Lajos Botz

Evaluating aspects of online medication safety in long-term follow-up of 136 Internet pharmacies: illegal rogue online pharmacies flourish and are long-lived.

Background A growing number of online pharmacies have been established worldwide. Among them are numerous illegal websites selling medicine without valid medical prescriptions or distributing substandard or counterfeit drugs. Only a limited number of studies have been published on Internet pharmacies with regard to patient safety, professionalism, long-term follow-up, and pharmaceutical legitimacy verification. Objective In this study, we selected, evaluated, and followed 136 Internet pharmacy websites aiming to identify indicators of professional online pharmacy service and online medication safety. Methods An Internet search was performed by simulating the needs of potential customers of online pharmacies. A total of 136 Internet pharmacy websites were assessed and followed for four years. According to the LegitScript database, relevant characteristics such as longevity, time of continuous operation, geographical location, displayed contact information, prescription requirement, medical information exchange, and pharmaceutical legitimacy verification were recorded and evaluated. Results The number of active Internet pharmacy websites decreased; 23 of 136 (16.9%) online pharmacies ceased operating within 12 months and only 67 monitored websites (49.3%) were accessible at the end of the four-year observation period. However, not all operated continuously, as about one-fifth (31/136) of all observed online pharmacy websites were inaccessible provisionally. Thus, only 56 (41.2%) Internet-based pharmacies were continuously operational. Thirty-one of the 136 online pharmacies (22.8%) had not provided any contact details, while only 59 (43.4%) displayed all necessary contact information on the website. We found that the declared physical location claims did not correspond to the area of domain registration (according to IP address) for most websites. Although the majority (120/136, 88.2%) of the examined Internet pharmacies distributed various prescription-only medicines, only 9 (6.6%) requested prior medical prescriptions before purchase. Medical information exchange was generally ineffective as 52 sites (38.2%) did not require any medical information from patients. The product information about the medicines was generally (126/136, 92.6%) not displayed adequately, and the contents of the patient information leaflet were incomplete in most cases (104/136, 76.5%). Numerous online operators (60/136, 44.1%) were defined as rogue Internet pharmacies, but no legitimate Internet-based pharmacies were among them. One site (0.7%) was yet unverified, 23 (16.9%) were unapproved, while the remaining (52/136, 38.2%) websites were not available in the LegitScript database. Contrary to our prior assumptions, prescription or medical information requirement, or the indication of contact information on the website, does not seem to correlate with “rogue pharmacy” status using the LegitScript online pharmacy verification standards. Instead, long-term continuous operation strongly correlated (P<.001) with explicit illegal activity. Conclusions Most Internet pharmacies in our study sample were illegal sites within the definition of “rogue” Internet pharmacy. These websites violate professional, legal, and ethical standards and endanger patient safety. This work shows evidence that online pharmacies that act illegally appear to have greater longevity than others, presumably because there is no compelling reason for frequent change in order to survive. We also found that one in five websites revived (closed down and reopened again within four years) and no-prescription sites with limited medicine and patient information are flourishing.

Characterization and TLC-bioautographic detection of essential oils from some Thymus taxa. Determination of the activity of the oils and their components against plant pathogenic bacteria

The chemical composition of the oils of four thyme (Lamiaceae) chemotypes (Thymus vulgaris L., Thymus serpyllum L., Thymus x citriodorus (Pers.) Schreb., and Thymus x citriodorus “Archer’s Gold”) has been determined by gas chromatography (GC). Thymol was the main component of the oils of Thymus vulgaris and Thymus serpyllum, geraniol was the main component of the oil of Thymus x citriodorus, and carvacrol was the main component of the oil of Thymus x citriodorus “Archer’s Gold”. The bioactivity of the volatile oil of Thymus vulgaris and of the three main components of the oils against Gram negative plant pathogenic bacteria was examined by direct bioautography. They had an inhibitory effect on all of the test microorganisms. Two bacterial strains (Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola) were most sensitive in the bioautographic system and use of these bacteria would considerably shorten the process of bioautographic detection. The two antibiotics gentamycin and streptomycin were used as controls.

Optimization of conditions for culture of the test bacteria used for direct bioautographic detection. 1. The gram-positive test bacterium Bacillus subtilis

The main purpose of this study was to determine optimum conditions for culture of a test microbe Bacillus subtilis (ATCC 6633) which enabled us to establish its use for direct bioautography. The viability of the bacteria on TLC plates was measured on the basis of their adenosine-5’-triphosphate (ATP) content as determined by bioluminescent luciferin/luciferase assay, the data being referred to values for total bacterial protein. In the first experiments, we used a ‘20-h’ culture of B. subtilis prepared by dilution of an optical density (OD) ≫ 0.4 culture to furnish a culture of OD = 0.4 (Method A). Later, on the basis of our optimization experiments we found that a ‘5–9-h’ broth culture of B. subtilis was suitable. Under these conditions the bacteria remained in the log phase (OD = 0.2–0.4) for 5–9 h (Method B) in immersion bacterial suspension. Because the test bacteria were in the log phase a much shorter incubation time (4–8 h) was sufficient for TLC plates instead of the original 18 h in a previous study. One advantage of this method, in addition to the shorter incubation time, is that we can use TLC plates coated with adsorbents other than silica.

