Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A simple high-performance liquid chromatography assay for on-line determination of catecholamines in adrenal gland by direct injection on an ISRP column

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development and validation of a rapid turbidimetric assay to determine the potency of cefuroxime sodium in powder for dissolution for injection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the double agar gel immunodiffusion test and of the enzyme-linked immunosorbent assay in the diagnosis and follow-up of patients with chronic pulmonary aspergillosis

Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES)

Effectiveness of hyperbaric oxygen for experimental treatment of schistosomiasis mansoni using praziquantel-free and encapsulated into liposomes: Assay in adult worms and oviposition

The treatment of schistosomiasis depends on a single drug: praziquantel (PZQ). However, this treatment presents limitations such as low and/or erratic bioavailability that can contribute to cases of tolerance. Improvements to the available drug are urgently needed and studies with a controlled system of drug release, like liposomes, have been gaining prominence. The present study evaluated the activity and synergy between liposomal-praziquantel (lip.PZQ) and hyperbaric oxygen therapy (HBO). Mice received doses of 60 or 100mg/kg PZQ or lip.PZQ, 50 days post-infection, and after the treatment, were exposed to HBO (3 atmosphere absolute - ATA) for 1h. The viability of adult worms and oviposition were analyzed, by necropsy and Kato-Katz examination performed after 15 days of treatment. A concentration of 100mg/kg of lip.PZQ+HBO was more effective (48.0% reduction of worms, 83.3% reduction of eggs/gram of feces) and 100% of the mice had altered of oograms (indicating interruption of oviposition) compared to other treatments and to the Control group (infected and untreated). It is known that PZQ requires participation of the host immune system to complete its antischistosomal activity and that HBO is able to stimulate the immune system. The drug became more available in the body when incorporated into liposomes and, used with HBO, the HBO worked as an adjuvant. This explains the decreases of oviposition and worms recovered form hepatic portal system.

Rapid turbidimetric assay to determine the potency of daptomycin in lyophilized powder

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Improvement of an enzyme immunosorbent assay for detecting antibodies against Dioctophyma renale

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-Dioctophyma renale antibodies in the sera of dogs using, detection of parasite eggs in urine sediment as a reference test. ELISA uses a soluble antigenic preparation of esophagus of D. renale and the optimal dilutions of the antigen, serum and conjugate were determined by means of checker board titration, using positive (n=13) and negative (n=27) reference serum. The specificity and sensitivity of the ELISA were 93.8% and 92.3% respectively and the kappa index was good (0.76). These results suggest that ELISA described may prove to be an effective serological test for detecting dogs infected and exposed to this parasite mainly dogs that are not eliminating parasite eggs through their urine.

Validation of HPLC-UV assay of caffeic acid in emulsions

An accurate, sensitive, precise and rapid reversed-phase high-performance liquid chromatographic method was successfully developed and validated for the determination of caffeic acid (CA) in emulsions. The best separation was achieved on a 250 × 4.6 mm, 5.0 µm particle size RP18 XDB Waters column using ethanol and purified water (40:60 v/v) adjusted to pH 2.5 with acetic acid as the mobile phase at a flow rate of 0.7 mL/min. Ultraviolet detection was performed at 325 nm at ambient column temperature (25°C). The method was linear over the concentration range of 10-60 µg/mL (r(2) = 0.9999) with limits of detection and quantification of 1.44 and 4.38 µg/mL, respectively. CA was subjected to oxidation, acid, base and neutral degradation, as well as photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of CA. The method was applied to the determination of CA in standard and pharmaceutical products with excellent recoveries. The method is applicable in the quality control of CA.

Development and validation of a rapid turbidimetric assay to determine the potency of ampicillin sodium in powder for solution for injection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pan-Assay interference compounds (PAINS): warning signs in biochemical-pharmacological evaluations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development and validation of a stability-indicative turbidimetric assay to determine the potency of doxycycline hyclate in tablets

The doxycycline (DOX) is a broad-spectrum antibiotic used in several countries. This drug is part of the list of medicines to the SUS (Unified Health System), a model of health care in Brazil. This study describes the development and validation of a microbiological assay, applying the turbidimetric method for the determination of DOX, as well as the evaluation of the ability of the method in determining the stability of DOX in tablets against acidic and basic hydrolysis, photolytic and oxidative degradations, using Escherichia coli ATCC 10536 as micro-organism test and 3×3 parallel line assay design, with nine tubes for each assay, as recommended by the Brazilian Pharmacopoeia. The developed and validated method showed excellent results of linearity, selectivity, precision, accuracy and robustness. The assay is based on the inhibitory effect of DOX using Escherichia coli ATCC 10536. The results of the assay were treated by analysis of variance and were found to be linear (r= 0.9986) in the range from 4.0 to 9.0μg/mL, precise (repeatability R.S.D.= 0.99 and intermediate precision was confirmed by statistical analysis the mean values obtained from analysis by different analysts) and exact (97.73%). DOX solution exposed to direct UV light, alkaline and acid hydrolysis and hydrogen peroxide causing oxidation were used to evaluate the specificity of the bioassay. Comparison of bioassay and liquid chromatography showed differences in results between methodologies. The results showed that the bioassay is valid, simple and useful alternative methodology for DOX determination in routine quality control.

A novel and rapid microbiological assay for ciprofloxacin hydrochloride

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Development and validation of a stability-indicative agar diffusion assay to determine the potency of flucloxacillin sodium in capsules

Flucloxacillin sodium (FLU) is a semi-synthetic penicillin active against many gram-positive bacteria such as streptococci and penicilinase-producing staphylococci, including methicillin-susceptible S. aureus. This study describes the development and validation of a microbiological assay, applying the diffusion agar method for the determination of FLU, as well as the evaluation of the ability of the method in determining the stability of FLU in capsules against acidic and basic hydrolysis, photolytic and oxidative degradations, using S. aureus ATCC 25923 as micro-organism test and 3 x 3 parallel line assay design (three doses of the standard and three doses of the sample in each plate), with six plates for each assay, according to the Brazilian Pharmacopoeia. The validation method showed good results including linearity, precision, accuracy, robustness and selectivity. The assay is based on the inhibitory effect of FLU using Staphylococcus aureus ATCC 25923. The results of the assay were treated by analysis of variance (ANOVA) and were found to be linear (r = 0.9997) in the range from 1.5 to 6.0 μg/mL, precise (repeatability: R.S.D. = 1.63 and intermediate precision: R.S.D. = 1.64) and accurate (98.96%). FLU solution (from the capsules) exposed to direct UVC light (254 nm), alkaline and acid hydrolysis and hydrogen peroxide causing oxidation were used to evaluate the specificity of the bioassay. Comparison of bioassay and liquid chromatography by ANOVA showed no difference between methodologies. The results demonstrated the validity of the proposed bioassay, which is a simple and useful alternative methodology for FLU determination in routine quality control.

Animal-side serologic assay for rapid detection of Mycobacterium Bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (n = 129), and wild boar (n = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.

Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.