Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics

Use of mueller-iinton broth and agar in the germ tue test / Utilização de ágar e caldo Mueller-Hinton na prova do tubo germinativo

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

Candida albicans é frequentemente isolada em amostras clínicas, assim a sua diferenciação presuntiva de outras espécies do gênero pode ser baseada na habilidade em formar o tubo germinativo em soro humano. Entretanto, existem outras duas espécies que também possuem essa característica, C. dubliniensis e C. africana. O objetivo foi comparar quatro diferentes substratos para a realização da prova do tubo germinativo (TG). Utilizou-se isolados de Candida spp. identificados através de meio manual (135 C. albicans, 24 C. tropicalis e um C. dubliniensis). A prova do tubo germinativo foi realizada utilizando soro previamente congelado e fresco, caldo e ágar Mueller-Hinton (MH). O TG através da técnica do soro a fresco foi observado em 96% (130/136), 94% (128/136) através do soro previamente congelado, 92% (125/136) no ágar e 90% (122/136) no caldo MH. A sensibilidade de cada teste foi maior que 90% e especificidade de 100%. Tanto o caldo quanto o ágar MH foram capazes de identificar apenas os verdadeiros positivos e não ocorrendo falsos positivos, porém deixaram de identificar alguns isolados de C. albicans. O ágar e o caldo MH podem ser utilizados na rápida e presuntiva identificação laboratorial de C. albicans, como uma alternativa para o teste do tubo germinativo.

Sensibilidad de hongos miceliares dematiaceos a diez antifungicos empleando un metodo de difusion en agar / In vitro susceptibiblity of dematiaceous fungi to ten antifungal drugs using an agar difussion test

Se ha evaluado la utilidad del metodo de difusion en agar NeoSensitabs para determinar la sensibilidad in vitro de 52 aislamientos de hongos filamentosos dematiaceos a diez antifungicos: anfotericina B.5-fluorocitosina, Ketoconasol, fluconazol, itraconazol, terbinafina, bifonazol, miconazol, clotrimazol, y griseofulvina. Para la preparacion del inoculo se ultilizo un metodo espectrofotometrico empleandose los medios de Shadomy y Casitone agar (CAS), simultaneamente. Todos los aislamientos fueron sensibles, al itraconazol, terbinafina y bifonazol. Al ketonazol el 90,4% resultaron sensibles al miconazol el 71% y al clotrimazol el 46%. El 63% de las cepas fueron sensibles a la anfotericina B y el 28,8% resistente. Por el contrario, el 94,2% de los aislamientos resultaron resistentes a la griseofulvina y el 96% al flucanazol. El 100% de las cepas fueron resistentes a la 5-fluorocitosina. Las zonas de inhibicion no mostraron variaciones en cuanto a la sensibilidad dependiendo del medio; sin embargo, hubo un mejor desarrollo fungico en el medio CAS. Las variaciones en la sensibiblidad observadas con especies como Exophiala spinifera y Fonsecaea pedrosi justificarian el estudio de la sensibiblidad in vitro para valorar el tratamiento clinico con antifungicos. Estos resultados demuestran que el metodo de difusion en agar NeoSensitabs es facil de realizar, rapido y economico por lo que esta al alcance de muchos laboratorios clinicos para el estudio de la sensibilidad in vitro en mochos dematiaceos