Behaviour analysis of patients who purchase medicines on the internet: can hospital pharmacists facilitate online medication safety?

Background Although there are numerous legal and regulated online pharmacies available on the internet, an abundance of illegitimate online pharmacies are offering medications without prescriptions and deliver products with unknown origins worldwide. Despite the fact that the problem has gained the attention of regulatory and health organisations, the awareness of patients and many healthcare professionals is relatively low. Objectives The purpose of this work is to assess the current situation of ordering medicines online, to survey the attitude of patients regarding online drug purchase, promoting the completion of the medication history worksheet and to recommend useful tools for hospital pharmacists to facilitate online medication safety. Methods The attitude of 422 patients regarding purchasing drugs online was evaluated in a hospital environment. Results 8.4% of Hungarian hospital patients have ordered drugs or dietary supplements online and 3.7% of the respondents are considering this option in the future. Most hospital patients (82.8%) are unaware of the quality of these products. Conclusions Patients are not fully aware of the risks of potential hazards associated with purchasing medicines online and presumably cannot differentiate between legal and illegal online pharmacies. Illegal and counterfeit medicines pose a serious public health risk because the origin and quality of these drugs are uncertain and patients typically take these preparations without the knowledge and supervision of physicians or pharmacists. Pharmacists can play an essential role in protecting patient safety and combating counterfeit medicines.

Pilot study for the characterization of pharmacogenetically relevant CYP2D6, CYP2C19 and ABCB1 gene polymorphisms in the Hungarian population

Polymorphisms of CYP450 metabolizer enzymes and transport proteins play crucial roles in the inter‐individual variability of drug efficiency. The aim of our study was to predict the frequency of functional variants of CYP2D6, CYP2C19 and ABCB1 genes in the Hungarian population. One hundred twelve unrelated healthy subjects donated DNA sample in the study. ABCB1 C3435T and G2677T/A single‐nucleotide polymorphisms (SNPs) were determined by LightCycler polymerase chain reaction. Because only limited amount of data is available on the rare allelic variants of CYP2D6 in the European populations, our study applied an expanded set of CYP2D6 and CYP2C19 alleles by using AmpliChip test. Our results show that the CYP2D6 phenotypes were 1.9% ultra‐rapid metabolizer, 6.5% intermediate metabolizer (IM), 8.3% poor metabolizer (PM) and 83.3% extensive metabolizer (EM), and the CYP2C19 phenotypes were 1.8% PM, 31.2% IM and 67% EM. The prevalence of the commonly observed CYP2D6 and CYP2C19 alleles in our study corresponds with that of other European populations. Nevertheless, our study confirms that extending the CYP2D6 allele set with loss‐of‐function variants such as CYP2D6*7, *9, *41 is worth considering. Frequency of the wild type ABCB1 3435C was 42.8% whereas the prevelance of 2677 G was 50.4%. Although frequency data of G2677T/A SNP in the European area are limited, some discrepancies with other studies were found. Copyright

Effect of rootstocks on floral nectar composition in apple cultivars

Abstract. Insect attraction of flowers in apple cultivars is an essential question from the viewpoint of fruit yield, since apple flowers are autosterile and entomogamous. Therefore, it is important to study the two most significant factors of primary attraction, pollen and nectar production. The composition of nectar in apple cultivars on different rootstocks was investigated, putting an emphasis on sugar components. It was stated that the composition of nectar and the ratio of mono- and disaccharides was almost identical and constant in trees on rootstock M.4, whereas on other rootstocks the effects of the season were more pronounced. In triploid cultivars on rootstocks M.9 and M.26 nectar can be characterised by the dominance of sucrose. Relationship was found between free fertilisation rate of cultivars and sugar amount of nectar. In triploid cultivars nectar sugar content was usually lower than in diploid ones, and reached the bee visitation threshold only in some years, influenced also by the effect of rootstocks.

