Population pharmacokinetic modelling and evaluation of different dosage regimens for darunavir and ritonavir in HIV-infected individuals.

OBJECTIVES: Darunavir is a protease inhibitor that is administered with low-dose ritonavir to enhance its bioavailability. It is prescribed at standard dosage regimens of 600/100 mg twice daily in treatment-experienced patients and 800/100 mg once daily in naive patients. A population pharmacokinetic approach was used to characterize the pharmacokinetics of both drugs and their interaction in a cohort of unselected patients and to compare darunavir exposure expected under alternative dosage regimens. METHODS: The study population included 105 HIV-infected individuals who provided darunavir and ritonavir plasma concentrations. Firstly, a population pharmacokinetic analysis for darunavir and ritonavir was conducted, with inclusion of patients' demographic, clinical and genetic characteristics as potential covariates (NONMEM(®)). Then, the interaction between darunavir and ritonavir was studied while incorporating levels of both drugs into different inhibitory models. Finally, model-based simulations were performed to compare trough concentrations (Cmin) between the recommended dosage regimen and alternative combinations of darunavir and ritonavir. RESULTS: A one-compartment model with first-order absorption adequately characterized darunavir and ritonavir pharmacokinetics. The between-subject variability in both compounds was important [coefficient of variation (CV%) 34% and 47% for darunavir and ritonavir clearance, respectively]. Lopinavir and ritonavir exposure (AUC) affected darunavir clearance, while body weight and darunavir AUC influenced ritonavir elimination. None of the tested genetic variants showed any influence on darunavir or ritonavir pharmacokinetics. The simulations predicted darunavir Cmin much higher than the IC50 thresholds for wild-type and protease inhibitor-resistant HIV-1 strains (55 and 550 ng/mL, respectively) under standard dosing in >98% of experienced and naive patients. Alternative regimens of darunavir/ritonavir 1200/100 or 1200/200 mg once daily also had predicted adequate Cmin (>550 ng/mL) in 84% and 93% of patients, respectively. Reduction of darunavir/ritonavir dosage to 600/50 mg twice daily led to a 23% reduction in average Cmin, still with only 3.8% of patients having concentrations below the IC50 for resistant strains. CONCLUSIONS: The important variability in darunavir and ritonavir pharmacokinetics is poorly explained by clinical covariates and genetic influences. In experienced patients, treatment simplification strategies guided by drug level measurements and adherence monitoring could be proposed.

Comparison of Methods for Evaluation of the Bactericidal Activity of Copper-Sputtered Surfaces against Methicillin-Resistant Staphylococcus aureus.

Bacteria can survive on hospital textiles and surfaces, from which they can be disseminated, representing a source of health care-associated infections (HCAIs). Surfaces containing copper (Cu), which is known for its bactericidal properties, could be an efficient way to lower the burden of potential pathogens. The antimicrobial activity of Cu-sputtered polyester surfaces, obtained by direct-current magnetron sputtering (DCMS), against methicillin-resistant Staphylococcus aureus (MRSA) was tested. The Cu-polyester microstructure was characterized by high-resolution transmission electron microscopy to determine the microstructure of the Cu nanoparticles and by profilometry to assess the thickness of the layers. Sputtering at 300 mA for 160 s led to a Cu film thickness of 20 nm (100 Cu layers) containing 0.209% (wt/wt) polyester. The viability of MRSA strain ATCC 43300 on Cu-sputtered polyester was evaluated by four methods: (i) mechanical detachment, (ii) microcalorimetry, (iii) direct transfer onto plates, and (iv) stereomicroscopy. The low efficacy of mechanical detachment impeded bacterial viability estimations. Microcalorimetry provided only semiquantitative results. Direct transfer onto plates and stereomicroscopy seemed to be the most suitable methods to evaluate the bacterial inactivation potential of Cu-sputtered polyester surfaces, since they presented the least experimental bias. Cu-polyester samples sputtered for 160 s by DCMS were further tested against 10 clinical MRSA isolates and showed a high level of bactericidal activity, with a 4-log(10) reduction in the initial MRSA load (10(6) CFU) within 1 h. Cu-sputtered polyester surfaces might be of use to prevent the transmission of HCAI pathogens.

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60ºC, for 30 min, and among them, 12 (18.75%) were resistant at 60ºC, for 60 min. Also, none strain was resistant at 100ºC. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity

Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.

