements was 100, 90 and 110 for a 100 microg/ml solution of leucine-enkephaline using acetonitrile, ethanol and n-propanol, respectively. Chromatographic efficiency in reversed phase separations was preserved for n-propanol compared to acetonitrile for the analysis of the whole protein cytochrome C and the peptide bacitracin on a column with particle and pore sizes of 5 microm and 300 A, but slightly deteriorated for the separation of the peptides leucine-enkephaline and bacitracin on a 3 microm and 90 A column as the peak width at half height for both peptides increased by a factor of two. The performance on the smaller dimensioned column could however be improved by running the separations at 40 degrees C.

Ultrafiltration separation of aquatic natural organic matter: chemical probes for quality assurance.

Characterization of molecular size of natural organic matter (NOM) is a valuable tool when assessing its effect on the performance of water treatment systems as well as its geochemical origin. Size fractionation can be accomplished by ultrafiltration (UF). Unfortunately, membrane manufacturing generates a range of pore sizes. Many membrane manufacturers use molecular weight cutoff (MWCO) metric based on a 90% retention of given solute after specified duration of filtration. The objective of this study was to characterize the ability of different commercially available UF membranes to separate different size fractions of NOM. The UF membranes characterized were YM (regenerated cellulose, negatively charged) and PB (polyethersulfone, negatively charged) product lines by Millipore. The probes used to represent the size, shape and charge of NOM were polymers (polyethylene glycols (PEGs), dextrans, polystyrene sulfonates (PSSs)), dyes (bromocresol green, congo red, methyl red, methyl orange) and biological molecules (vitamin B-12 and bacitracin). The results show that MWCO definition does not hold for membranes of 5kDa and 10kDa pore openings using most polymers and dyes. The MWCO definition holds for 1kDa membrane for all tested probes. Under natural water conditions PSSs assume random coil configurations that are nearly identical to Suwannee fulvic acid. The results show that PSS agrees with stated MWCOs. The study demonstrates that ultrafiltration is not a simple mechanical sieving process, but that charges on the membrane and the constituent play a significant role in the rejection process. Effective probe size was increased seven- to fourteen-fold by charge interactions between the negative probes and negatively charged membrane. Uncharged molecules larger than specified MWCOs are able to pass through pores (PEGs), while small charged molecules (dyes) do not pass. For probes with low or neutral charges, shape becomes an important factor, with globular being favored over linear structure. Thus, MWCOs cannot be trusted for purposes of NOM size characterization. The study recommends the use of YM 1K, PB 5K and YM 10kDa membranes for comparative-only NOM size ultrafiltration characterization within the 1-10kDa size range.

Sposensor: a whole-bacterial biosensor that uses immobilized Bacillus subtilis spores and a one-step incubation/detection process.

A generic whole-cell bacterial sensor called sposensor was developed with immobilized spores from engineered Bacillus subtilis. Sposensor contains two different types of spores: reporting spores that contain a reporter gene fused to a promoter responding to a compound to be detected, and control spores use to monitor cell germination and viability. A one-step incubation/detection process was developed to meet the constraints of on-site analysis. Spores were directly incubated with culture medium containing the compound to be detected. beta-Galactosidase was chosen as a reporter protein in both cases and its activity followed by a colorimetric assay. Results showed that sposensor was efficient in detecting two different compounds, a metal (Zn(2+)) and a peptidic antibiotic (bacitracin). Owing to the stability and robustness of spores, sposensor is a very efficient and easy tool to manipulate for analyzing the presence of toxic compounds in natural settings.

Collaborative study of a microbiological screening method (three-plate) for the banned antimicrobial growth promotors tylosin, virginiamycin, spiramycin, zinc bacitracin and avoparcin in animal feed.

A microbiological screening method (three-plate) for the detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin, and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg(-1) (false-positives, 2%; false-negatives, 3, 0, and 6%, respectively). Avoparcin could be detected at 1 mg kg(-1) in feed in general (false-positives, 2%; false-negatives, 0%). However, in calf feed the sensitivity was lower. The percentages of false-negatives were found to be 12%, 7%, and 0% at 1, 3, and 5 mg kg(-1), respectively (false-positives, 4%). The limit of detection for zinc bacitracin was 3-5 mg kg(-1) (false-positives, 5-10%; false-negatives, 77% at 1 mg kg(-1), 45% at 2 mg kg(-1), 12% at 3 mg kg(-1), and 4% at 5 mg kg(-1)). The method allowed for a distinction to be made between the groups of antibiotics: avoparcin/zinc bacitracin versus tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis.

Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed.

An MEKC procedure was developed for the separation of zinc bacitracin (Zn-BC) and nystatin (NYS) in mixtures and in animal feedstuff. The running buffer was 15 mM borate/19 mM phosphate, pH 8.2, containing 20 mM SDS and 10% v/v methanol. Samples were run at 25 degrees C, the applied voltage was 25 kV, and an additional pressure of 5 mbar was applied. Both analytes were detected by UV simultaneously at 215 nm, Zn-BC alone at 192 and 254 nm, and NYS alone at 305 nm. The method was shown to be specific, accurate (recoveries were 100.0 +/- 0.6% and 100.1 +/- 0.6% for Zn-BC and NYS, respectively), linear over the tested range (correlation coefficients 0.9991 and 0.9994), and precise (RSD below 1.3% for both analytes). The method was applied to determine Zn-BC and NYS as additives in animal feed.

