Microbial safety implications of in-use topical diagnostic ophthalmic medications in eye clinics in Ghana / Implicaciones sobre seguridad microbiana de los fármacos oftálmicos diagnósticos tópicos utilizados en las clínicas oftalmológicas de Gana

PURPOSE: To determine the microbial contaminants and its clinical importance in topical diagnostic ophthalmic medications (cycloplegics/mydriatics and miotics) in eye clinics in Ghana. Method: A cross-section of eye clinics was sampled for the diagnostic agents (Atropine, Phenylephrine, Tropicamide and Cyclopentolate, Pilocarpine). Standard laboratory procedures and protocols were observed in culturing the samples on different Agars. Microscopy and various biochemical tests were performed to identify microbial species. Antimicrobial susceptibility testing was also performed to ascertain the clinical importance of the isolated microbes. RESULTS: A total of 113 samples were obtained, from which 334 bacteria were isolated which included Bacilli spp. 91(27.25%), Coagulase Negative Staphylococci spp. 59(17.66%), Moraxella spp. 47(14.07%), Staphylococcus aureus 41(12.27%), Streptococcus spp. 21(6.29%), Klebsiella spp. 20(5.99%), Pseudomonas spp. 13(3.89%), Proteus spp. 12(3.59%), Escherichia coli. 12 (3.59%), Serratia spp. 10(2.99%), Shigella spp. 7(2.09%), Salmonella spp. 1(0.3%). There were 96 isolated fungal contaminants mainly Penicillium spp. 41(42.71%), Cephalosporium spp. 19(19.79%), Cladosporium spp. 15(15.63%), Aspergillus spp. 13(13.54%), Cercospora spp. 8(8.33%). The diagnostic agent with the most bacteria contamination was Phenylephrine 90 (26.95%) and the least being Pilocarpine 49 (14.67%). Also, the diagnostic agent with the most fungal contamination was Cyclopentolate 29 (30.2%) and the least was Tropicamide and Pilocarpine with 15 (15.63%) each. Gentamicin and Ciprofloxacin were the only antibiotics that showed 100% activity against all the bacterial isolates. Fungal contaminants were more susceptible to Ketoconazole as compared to Fluconazole. Conclusion: Topical diagnostic ophthalmic preparations used in clinical settings in Ghana are contaminated with clinically important bacteria and fungi

OBJETIVO: Determinar los contaminantes microbianos y su importancia clínica en los fármacos oftálmicos diagnósticos tópicos (ciclopléjicos/midriáticos y mióticos) en clínicas oftalmológicas de Gana. MÉTODO: Se realizó una muestra transversal de clínicas oftalmológicas para los agentes diagnósticos (Atropina, Fenilefrina, Tropicamida y Ciclopentolato, Pilocarpina). Se observaron procedimientos y protocolos de laboratorio estándar en cuanto al cultivo de muestras en diferentes soluciones de Agar. Se realizaron diversas pruebas microscópicas y bioquímicas para identificar las especies microbianas. También se realizó la prueba de susceptibilidad antimicrobiana para comprobar la importancia clínica de los microbios aislados. RESULTADOS: Se obtuvieron un total de 113 muestras, de las cuales se aislaron 334 bacterias que incluyeron Bacilli spp. 91(27,25%), Staphylococci spp. Coagulasa negativos 59(17,66%), Moraxella spp. 47(14,07%), Staphylococcus aureus 41(12,27%), Streptococcus spp. 21(6,29%), Klebsiella spp. 20(5,99%), Pseudomonas spp. 13(3,89%), Proteus spp. 12(3,59%), Escherichia coli. 12(3,59%), Serratia spp. 10(2,99%), Shigella spp. 7(2,09%), Salmonella spp. 1(0,3%). Se encontraron 96 contaminantes fúngicos aislados, principalmente Penicillium spp. 41 (42,71%), Cephalosporium spp. 19 (19,79%), Cladosporium spp. 15 (15,63%), Aspergillus spp. 13 (13,54%), Cercospora spp. 8 (8,33%). El agente diagnóstico con mayor contaminación bacteriana fue Fenilefrina 90(26,95%), siendo Pilocarpina 49 (14,67%) el que reflejó una menor contaminación bacteriana. De igual modo, el agente diagnóstico con mayor contaminación fúngica fue Ciclopentolato 29 (30,2%), siendo Tropicamida y Pilocarpina, con 15 (15.63%) cada uno, los que reflejaron menos contaminación fúngica. Gentamicina y Ciprofloxacina fueron los únicos antibióticos que reflejaron un 100% de actividad frente a todos los aislados bacterianos. Los contaminantes fúngicos fueron más susceptibles a Ketoconazol, en comparación con Fluconazol. CONCLUSIÓN: Los preparados oftálmicos diagnósticos tópicos en entornos clínicos en Gana están contaminados por bacterias y hongos clínicamente importantes