Acute haematogenous prosthetic joint infection: prospective evaluation of medical and surgical management

The optimum treatment for prosthetic joint infections has not been clearly defined. We report our experience of the management of acute haematogenous prosthetic joint infection (AHPJI) in patients during a 3-year prospective study in nine Spanish hospitals. Fifty patients, of whom 30 (60%) were female, with a median age of 76 years, were diagnosed with AHPJI. The median infection-free period following joint replacement was 4.9 years. Symptoms were acute in all cases. A distant previous infection and/or bacteraemia were identified in 48%. The aetiology was as follows: Staphylococcus aureus, 19; Streptococcus spp., 14; Gram-negative bacilli, 12; anaerobes, two; and mixed infections, three. Thirty-four (68%) patients were treated with a conservative surgical approach (CSA) with implant retention, and 16 had prosthesis removal. At 2-year follow-up, 24 (48%) were cured, seven (14%) had relapsed, seven (14%) had died, five (10%) had persistent infection, five had re-infection, and two had an unknown evolution. Overall, the treatment failure rates were 57.8% in staphylococcal infections and 14.3% in streptococcal infections. There were no failures in patients with Gram-negative bacillary. By multivariate analysis, CSA was the only factor independently associated with treatment failure (OR 11.6; 95% CI 1.29-104.8). We were unable to identify any factors predicting treatment failure in CSA patients, although a Gram-negative bacillary aetiology was a protective factor. These data suggest that although conservative surgery was the only factor independently associated with treatment failure, it could be the first therapeutic choice for the management of Gram-negative bacillary and streptococcal AHPJI, and for some cases with acute S. aureus infections.

SYNTHESIS AND BIOLOGICAL EVALUATION OF SPIRO[INDOLINE-3,4’-PYRANO[2,3-C:6,5-C’]DIPYRAZOL]- 2-ONES IN THE PRESENCE OF SBA-Pr-SO3H AS A NANOCATALYST

Sulfonic acid functionalized SBA-15 nanoporous material (SBA-Pr-SO3H) with a large pore size of 6 nm, a high surface area, high selectivity, and excellent chemical and thermal stability was applied as an efficient heterogeneous nanoporous acid catalyst in the reaction of isatin with pyrazolones under mild reaction conditions. A novel class of symmetrical spiro[indoline-3,4'-pyrano[2,3-c:6,5-c']dipyrazol]-2-one derivatives was successfully obtained in high yields. Comparison of these results with those reported in the literature shows that the current method is efficient, and results in better reaction times and yields of the desired products. Other advantages of this new method are its operational simplicity, easy work-up procedure, and the use of SBA-Pr-SO3H as a reusable and environmentally benign nanoreactor, such that the reaction proceeds easily in its nanopores. We also tested the antimicrobial activity of the prepared compounds using the disc diffusion method, and some of the synthesized compounds exhibited the best results against B. subtilis and S. aureus.

Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus)

The aim of this study was to research the occurrence of Salmonella spp. and Escherichia coli in feces samples of sparrows, as well as to identify the pathogenicity, cytotoxicity and sensitivity profile of the isolates to antimicrobial use. Two hundred and twenty eight sparrows were captured in eight farms. The in vitro pathogenicity test was performed by the isolates culture on congo red-magnesium oxalate Agar, whilst the in vivo pathogenicity test was performed in one day-old chicks. In order to study the cytotoxic effects of indicators, samples were inoculated into Vero cells. The results obtained for Escherichia coli isolation confirmed the presence of this microorganism in 30 (13.2%) of the evaluated samples. Out of those isolates, 10 (33.3%) presented the capacity of absorbing ongo red. As for in vivo pathogenicity a 68.0% of mortality rate of the evaluated samples was observed. Out of 20 isolates tested for cytotoxin production, none of them presented cytotoxic effect in the Vero cells. The Salmonella spp was isolated only in one sample (0.04%), and it was identified as Salmonella enterica subspecies houtenae. Results obtained through this research indicate the need for new studies to identify other virulence factors of E. coli samples and to delineate the phylogenetic profile of the isolates in order to establish a relation with colibacillosis outbreaks in chickens and broilers in the studied region, as well as to analyze the critical points in the aviculture productive chain to identify the source of Salmonella enterica subspecies houtenae.

Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis

The objective of this study was to evaluate herd management practices and mastitis treatment procedures as risk factors associated with Staphylococcus aureus antimicrobial resistance. For this study, 13 herds were selected to participate in the study to evaluate the association between their management practices and mastitis treatment procedures and in vitro antimicrobial susceptibility. A total of 1069 composite milk samples were collected aseptically from the selected cows in four different periods over two years. The samples were used for microbiological culturing of S. aureus isolates and evaluation of their antimicrobial susceptibility. A total of 756 samples (70.7%) were culture-positive, and S. aureus comprised 27.77% (n=210) of the isolates. The S. aureus isolates were tested using the disk-diffusion susceptibility assay with the following antimicrobials: ampicillin 10mg; clindamycin 2μg; penicillin 1mg; ceftiofur 30μg; gentamicin 10mg; sulfa-trimethoprim 25μg; enrofloxacin 5μg; sulfonamide 300μg; tetracycline 30μg; oxacillin 1mg; cephalothin 30μg and erythromycin 5μg. The variables that were significantly associated with S. aureus resistance were as follows: the treatment of clinical mastitis for ampicillin (OR=2.18), dry cow treatment for enrofloxacin (OR=2.11) and not sending milk samples for microbiological culture and susceptibility tests, for ampicillin (OR=2.57) and penicillin (OR=4.69). In conclusion, the identification of risk factors for S. aureus resistance against various mastitis antimicrobials is an important information that may help in practical recommendations for prudent use of antimicrobial in milk production.

