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.

Determination of atropine enantiomers in ophthalmic solutions by liquid chromatography using a Chiral AGP column.

Many therapeutic agents are commercialized under their racemic form. The enantiomers can show differences in the pharmacokinetic and pharmacodynamic profile. The use of a pure enantiomer in pharmaceutical formulations may result in a better therapeutic index and fewer adverse effects. Atropine, an alkaloid of Atropa belladonna, is a racemic mixture of l-hyoscyamine and d-hyoscyamine. It is widely used to dilate the pupil. To quantify these enantiomers in ophthalmic solutions, an HPLC method was developed and validated using a Chiral AGP column at 20 degrees C. The mobile phase consisted of a buffered phosphate solution (containing 10 mM 1-octanesulfonic acid sodium salt and 7.5 mM triethylamine, adjusted to pH 7.0 with orthophosphoric acid) and acetonitrile (99 + 1, v/v). The flow rate was 0.6 ml/min, with UV detection at 205 nm. In the concentration range of 14.0-26.0 microg/mL, the method was found to be linear (r > 0.9999), accurate (with recovery of 100.1-100.5%), and precise (RSD system < or = 0.6%; RSD intraday < or = 1.1%; RSD interday < or = 0.9%). The method was specific, and the standard and sample solutions were stable for up to 72 h. The factorial design assures robustness with a variation of +/- 10% in the mobile phase components and 2 degrees C of column temperature. The complete validation, including stress testing and factorial design, was studied and is presented in this research.

Determination of tropicamide and its major impurity in raw material by the HPLC-DAD analysis and identification of this impurity using the off-line HPLC-FT-IR coupling.

This study presents TLC, HPLC-DAD and HPLC-FT-IR analyses concerning the detection, identification and determination of organic impurities of commercial tropicamide ((R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(4-pyridylmethyl)propanamide) as a medical substance designed for the production of eye drops. In the examined samples from several random production batches, only one impurity (defined by Ph. Eur. 6th Ed.) was discovered in the amount sufficient for the quantitative analysis. On the basis of comparison of retention times, UV and IR spectra of the impurity and its synthesized standard, this impurity was identified as apotropicamide (N-ethyl-2-phenyl-N-(4-pyridylmethyl)prop-2-enamide). For the chemical identification of organic compounds occurring in the tropicamide samples, an off-line coupling of HPLC with FT-IR was used. The structure of a standard of apotropicamide was confirmed by (1)H NMR and (13)C NMR analysis. Validation of the HPLC-DAD method of determination both tropicamide and apotropicamide in the tropicamide medical substance was performed. This method is suitable for use in the quality control of tropicamide during its production.

[Enantioseparation of tropicamide by capillary electrophoresis with square wave amperometric detection].

An enantioseparation method for tropicamide by high performance capillary electrophoresis with square wave amperometric detection (SWAD) was developed. The enantiomers of tropicamide were baseline separated in 16 min with an uncoated fused-silica capillary (75 microm i.d. x 50 cm) under the optimum conditions: 7 mmol/L Tris-10 mmol/L citric acid-2 mmol/L H3BO(3)-15 mmol/L beta-cyclodextrin (beta-CD) (pH 3.0) as background electrolyte, SWAD balance potential (E(b)) + 0.80 V (vs. Ag/AgCl), separation voltage 15 kV, injection height 20 cm, and injection time 10 s. The calibration curve of the enantiomers showed good linearity in the range from 5 micromol/L to 750 micromol/L with detection limit of 2 micromol/L. The average recoveries of added standards were 96% - 103%. The effects of the concentrations of beta-CD and the boric acid, the pH of the background electrolyte on resolutions (Rs) of the enantiomers were discussed in details. The proposed method was applied to the determination of a commercial tropicamide eye-drops sample without pre-treatment, and satisfactory results were obtained.

High-performance liquid chromatographic determination of phenylephrine and tropicamide in human aqueous humor.