Microbial safety implications of in-use topical diagnostic ophthalmic medications in eye clinics in Ghana.

PURPOSE: To determine the microbial contaminants and its clinical importance in topical diagnostic ophthalmic medications (cycloplegics/mydriatics and miotics) in eye clinics in Ghana. METHOD: A cross-section of eye clinics was sampled for the diagnostic agents (Atropine, Phenylephrine, Tropicamide and Cyclopentolate, Pilocarpine). Standard laboratory procedures and protocols were observed in culturing the samples on different Agars. Microscopy and various biochemical tests were performed to identify microbial species. Antimicrobial susceptibility testing was also performed to ascertain the clinical importance of the isolated microbes. RESULTS: A total of 113 samples were obtained, from which 334 bacteria were isolated which included Bacilli spp. 91(27.25%), Coagulase Negative Staphylococci spp. 59(17.66%), Moraxella spp. 47(14.07%), Staphylococcus aureus 41(12.27%), Streptococcus spp. 21(6.29%), Klebsiella spp. 20(5.99%), Pseudomonas spp. 13(3.89%), Proteus spp. 12(3.59%), Escherichia coli. 12 (3.59%), Serratia spp. 10(2.99%), Shigella spp. 7(2.09%), Salmonella spp. 1(0.3%). There were 96 isolated fungal contaminants mainly Penicillium spp. 41(42.71%), Cephalosporium spp. 19(19.79%), Cladosporium spp. 15(15.63%), Aspergillus spp. 13(13.54%), Cercospora spp. 8(8.33%). The diagnostic agent with the most bacteria contamination was Phenylephrine 90 (26.95%) and the least being Pilocarpine 49 (14.67%). Also, the diagnostic agent with the most fungal contamination was Cyclopentolate 29 (30.2%) and the least was Tropicamide and Pilocarpine with 15 (15.63%) each. Gentamicin and Ciprofloxacin were the only antibiotics that showed 100% activity against all the bacterial isolates. Fungal contaminants were more susceptible to Ketoconazole as compared to Fluconazole. CONCLUSION: Topical diagnostic ophthalmic preparations used in clinical settings in Ghana are contaminated with clinically important bacteria and fungi.

[Preparation and testing of buffered eye drops containing pilocarpine chloride with timolol maleate].