Evaluation of the efficacy of commercial sanitizers against adhered and planktonic cells of Listeria monocytogenes and Salmonella spp.

Antimicrobial activities of two commercial disinfectants, alone or combined with heat, against three Salmonella strains and three Listeria monocytogenes strains were studied. The efficacy of disinfectants against planktonic bacteria and bacteria attached to three food contact industrial surfaces (stainless steel, polytetraflourethylene, and rubber) was investigated. The tests were conducted using the sanitizer (quaternary ammonium compounds, and alquyldiethylenediamineglycine and di-alquyldiamineethylglycine) concentrations recommended by the manufacturers, and concentrations twice and four times higher than those values. The recommended concentrations were not effective to kill bacteria, especially when they were attached to surfaces. Concentrations of disinfectants twice and four times higher than those recommended were needed to fully eliminate planktonic bacteria. These same sanitizer concentrations were not sufficient to remove attached bacteria. To remove them from the surfaces, a treatment with recommended concentrations in combination with heat was needed. Our results indicate that these two pathogenic bacteria could survive common sanitation programs used in the food industry.

Synthesis of antimicrobial peptides derived from BP100 and BPC194

In the present PhD thesis we studied the solid-phase peptide synthesis of antimicrobial peptides derived from the lead peptides BP100 and BPC194. First, peptides derived from BP100 containing D-amino acids at different positions of the sequences were prepared. Moreover, peptidotriazoles derived from BP100 were also synthesized containing the triazole ring at the side-chain of different amino acids. Then, we proceeded to perform studies for the synthesis of multivalent peptides derived from BPC194. To achieve this objective, the synthesis of cyclic peptides containig a triazole ring at amino acids side-chain with different elongations was carried out. Finally, we prepared various carbopeptides containing 2 and 4 units of BP100 and/or its derivatives. The evaluation of the biological activity allowed the identification of active sequences against the economically important phytopathogenic bacteria and fungi and not toxic against eukaryotic cells.

In vitro evaluation of the fermentation properties and potential prebiotic activity of caprine cheese whey oligosaccharides in batch culture systems

The prebiotic effect of oligosaccharides recovered and purified from caprine whey, was evaluated by in vitro fermentation under anaerobic conditions using batch cultures at 37ºC with human faeces. Effects on key gut bacterial groups were monitored over 24h by fluorescence in situ hybridisation (FISH), which was used to determine a quantitative prebiotic index score. Production of short-chain fatty acids (SCFAs) as fermentation end products was analysed by high-performance liquid chromatography (HPLC). Growth of Bifidobacterium spp was significantly higher (p ≥ 0.05) with the purified oligosaccharides compared to the negative control. Lactic and propionic acids were the main SCFAs produced. Antimicrobial activity of the oligosaccharides was also tested, revealing no inhibition though a decrease in Staphylococcus aureus and Escherichia coli growth. These findings indicate that naturally extracted oligosaccharides from caprine whey could be used as new and valuable source of prebiotics.

Assessment of antimicrobial effect of Biosilicate (R) against anaerobic, microaerophilic and facultative anaerobic microorganisms

This study assessed the antimicrobial activity of a new bioactive glass-ceramic (Biosilicate (R)) against anaerobic, microaerophilic, and facultative anaerobic microorganisms. Evaluation of the antimicrobial activity was carried out by three methods, namely agar diffusion, direct contact, and minimal inhibitory concentration (MIC). For the agar diffusion technique, bio glass-ceramic activity was observed against various microorganisms, with inhibition haloes ranging from 9.0 +/- 1.0 to 22.3 +/- 2.1 mm. For the direct contact technique, Biosilicate (R) displayed activity against all the microorganisms, except for S. aureus. In the first 10 min of contact between the microorganisms and Biosilicate (R), there was a drastic reduction in the number of viable cells. Confirming the latter results, MIC showed that the Biosilicate (R) inhibited the growth of microorganisms, with variations between <= 2.5 and 20 mg/ml. The lowest MIC values (7.5 to <= 2.5 mg/ml) were obtained for oral microorganisms. In conclusion, Biosilicate (R) exhibits a wide spectrum of antimicrobial properties, including anaerobic bacteria.