A high-performance liquid chromatographic method for the simultaneous determination of phenylephrine and tropicamide in human aqueous humor was developed. After centrifugation, an aliquot of the supernatant was injected onto the column and the eluent was monitored at 280 nm then 254 nm after 5 min. Separation was performed on a CN column with 0.01 M Pic B8 (octane sulfonic acid)-acetonitrile (65:35, v/v) as mobile phase. The standard curves were linear in the detection range. The precision of the method (expressed by relative standard deviation) and the accuracy (mean error in per cent) were <5% for both intra- and interassays.

Determination of atropine in biological fluids by micellar electrokinetic capillary chromatography in the presence of strychnine and tetracaine.

The identification and quantitation of atropine, in whole blood and gastric contents in the presence of strychnine and tetracaine is described. This method uses liquid-liquid extraction and micellar electrokinetic chromatography (MECC). Separations are made using a 50 cm long capillary and a borate/phosphate buffer at pH 9.2 with 50 mM sodium dodecyl sulfate (SDS). Linearity was established for the three compounds between 1.0 and 100 microg/mL, using scopolamine as internal standard. The limit of detection for atropine was estimated at 0.06 microg/mL and the limit of quantitation at 0.2 microg/mL. The run time is less than 30 min. Alternate parameters are proposed to reduce the run time to under 10 min. The method was applied to a forensic post-mortem case.

The analysis of aqueous humor constituents using capillary zone electrophoresis.

One of the difficulties encountered in the study of aqueous humor is the relatively small volume generally available for analysis. The purpose of this study was to investigate the potential use of capillary zone electrophoresis (CE) for the analysis of nanolitre quantities of this fluid. Twelve samples of aqueous humor were obtained from patients undergoing cataract surgery and a further three samples were from non cataract post mortem subjects within 6 hr of death. CE was carried out in an uncoated fused silica glass capillary, 75 mu internal diameter and 100 cm long using a run buffer of 40 mM borate pH 9.4 containing 0.4 g l-1 methylcellulose. Detection of the separated zones was by ultra violet absorption at 200 nm. Preliminary identification of peaks was achieved by enzymatic hydrolysis and spiking with purified analytes. A number of very well resolved peaks were obtained from both cataract and post mortem samples using nanolitre quantities of unmodified fluid. Additional peaks were noted in the post mortem samples, most of which were likely to be due to a partial breakdown of the blood aqueous humor barrier. The profiles obtained were not significantly affected by various drugs routinely administered during cataract surgery. This preliminary study has demonstrated the potential value of CE in the analysis of aqueous humor in health and disease.

[Mydriasis caused by plant contact]. / Mydriasis durch Pflanzenkontakt.

Uni- or bilateral dilatation of pupils that are not reactive to light and lack miosis in response to 1% pilocarpine may be caused by contact with plants containing alkaloids such as scopolamine and atropine. Other causes of a non-light-reactive dilated pupil, such as Adie's tonic pupil, third nerve palsy and lesion of the mesencephalic pretectal region, must be excluded before testing the iris sphincter reaction to 1% pilocarpine. Among the naturally growing flowers in Germany, deadly nightshade (Atropa belladonna), jimson weed (thornapple, Datura stramonium) and black henbane (Hyoscyamus niger) contain enough alkaloids to cause mydriasis by direct contact. However, in most cases an accidental mydriasis by plants in Germany is caused by Datura arborea taxa, e.g. Datura suaveolens, D. candida, D. aurea and D. sanguinea. They contain up to 0.6% dry weight scopolamine. These plants can grow very large and are often planted in tubs. They have to be cut back each year before the winter. This is typically how the eye is contaminated by parts of the plants, which can cause dilatation of the pupil mimicing a neuroophthalmological disorder.

[Acute psychosis after cyclopentolate-HCL (Zyklolat) (author's transl)]. / Intoxikationspsychose nach Cyclopentolat-HCL (Zyklolat).

Symptoms of poisoning were noticed in a eight-year-old boy after prescribed application of cyclopentolate 1 per cent (as declared by manufacturing firm). Acute psychosis disappeared spontaneous 3,5 hours later. Pharmacological analysis of the eye-drops showed that concentration of active agent wasn't 1 per cent but 1.31 per cent.