Detection of residual bacitracin A, colistin A, and colistin B in milk and animal tissues by liquid chromatography tandem mass spectrometry.

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was applied to the determination of residual bacitracin A, colistin A, and colistin B in milk and animal tissue samples. Prior to instrumental analysis, samples were subjected to acid extraction followed by solid-phase cleanup using Strata-X cartridges. Mass spectral acquisitions were performed under selective multiple reaction monitoring (MRM) mode at m/z 199 and 670 from triply charged precursors of bacitracin A (m/z 475); m/z 385 and 379 from triply charged precursors of colistin A (m/z 391); and m/z 380 and 374 from triply charged precursors of colistin B (m/z 386). Method precision was evaluated from spike recovery of samples fortified at concentrations corresponding to 2/5 of the maximum residue limits (MRLs) for each of the analytes under study. Intra-day and inter-day variations were found to range from 90.9 to 104% with relative standard deviation (RSD) <6.5%, and from 90.1 to 106% with RSD <9.1%, respectively. Limits of quantification (LOQs) were defined as the spiking concentrations at 2/5 MRL, and limits of detection (LODs) were 10-47 microg kg(-1) for bacitracin A, 1-16 microg kg(-1) for colistin A, and 6-14 microg kg(-1) for colistin B in milk and animal tissues. The presented method has good precision and high sensitivity and was applied as a fast screening protocol and a quantitative tool for monitoring of the concerned polypeptides in foods as part of a surveillance program.

Diagnóstico de la faringitis estreptocócica en la consulta de atención primaria: una antigua solución reconsiderada / Diagnosis of streptococcal pharyngitis in the office: an old solution reconsidered

Introducción: La faringitis aguda es muy prevalente en pediatría primaria. La etiología más frecuente es viral (70%-80%), siendo Streptococcus pyogenes (SP) la causa bacteriana más importante. El objetivo de este trabajo es evaluar tres scores clínicos publicados para predecir la faringitis estreptocócica, en comparación con el resultado del cultivo faríngeo (CF) realizado en la consulta. Material y métodos: Se estudiaron 697 niños de entre 6 meses y 14 años de edad, con clínica de faringitis. En todos los casos se efectuó un CF mediante siembra del exudado faríngeo en agar-sangre e identificación de SP por la prueba de la bacitracina. Se calculó el coste y el tiempo invertido en cada determinación. Se valoraron tres scores, comparando cada uno de ellos con el resultado del CF. Resultados: Un total de 174 (25%) de los niños estudiados tuvo un cultivo faríngeo positivo para SP. El valor predictivo positivo (VPP) en los tres scores es inferior al 40% y su sensibilidad (S) varía entre un 25 y un 39%. El coste de cada CF fue de 0,50 euros. El tiempo medio invertido en cada CF fue de 4 minutos. Conclusiones: Según los resultados obtenidos, los bajos VPP y la escasa S sugieren que no existen criterios clínicos fiables que permitan el diagnóstico de la faringitis estreptocócica en la mayor parte de los casos. La triple disyuntiva que supone optar por realizar CF en los laboratorios de bacteriología, usar métodos rápidos o emplear criterios clínicos queda superada por la realización del CF en la consulta. En nuestra experiencia, la práctica del CF y la prueba de la bacitracina como técnica diagnóstica es idónea en la consulta de atención primaria, por ser segura, poco costosa, práctica y de fácil realización

Introduction. Acute pharyngitis is highly prevalent in pediatric primary careo It is usually viral in origin (70%-80%), Streptococcus pyogenes being the leading bacterial cause. The objective of this report is to evaluate three published clinical scoring systems for the prediction of streptococcal pharyngitis, comparing them with the results of throat culture performed in the office. Material and methods. We studied 697 children between the ages of 6 months and 14 years with the clinical signs and symptoms of acute pharyngitis. Throat swab samples from each child were inoculated on blood agar plates and incubated, using the bacitracin test for the identification of S. pyogenes. The cost and time spent for each determination were calculated. Three scores were assessed and compared with the results of the throat culture. Results. One hundred seventy-four (25%) of the children studied had throat cultures positive for S. pyogenes. The positive predictive value (PPV) of the three scores was under 40%, and their sensitivity ranged between 25% and 39%. Each throat culture cost 0.50 euros and required a mean time of 4 minutes. Conclusions. According to our findings, the low PPV and sensitivity suggest the nonexistence of reliable clinical criteria for the diagnosis of streptococcal pharyngitis in the majority of cases. The performance of throat culture in the microbiology laboratory, the use of "rapid methods" and the application of clinical criteria are less advantageous than the performance of throat culture in the office. In our experience, throat culture using the bacitracin test is safe, inexpensive and easy to perform and, thus, is the diagnostic technique of choice in the primary care office setting

Batch variability of bacitracin: HPLC versus MEKC.

10 lots of bacitracin collected from the European market were studied applying the European Pharmacopoeia HPLC method, and the results were compared to the findings of an orthogonal micellar electrokinetic chromatographic method. The latter method exhibited a far higher selectivity, resulting in a baseline separation of all bacitracin components, which could not be achieved with HPLC. Considering the contents of bacitracin A, B1, and B3, the lots could be organised into 2 groups. Due to high amounts of bacitracin B1 and B3, one lot did not fit into either group. All lots met the requirements of the European Pharmacopoeia.