HPTLC and HPLC determination of alkaloids in poppies subjected to stress

Papaver somniferum produces secondary metabolites which have important roles in self-defense processes, in plant biochemistry, and in allelochemistry. By performing experiments to determine how irregular stress effects changed the alkaloid content of poppies we have shown that different types of stress affect the quantities of alkaloids. Papaver somniferum (cv. ‘Kék Duna’, Budakalasz, Hungary) plants were grown for 2 months, from seeds, in quartz-sand (natural light, temperature 24–28°C, in Knopf’s nutritive solution). In this work we studied alkaloids in poppies subjected to two kinds of stress - mycotoxin and drought. Amounts of alkaloids were measured by different separation and detection procedures - thin-layer chromatography (TLC and HPTLC) with fluorescence detection, and high-performance liquid chromatography (HPLC). HPLC proved superior for identification and approximate estimation of the morphine alkaloids, but the effects of stress on poppy plants can be detected by use of either method. Drought stress resulted in higher levels of the alkaloids whereas mycotoxin stress did not result in significant differences.

Optimization of bioassay method for the quantitative microbiological determination of amphotericin B.

The aim of this study was to find the optimal bioassay parameters for the quantitative analysis of an amphotericin B nasal spray solution as the bioassay conditions recommended by the Ph. Eur. 6. were less sensitive and were only applicable for the measurement of a narrow concentration range, which makes the method unsuitable in case of a stability test. We evaluated five commonly used assay media with Candida albicans and Saccharomyces cerevisiae as test organisms. Our results showed that Mueller Hinton Agar supplemented with 2% glucose and 0.5 μg ml−1 methylene blue inoculated with C. albicans gave the best bioassay circumstance as a wide concentration range (1.54–60.0 μg ml−1 amphotericin B) could be measured and the inhibition zone borders were distinct and easy to read.

Optimization of conditions for culture of test bacteria used for direct bioautographic TLC detection. 2. Gram-negative test bacterium: Escherichia coli

Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a complex mixture. In this process the developed TLC plate is dipped into a broth culture of a test bacterium and the bacterium will grow directly on the plate. Optimum experimental conditions must, however, be used for each test bacterium. The main purpose of this study was to find optimum culture conditions for a Gram-negative test bacterium, Escherichia coli (ATCC 25922) enabling us to establish a direct bioautographic method with the shortest possible performance time. Because the intracellular adenosine-5’-triphosphate (ATP) level is a direct and sensitive measure of bacterial well-being, ATPassay was used for this purpose. As far as we know this is the first report of the use of an ATP method for optimization of direct bioautography with E. coli. Our optimizing experiments on E. coli culture showed that the bacteria had to be in the log phase (optical density, OD600nm = 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the logphase culture needed a shorter incubation time for bacterial growth on the TLC plate (3 h) than for the original ‘overnight’ culturing suggested in studies by others. In this paper we will show that: – ATP assay is a valid method for optimizing E. coli direct bioautography. – Bacterial ATP level oscillates during the growth phase in culture media. – TLC plates should be immersed in E. coli dipping suspension with OD600nm = 0.1–0.4. – Dipping a developed TLC plate for 10 s gave acceptable results. – Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum. – An ATP/protein ratio of 10–15 nmol mg−1 in dipping culture and ~5 nmol mg−1 on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction. – With our optimized coditions the total performance time of E. coli direct bioautography is 9.6 h instead of the originally reported 11.5 h. – Our procedure results in much sharper contrast of the inhibition zone than that without optimization. – ATP assay is a valid method for optimizing E. coli direct bioautography. – Bacterial ATP level oscillates during the growth phase in culture media. – TLC plates should be immersed in E. coli dipping suspension with OD600nm = 0.1–0.4. – Dipping a developed TLC plate for 10 s gave acceptable results. – Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum. – An ATP/protein ratio of 10–15 nmol mg−1 in dipping culture and ~5 nmol mg−1 on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction. – With our optimized coditions the total performance time of E. coli direct bioautography is 9.6 h instead of the originally reported 11.5 h. – Our procedure results in much sharper contrast of the inhibition zone than that without optimization.

Optimization of Growth Conditions for Test Fungus Cultures used in Direct Bioautographic TLC Detection. 3. Test Fungus: Candida albicans

Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones in direct bioautographic TLC. Bioluminescent ATP assay is a highly sensitive method for optimizing the viability of Candida albicans test fungus in bioautographic TLC detection. A suspension of microbes (OD600nm = 0.5-0.7, in Mueller-Hinton broth with 5% glucose) in the log phase of growth can be used for dipping TLC plates. On the basis of our results with Candida albicans, we can differentiate between microbiostatic (bacteriostatic or fungistatic) and microbio-cidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioauto-grams. This technique leads to new possibilities in studies of the interactions between microbes and antimicrobial compounds on bioautographic silica gel TLC plates by using scanning electron microscopy. In this paper we describe an optimization procedure for bioautographic TLC detection.