BACKGROUND/AIM: In glaucoma therapy beta-blocator, timolol-maleate, is the first-line medicine of selection, whereas a miotic, pilocarpine chloride, is one of the oldest medicines used for the treatment of this illness. They are applied as eye drops. In order to achieve a better therapeutic effect and improve life quality of the ill, we produced and tested eye drops formulations based on combining pilocarpinechloride and timolol maleate in buffers of diferent pH values. METHODS: Following the general pharmacopoeial eye drops preparation regulation, we prepared formulations, of solution type, of pilocarpine chloride and timolol maleate combination, of pilocarpine choloride alone and of timolol maleate alone. A modified phosphate buffer according to Sorensen at 7.4, 7.7 and 8.0 pH values was used as a solvent. The quality of the produced formulations was examined using physical and physico-chemical methods and biological tests. Following pharmacopoeial regulations, we examined clarity, pH value and sterilty. Pilocarpine chloride level was determined by means of ion-par (High Performance Liguid Chromatography) RP-HPLC method, whereas UV/VIS absorption spectrophotometry was used for determining the level of both timolol maleate, and timolol. RESULTS: Results showed that monocomponent and combined preparations complied with the regulation demands. With the increase in the pH value of the solution pilocarpine chloride level decreased in relation to its initial content, whereas timolol level showed a tendency of moderate increase. CONCLUSION: Magistrally prepared pilocarpine chloride eye drops with timolol maleate have satisfied all the required conditions for an ophtalmological preparation. A modified phosphate buffer according to Sorensen at 7.4 pH value proved to be the most optimal solvent.

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products.

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.

Physico-chemical, in vitro and in vivo characterisation of polymers for ocular use.

The influence of artificial tear fluid (AT) on ionic and nonionic ophthalmic polymer excipients was rheologically established. In usual concentrations polyvinylalcohol, polyvinylpyrrolidone, dextran, hydroxypropylmethylcellulose, hydroxyethylcellulose and methylcellulose did not show any changes. In contrast, solutions of polyacrylic acid, sodium hyaluronate (S-Hya), sodium alginate (S-Alg) and chitosan decrease the apparent viscosity in contact with AT, while gellan solution increases the viscosity and shows thixotropy. The adhesion of selected polymers (polysaccharides) on mucin was evaluated using a rheological method and resulted in the order S-Hya > Gellan > S-Alg > dextran. Miosis testing of Gellan containing pilocarpine HCl formulations in rabbits shows a possible reduction of drug concentration from 2% to 0.5% obtaining the same bioavailability.

Neurogenic calcitonin gene-related peptide: a neurotrophic factor in the maintenance of acetylcholinesterase molecular forms in adult skeletal muscles.

This work addresses the role of calcitonin gene-related peptide (CGRP) in the physiological maintenance of acetylcholinesterase (AChE) molecular forms in motor endplate regions of adult Sprague-Dawley rat fast-twitch anterior gracilis muscles. Results show that: (a) CGRP is present in obturator nerve motor neurons which supply the gracilis muscle, as well as in the corresponding motor endplate regions where high levels of both AChE activity and acetylcholine receptors (AChRs) are detected; (b) endplate-associated CGRP declines with muscle denervation several hours before any changes in AChE forms are detected; (c) a single subcutaneous injection of CGRP reversibly reduces the activities of all AChE forms in endplate regions of normally innervated and otherwise untreated gracilis muscles; and (d) similar treatment with hCGRP(8-37), a potent and selective CGRP antagonist, produces the opposite effects, i.e., it reversibly elevates the activities of all AChE forms. These and other findings indicate that CGRP and hCGRP(8-37) influence the mechanism(s) by which AChE forms are maintained in intact adult gracilis muscles. Indeed, the findings lend strong support to the hypothesis that nerve-derived CGRP plays a key role in the trophic regulation of AChE forms at the neuromuscular junction.

Evaluation of pilocarpine loaded gelatin particles for topical ophthalmic use.

Gelatin particles containing pilocarpine HCl were prepared using a desolvation method. The influence of the gelatin type and the preparation pH on particle properties was studied. The drug entrapment amounted 44 to 57%, zetapotential was slightly negative. Particle size varied from 312 nm to 500 nm depending on preparation pH. Franz diffusion cell experiments showed a release approaching zero-order kinetics and a sustained release compared to an aqueous pilocarpine HCl solution.

Fractional factorial design optimization of the separation of pilocarpine and its degradation products by capillary electrophoresis.

The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, its trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.